Planta 139, 289- 299 (1978)
P l a n t a 9 by Springer-Verlag 1978
Some Properties of Giutamine Synthetase from Anabaena cylindrica S.K. Sawhney and D.J.D. Nicholas Department of Agricultural Biochemistry, Waite Agricultural Research Institute, University of Adelaide, Glen Osmond, South Australia, 5064
Abstract. Some properties of the biosynthetic and 7-glutamyltransferase activities of glutamine synthetase (EC 6.3.1.2) from A n a b a e n a c y l i n d r i c a are described, including requirement for divalent cations, pH optimum and K m for substrates. The y-glutamyltransferase reaction was inhibited by L-glutamate, ammonia and ATP. The inhibition by L-glutamate and ammonia was competitive for L-glutamine and non-competitive for hydroxylamine. Both the biosynthetic and the 7-glutamyltransferase activities of the desalted enzyme were much more sensitive to inactivation by treatments such as urea, hydroxylamine and incubation at 50~ than the preparation which contained a divalent cation. The effects of some substrates of these reactions on protection against thermal denaturation and hydroxylamine were examined. An interpretation of these results in terms of the sequence of binding of substrates both in the biosynthetic and the 7-glutamyltransferase reactions are discussed. Key words: A n a b a e n a - Biosynthetic and transferase activities - Glutamine synthetase.
Introduction
Glutamine synthetase (L-glutamate:ammonia tigase, ADP, EC 6.3.1.2) plays an important role in nitrogen metabolism of bacteria (Nagatani et al., 1971 ; Brown et al., 1974), algae (Dharmawardene et al., 1972; Stewart and Rowell, 1975 ; Wolk et al., 1976 ; Thomas et al., 1977) and plants (Stewart and Rhodes, 1976; Miflin and Lea, 1976) since glutamine formed in the reaction is utilized for a variety of nitrogenous metabolites of a cell. In addition to catalyzing biosynthetic reaction (reaction I), highly purified preparations of glutamine synthetase from various organisms exhibit
several other activities (Meister, 1974; Stadtman and Ginsburg, 1974). Among these, 7-glutamyltransferase activity (reaction II) has been used extensively during purification of the enzyme by various workers : L-Glutamate + ATP + NH 3 -
Mez+
L-Glutamine + ADP + Pi
(i)
L-Glutamine + NH2OH Me2+'ADP, arsenate or Pi
7-Glutamylhydroxamate+ NH 3
0I)
The relationship between these two activities of glutamine synthetase is not known. Investigations by Levintow et al. (1955) discounted the possibility that 7glutamyltransferase reaction is the reversal of biosynthetic activity in which hydroxylamine substitutes for ammonia. It has been stated that y-glutamyltransferase activity might represent a simple uncoupling of the reverse biosynthetic reaction (Hunt et al., 1975). Hubbard and Stadtman (1967b) mentioned that the substrate-binding sites for these two activities are probably shared by a corresponding pair of substrates like glutamate and glutamine, ammonia and hydroxylamine and ATP and ADP. There is, however, no experimental evidence for these common sites. Although glutamine synthetase from bacteria (Hubbard and Stadtman, 1967a; Deuel and Stadtman, 1970; Stadtman and Ginsburg, 1974; Hachimori et al., 1974), plants (Elliot, 1953; Kanamori and Matsumoto, 1972; O'Neal and Joy, 1973) and animal tissues (Ronzio et al., 1969; Tate et al., 1972; Seyama et al., 1972; Tare and Meister, 1971) has been extensively characterized, little information (Dharmawardene et al., 1973) is available about this enzyme in algae. In this paper the properties as well as the relation between the biosynthetic and 7-glutamyl-
290
S.K. Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena
transferase activities of glutamine synthetase are described for a blue-green algae, Anabaena cylindrica.
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Materials and Methods ~0.6
Materials
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A culture of the blue-green alga, Anabaena eylindriea (Lemmerman strain 1403/2A) was obtained from the Culture Collection of Algae and Protozoa, The Botany School, Cambridge, U.K. L-glutamate, L-glutamine, 7-glutamylhydroxamate and ATP were purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A. /7mercaptoethanol was from B.D.H., Poole, England, Hydroxylamine hydrochloride was bought from Ajax Chemical Ltd., Sydney. All the other chemicals used in these investigations were of analytical grade.
Methods Preparation of Cell-Free Extract. Anabaena cylindrica was grown, in a medium containing 5 mmol 1- ~ KNO3, as described by Brownell and Nicholas (1967). All the operations during harvesting and preparation of cell-free extracts were done at 4~C. The sevendays old cells were collected by centrifuging at ),000 x g for 10 rain. The cells were broken by passing them through an Aminco French pressure cell at 16,000 p.s.i. The homogenate was centrifuged at 6,000 x g for 20 min and the supernatant fraction ($6) obtained was used for the purification of glutamine synthetase.
DesaIting Procedure. In some of the experiments the purified enzyme preparation was used after desalting. About 5 ml of the enzyme was passed through a sephadex G-10 column (2.5 • 20 cm) which had been previously equilibrated with 0.1 mol 1 1 Tris-HC1 pH 7.5. The enzyme was eluted from the column with the same buffer. The desalted enzyme was always examined for both the biosynthetic and the y-glutamyltransferase activities, in the absence of divalent cation, to ascertain complete removal of MgC12 from the preparation. Enzyme Assays. Both the biosynthetic and 7-glutamyltransferase activities of glutamine synthetase were determined by the procedures described by Shapiro and Stadtman (1970). The reaction mixture for ~/-glutamyltransferase activity, in a final volume of 1 ml contained, in ~tmol: Imidazole-HC1 buffer (pH 7.0), 40; L-glutamine, 30; hydroxylamine hydrochloride (neutralized with NaOH), 30; MnCI2, 3.0; sodium arsenate, 2.0; ADP, 0.4 and an appropriate amount of the enzyme. Control tubes from which L-glutamine was omitted were always included. The amount of 7-glutamylhydroxamate produced after 15 rain at 37~ was determined colorimetrically using a reference curve prepared from authentic ,/-glutamylhydroxamate (Sigma Chemical Co~, USA). For the biosynthetic acti+ity, the assay mixture in a final volume of 0.3 ml contained in lamol: Imidazole-HC1 buffer (pH 7.0), 10; .Lglutamate, 10; MgC12, 20; NH4C1, 10; ATP, 3,0 and the enzyme preparation. From the control tubes L-glutamate was omitted. The amount of Pi produced after 30 rain was determined as described by Shapiro and Stadtman (1970). For every batch of purified glutamine synthetase, the amount of enzyme for the biosynthetic and y-glutamyltransferase activities was determined so that, under the conditions of the assay, the activity was linear over a period of 45 min.
Protein Content. Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
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1"4 :~
30
40
50
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60
70
80
90
100
number
Fig. 1. Elution profile for glutamine synthetase from a Sepharose 6B column. Experimental details are given in the text. Enzyme activity was determined by measuring the rate of 7-glutamylhydroxamate (v-GH) production. Reaction mixture in a final volume of 1 ml contained, in gmol: Imidazole-HC1 buffer (pH 7.0) 40; L-glutamine, 30; hydroxylamine, 30; ADP, 0.4; sodium arsenate, 2; MnC12, 3 and 0.2 ml of enzyme. Reaction carried out at 37 ~ C for 15 rain. Absorbance at 280 nm, e - - e ; Enzyme activity, i - - i
Enzyme Purification Glutamine synthetase from Anabaena cylindrica was purified by the procedure described below. Unless otherwise stated all the operations were carried out at 4~
Heat Treatment. Cell-free extract (S 6 fraction) was kept in a water bath at 50~ for 20 min. After cooling, the precipitated proteins were removed by centrifuging at 30,000 x g for 30 min.
p H Precipitation. The pH of the supernatant ($30 heated) from the preceding step was adjusted to 5.2 with M-acetic acid. After standing it on ice for 30 min, it was centrifuged at 30,000 x g for 30 min. The pellet was discarded and pH of the supernatant fraction was brought to 4.0 by further addition of M-acetic acid. After 30 rain, the preparation was centrifuged at 30,000 x g for 30 min and the pellet dissolved in 17 ml of 0.1 mol 1-1 Tris-HC1 buffer (pH 7.5) containing 10 mmol 1-1 MgC12.
Ammonium Sulphate Precipitation. Solid ammonium sulphate was added gradually, to the above preparation, to obtain 30% saturation. The precipitated proteins were removed after 20 rain by centrifuging at 30,000 x g for 30 min. The ammonium sulphate concentration was then raised to 60% saturation. The pellet, after centrifuging at 30,000 x g for 30 rain, was dissolved in 6 ml of extraction buffer and dialyzed overnight against the same buffer. Gel Filtration, The dialyzed enzyme was then loaded onto a Sepharose 6B column (3 x 70 cm) which had been previously equilibrated with 0.1 mol 1 1 Tris-HCl (pH 7.5) containing 10 mmol 1-1 MgC12. The sample was eluted with the same buffer at a flow rate of 12 ml/h and 5 ml fractions were collected. The elution profiles for protein and glutamine synthetase are shown in Figure 1. The enzyme was recovered in the fractions just prior to the bluishcoloured second protein peak. The pooled fractions from the column had 20-25-fold higher activity than the $6 preparation and about 13% of the original activity was recovered by this procedure (Table 1). This partially purified enzyme was stable for several weeks at 0~
S.K. Sawhney a n d D.J.D. Nicholas: Glutamine Synthetase from Anabaena
291
Table 1. Purification of glutamine synthetase from Anabaena cylindrica Experimental details given in the text. Enzyme activity was determined as described in Figure 1. One unit of enzyme activity represents 1 ~tmol of 7-glutamylhydroxamate produced per min Purification step
Total volume (ml)
Total activity (units)
Total protein (rag)
% recovery
$6 Supernatant of broken cells centrifuged at 6000 x g for 10 min
75
307.5
517.5
100
0.6
1
S30(heated) S 6 heated to 50~ for 20 rain and centrifuged at 30000 x g for 30 min
71
305.3
216.5
99
1.4
2
Pellet pH 4.0
20
243.0
61.0
79
4.0
7
6
125.0
26.1
41
4.8
8
88
39.8
13
15.1
26
30 60% a m m o n i u m sulphate fraction Pooled Sepharose 6B fractions
2.64
Specific activity units/mg protein
Purification (fold)
Results
Table 2. Biosynthetic and y-glutamyltransferase activities with various divalent cations
Properties of Biosynthetic and y-Glutamyhransferase Activities
Biosynthetic and ~/-glutamyltransferase activities were determined from the rates of formation of Pi and y-glutamylhydroxamate, respectively. Purified enzyme preparation was used after desalting through a Sephadex G-10 column (2.5 x 20 cm). For the biosynthetic activity, the reaction mixture in a final volume of 0.3 ml contained, in Ixmol: hnidazole-HCl buffer (pH 7.0), 10; L-glutamate, 20; a m m o n i u m chloride, 10; ATP, 3; enzyme preparation 0.25 ml (I0 ~tg protein) and the specified concentrations of various cations. The a m o u n t of Pi produced after 45 min at 37~ was determined as in Methods. The assay mixture for q/-glutamyltransferase activity was as described in Figure 1 except that M n 2 § was replaced by other cations as indicated in the table
Both the biosynthetic and y-glutamyltransferase reactions were catalyzed by the 25-fold purified glutamine synthetase preparation. The properties of these two activities of the enzyme were compared.
Divalent Cation Specificity. The relative rates of biosynthetic activity, at p H 7.0, in the presence of various divalent cations are given in Table 2. At 70 mmol 1- t, m a x i m u m activity was obtained with Mg 2 +. The observed order of effectiveness of the different cations was Mg 2+ > Co 2+ > Cu 2+ > Fe z+ > Mn 2+ = Z n 2+ . The lower activity at 14 mmol 1-1 Mg 2+ than with Co 2+ was due to a relatively higher concentration of Mg 2+ required to saturate the enzyme. This effect is illustrated in Figure 2A; thus at lower concentrations of either Mg z+ or Co z+, enzyme activity with the latter was higher than with Mg 2+. However, at saturating concentrations of the metal ions, activity with Mg 2+ was significantly greater than with Co 2+. For 7-glutamyltransferase activity, Mn z+ was the most effective cation followed by Mg 2+ and Co 2+ (Table 2). A relatively small activity was obtained with Cu 2+ and the other cations Zn z+, Fe 2+, Ba 2+, Ca 2+ were ineffective. Marked differences were observed in the concentrations of Mn 2+, Co 2+ and Mg 2+ required for m a x i m u m enzyme activity (Figs. 2B, C, D). As found for the biosynthetic reaction, relatively higher amounts of Mg 2+ than Co 2+ were required to saturate the enzyme.
Effect ofpH. The p H optimum for the biosynthetic reaction was influenced by the type of activating metal ion used. Thus m a x i m u m activities with Mg 2+
Metal ion
Mg 2+ M n 2+ Co 2+ Cu 2+ Fe 2+ Zn 2+ Ni 2+ Ca 2+ a b
Biosynthetic activity a Metal ion (mmol i- 1)
7-Glutamyltransferase activity u Metal ion (mmol 1 1)
1.4
14
70
0.5
5.0
50.0
20 20 20 20 20 20 7 7
160 73 184 20 80 50 20 11
280 80 157 123 107 80 34 34
0.24 1.60 0.22 0.07 0 0 0 0
0.35 1.43 0.29 0.16 0 0 0 0
0.43 0.60 0.21 0.07 0 0 0 0
nmol Pi produced in 45 min gmol ~/-glutamylhydroxamate produced in 30 rain
( 1 0 0 m m o l l - 1 ) , Co 2+ ( 3 5 m m o l l -~) and Mn 2+ (20 m m o l l 1) w e r e obtained at pH 7.3, 6.9 and 6.0 respectively. However, an identical p H o p t i m u m of 8.0 was observed for the y-glutamyltransferase reaction with all three cations (concentrations used were 10 m m o l 1-1 of either Mg 2+ or Co 2+ and 3 m m o l 1-a Mn2+). At optimal conditions of p H and metal ion concentration, the biosynthetic activities with Co 2+ and Mn 2+ were 50 and 25% respectively of that with
292
S.K. S a w h n e y a n d D . J . D . N i c h o l a s : G l u t a m i n e S y n t h e t a s e f r o m A n a b a e n a
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Fig. 2 A - D , Biosynthetic a n d 7 - g l u t a m y l t r a n s f e r a s e activities in the presence o f v a r i o u s c o n c e n t r a t i o n s of divalent cations. E x p e r i m e n tal details as given in T a b l e 2. A B i o s y n t h e t i c activity with M g 2+, 9 9 OF C o 2 +, A - I k . B, C, D. y-glutamyltransferase activity with M n 2 +, Co 2 + a n d M g z+, respectively. ( ? - G H = y - g t u t a m y l h y d r oxamate)
Fig. 3. Effect of v a r y i n g c o n c e n t r a t i o n s of L - g l u t a m a t e on the M g 2+ a n d C o Z + - d e p e n d e n t b i o s y n t h e t i c activity. D e t a i l s of the r e a c t i o n m i x t u r e as given in T a b l e 2 except t h a t the c o n c e n t r a t i o n of L - g l u t a m a t e was v a r i e d as i n d i c a t e d a n d the p H of the assay m i x t u r e s for d e t e r m i n i n g the activities w i t h M g 2+ a n d C o 2+ were 7.5 a n d 6.5 respectively. Purified e n z y m e p r e p a r a t i o n was used after d e s a l t i n g t h r o u g h a S e p h a d e x G-10 column. C o n c e n t r a t i o n s of d i v a l e n t cations u s e d were: M g 1+, 70 m m o l l - 1 9 9 C o 2+ 30mmoll • 9 9
Mg 2+ and for the 7-glutamyltransferase reaction Mg 2+ and Co 2+ gave 35 and 30% of the activity obtained with Mn 2+.
catalyzed by Mn 2 + did not have an absolute requirement for A D P but was stimulated by about 35% on adding this nucleotide. M a x i m u m activities with all the three cations were obtained with 0.05 m m o l 1ADP.
Requirements for Enzyme Activity. The biosynthetic activity, with either Mg 2+, Co 2+ or Mn 2+ was dependent on the presence of L-glutamate, a m m o n i u m chloride and ATP. Negligible amounts of P~ were liberated when any of these components were omitted from the reaction mixture. When hydroxylamine substituted for a m m o n i u m chloride, then only 30% of the activity was recorded. All the components of the standard 7-glutamyltransferase reaction mixture were required for the Mg 2+- and Co2+-dependent activities. Less than 5% 7-glutamylhydroxamate was produced in the absence of any of the following constituents: L-glutamine, hydroxylamine, metal ion, A D P or arsenate. Similar results were obtained with the Mn2+-activated transferase reaction except that activity in the absence of A D P was about 65% of that of the complete assay mixture. Thus, unlike the Mg 2+- and Co2+-dependent activities, the reaction
Effect of Concentration of Substrates. Because the biosynthetic activity with Mn 2+ was small, only Mg 2+and Co2+-dependent activities were studied in the subsequent experiments. The rate of reaction increased proportionally with increasing amounts of a m m o n i u m chloride up to 1 m m o l 1- 1 and an identical K m value of 0.7 m m o l 1 - 1 for the a m m o n i u m ion was obtained for both the Mg 2+- and Co2+-depen dent activities. M a x i m u m Mg2+-dependent activity occurred at 6.6 mmol 1-1 L-glutamate and concentrations exceeding 25 mmol 1-~ were inhibitory. In contrast, the Co 2 +-dependent activity increased progressively up to 50 m m o l 1-1 L_glutamate and it was still increasing at the highest concentration used, viz. 100 m m o l 1- 1 L-glutamate (Fig. 3 inset.) It should be noted that under these conditions the Co2+-depen -
S.K. Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena
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B
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293
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Fig. 4A-C. Effect of varying concentrations of A T P on the biosynthetic activity in presence of various a m o u n t s of divalent cations. Experimental details as described in Table 2 except that the concentration of A T P was varied as indicated and the pH of the assay mixtures for Mg 2+, Co z+ and M n 2+ were 7.5, 6.5 and 6.0, respectively. The concentrations of divalent cations used were (mmol 1 1). AMg2+:33.3, 9149 50, J, A; 66.6, 9 =; 100, o o ; B Co2+: 16.6, 9 9 33.3, s - - A ; 66.6, 9 C Mn2+: 6.6, 9 9 16.6, A A; 33.3, . - - ; 66.6, 9 1 6 9
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Fig. 5A and B. ?-Glutamyltransferase activity with different concentrations of L-glutamine and hydroxylamine in the presence of various divalent cations. Reaction mixture for assaying 7-glutamyltransferase activity was as given in Table 2 except that the a m o u n t s of L-glutamine A and hydroxylamine B were varied as shown. Purified enzyme preparation, desalted by passing through a Sephadex G-10 column was used. The concentrations of divalent cations in the assay mixture were : M n 2 + 3 mmol l - 1, 9 9 ; Mg 2 + 50 mmol l - 1 9 9; Co 2+ 5 mmol 1 1 A - - A . -/-GH=)~-glutamylhydroxamate and l / V represents the reciprocal of gmol of y - G H produced in 30 min
dent activity was about 80% of the m a x i m u m activity with Mg z+. The K m values for L-glutamate for the Mg 2+ and Co 2+ catalyzed activites were 2.3 and 23.2 m m o l 1-1 respectively (Fig. 3). The results in Figures 4A, B, C show that the o p t i m u m amount of A T P required varied with the divalent cation used. Thus with Mg 2 + (Fig. 4A) the amount of Pi produced
increased progressively up to 33 m m o l 1-1 A T P but above this concentration, irrespective of the level of Mg 2+, A T P inhibited the enzyme. In contrast, the response of Co 2 + = (Fig. 4 B) and Mn 2 + = (Fig. 4 C) dependent reactions to varying amounts of ATP, was determined by the concentration of the cation. The enzyme activity increased with higher amounts of
294
S.K, Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena
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0.1
0.2
l
0.3
0.4
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Fig. 6A and B. Inhibition of y-glutamyltransferase activity by a m m o n i u m chloride. Assay procedure, using a desalted enzyme preparation, for determining ~?-glutamyltransferase activity (with 3 mmol 1 1 Mn2+) as described in Table 2 was used with the following modifications: A (inset): normal reaction mixture was supplemented with various a m o u n t s of a m m o n i u m chloride. Concentration of glutamine was varied as shown and different a m o u n t s of a m m o n i u m chloride used were: without a m m o n i u m chloride, e - - - e ; 5 mmol I 1, . . _ _ . . ; l0 m m o l I 1, 9 A. B concentration of hydroxylamine was varied as shown and the following a m o u n t s of a m m o n i u m chloride were included in the assay mixture: Without a m m o n i u m chloride, 9 e; 5 mmol1-1, 9 ..; 1 0 m m o l l -~, 9 9 15mmol1-1, [] D. 1IV represents reciprocal of gmol of y-glutamylhydroxamate formed in 30 min
ATP until the concentration of this nucleotide was approximately equivalent to that of the cation. Relatively higher concentrations of ATP to that of the cations inhibited the activity. The effects of various divalent cations (Figs. 2 B, C, D) and ADP on 7-glutamyltransferase activity have been presented earlier in this paper. Enzyme activities with Mn 2+, Co 2+ and Mg a+ respectively, with varying concentrations of L-glutamine are shown in Figure 5A (inset). The production of 7-glutamylhydroxamate with either of the three cations increased only slightly beyond 20 mmol 1- 1 glutamine. Double-reciprocal plots (Fig. 5A) gave an identical Km value of 5 mmol 1-1 glutamine with all three cations. The effects of varying the concentration of hydroxylamine on 7-glutamyltransferase activity are presented in Figure 5B (inset). With either Mn 2+ or Mg 2+, the enzyme was saturated with 12 mmol 1- 1 hydroxylamine whereas with Co 2+, no further increase in the activity occurred beyond 6 mmol 1-1 hydroxylamine. The activity with Mn 2§ or Mg 2+ was inhibited by 38 and 10% respectively at 60 mmol 1 - 1 hydroxylamine while that for Co 2+ was not affected. The Km values for hydroxylamine with Mg 2+ and Mn 2§ were 5.88 and 6.25 mmol 1-1 respectively and a lower value of
2.70 mmol 1-1 was obtained for the CoZ+-dependent activity (Fig. 5 B).
Effects of Substrates of the Biosynthetic Reaction on 7-Glutamyltransferase Activity. The effects of various substrates of the biosynthetic reaction on the Mn 2+dependent y-glutamyltransferase activity were examined. 7-glutamyltransferase activity was inhibited progressively in the presence of increasing amounts of ammonium chloride (Fig. 6A inset), and its Ki value was 23.0 mmol 1 - 1 Kinetic experiments on the inhibition of enzyme activity by ammonium ions with varying concentrations of glutamine (Fig. 6A) and hydroxylamine (Fig. 6B) indicated that it is a competitive inhibitor for glutamine and is non-competitive for hydroxylamine. The inhibitor effects on 7-glutamyltransferase activity of L-glutamate, in the presence of three concentrations of glutamine, are illustrated in Figure 7A (inset). Double-reciprocal plots of the inhibition by Lglutamate indicated that it was a competitive inhibitor of L-glutamine (Fig. 7A) and non-competitive with hydroxylamine (Fig. 7 B). 7-Glutamyltransferase activity was also inhibited by ATP and this effect was reversed with higher
S.K. Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena
2.0
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0.1
0.2
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Fig. 7A and B. Effect of L-glutamate on ,/-glutamyltransferase activity in the presence of varying concentrations of L-glutamine and hydroxylamine. 7-glutamyltransferase activity with 3 mmol 1 1 Mn2+, using a desalted enzyme preparation, was determined as in Table 2 with the following modifications: A Inset : effect of L-glutamate was examined in presence of three concentrations of glutamine: 5 mmol 1- t, 9 9 10 mmol 1 1, 9 9 20 mmol 1-~, 9 9 Concentration of L-glutamine was varied as shown and the reaction mixture contained the following amounts of glutamate: without glutamate, 9 9 ; 10 mmol 1-1, 9 9 ; 20 mmol 1-1 9 9 ; 30 mmol 1- a, z x - - z x . B Reaction mixture contained 5 mmol 1 1 L-glutamine, various amounts of hydroxylamine and the following concentrations of L-glutamate: without glutamate, 9 9 ; 20 mmol 1-1, 9 9 ; 30 mmol 1-1, 9 9 1/Vrepresents reciprocal of gmol of ?-glutamylhydroxamate produced in 30 rain
concentrations of ADP. Thus in the presence of 0.05 mmol 1- ~ ADP, a 40% inhibition of 7-glutamyltransferase activity by 2 m m o l 1- a ATP, was reduced to 30 and 15% on increasing A D P to 0.1 and 0.4 mmol 1 1 respectively. As mentioned earlier, a substantial ?-glutamyltransferase activity with Mn 2 + was obtained even in the absence of ADP. This apparently ADP-independent activity was also inhibited by A T P but this effect did not exceed 20% even at saturating concentration of ATP. This should be compared with a 40% inhibition of the activity in the presence of 0.05 m m o l 1- ~ ADP.
Effects of Various Denaturing Treatments on the Biosynthetic and 7-Glutamyltransferase Activities. Results in Table 3 demonstrate that treatment of the enzyme with urea caused a relatively greater loss of 7-glutamyltransferase than the biosynthetic activity. Thus 2 tool 1-~ urea reduced the biosynthetic activity by 17%, whereas 7-glutamyltransferase activity was inhibited by 35%. The denaturation of the enzyme by urea was significantly suppressed by adding metal ions (Table 3). Thus a loss of 48% of the biosynthetic activity with 3 tool 1 1 urea was reduced to 5% when
the enzyme was incubated with urea in the presence of Mg 2§ In similar experiments (Table 3) Mn 2§ lowered the extent of inhibition of 7-glutamyltransferas 9 activity by 3 mol 1-1 urea from 59 to 18%. The data in Table 4 indicate that both the biosynthetic and v-glutamyltransferase activities were lost on incubating the desalted enzyme preparation at 50~ for 1 h. This thermal denaturation was prevented significantly and to the same extent, by including ]~-mercaptoethanol in the incubation mixture during the heat treatment. However, both enzyme activities were fairly stable to the heat treatment when the preparation was used without desalting, when the enzyme preparation contained 1 0 m m o l 1 - 1 MgC12. Under these conditions the addition of /~-mercaptoethanol induced loss of enzyme activity. The biosynthetic activity was affected to a greater extent since with 0.05 m m o l 1 - 1 fi-mercaptoethanol its activity was reduced by 24% whereas the corresponding loss of 7-glutamyltransferase activity was only 7%. A preliminary experiment showed that Mn 2§ Mg 2§ and Co 2 + dependent ?-glutamyltransferase and Mg 2+ dependent biosynthetic activities were lost concomitantly on incubating the enzyme at 50~ over
296
S.K. Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena
Table 3. Effect of urea on biosynthetic and ,/-glutamyltransferase activities
Table 5. Effect of various substrates on thermal stability of biosynthetic and y-glutamyltransferase activities
Desalted enzyme preparation was preincubated either in the presence or absence of divalent cation (70 m m o l l - 1 Mg 2+ for the biosynthetic and 2.5 mmol 1-1 Mn2+ for 7-glutamyltransferase activities, respectively) with various concentrations of urea for 15 min at 37 ~C. Both the biosynthetic and 7-glutamyltransferase reactions were started by adding the corresponding assay mixtures, as described in Table2, except that 23 p,mol of Mg 2§ and 2.50 gmol of Mn 2+ were used in the reaction, mixtures for the biosynthetic and 7-glutamyltransferase activities respectively. Cation-free reaction mixtures were added to the tubes which already contained the metal ions during preincubation. The biosynthetic and 7-glutamyltransferase activities of the controls were 0.26 and 2.75 gmol of Pi and 7-glutamylhydroxamate, respectively, produced in 30 min
For biosynthetic activity (Table5a), 125 ~tl of desalted enzyme was preincubated at 50~ for 30 min along with the following concentrations of the compounds indicated in the table (in ~tmol): ATP, 3; L-glutamate, 10; ammonium chloride, 10; Mg 2+, 20; L-glutamine, 10; ADP, 0.5; sodium arsenate, 10. After preincubation the tubes were cooled and 10 gmol of imidazole-HC1 buffer (pH 7.0) added to each tube. Components of the biosynthetic assay mixture other than the ones already present during preincubation, were added, the final volume made to 0.3 ml and the enzyme activity determined as described in Methods. Controls, containing each component individually, were run simultaneously to determine their heat stability and also to correct for any formation of Pi from ATP or A D P at higher temperatures. The results are expressed as per cent of the biosynthetic activity of the enzyme without heat treatment except where either L-glutamine, A D P or arsenate were included. In the latter case, per cent activities were calculated from corresponding control samples assayed in the presence of these compounds. The conditions for preincubating the enzyme for ),-glutamyltransferase (Table 5b) were the same as for the biosynthetic activity, except that the concentrations of various compounds used were, in lamol; glutamine, 20; sodium arsenate, 10; ADP, 2.0; hydroxylamine, 15; and M n z+ 1.5. After preincubation the enzyme activity was determined by adding the remaining components of ?~-glutamylhydroxamate assay mixture, at the concentrations specified above, in a final volume of 0.3 ml. The biosynthetic and 7-glutamylhydroxamate activities of the controls (without heat treatment) were 0.25 gmol of P~ and 2.89 gmol of 7-glutamylhydroxamate produced respectively in 30 min
Urea
Biosynthetic
mol 1 1
Preincubation at 37~ Without Mg 2+
y-glutamyttransferase for 15 min
With Mg 2+
Without Mn 2+
With Mn 2+
100 92 65 41 33 24 11
100 87 84 82 68 52 37
%activity of control 0 t 2 3 4 5 6
100 i00 83 52 22 8 3
100 103 100 95 86 69 59
Table 4. Biosynthetic and 7-glutamyltransferase activities after preincubating enzyme with fi-mercaptoethanol Purified enzyme without desalting (containing 10 mmol l - 1 MgC12) or after desalting was incubated with various concentrations of fl-mercaptoethanol at 50 ~C for 1 h. After cooling, the biosynthetic activity (with 70 mmol 1-~ Mg 2+) and y-glutamyltransferase activity (with 2.5 mmol 1-1 Mn 2+) were started by adding the corresponding assay mixtures as given in Table 2 Activities of the control sample (without heat treatment and in absence of fi-ercaptoethanol) without desalting were 0.25 p,mol Pi and 3.30 gmol of 7-glutamylhydroxamate and those after desalting were 0.20 btmol Pi and 2.45 gmol ~/-glutamylhydroxamate produced in 30 min for the biosynthetic and ~,-glutamyltransferase activities respectively fi-mercaptoethanol (Ixmol I- t)
After desalting
Without desalting
Bioy-glutamylsynthetic transferase
Biosynthetic
~-glutamyitransferase
100 94 81 76 68
100 100 100 93 83
%activity of control 0 10 25 50 100
21 32 41 69 57
14 38 48 72 53
a) Biosynthetic activity
b) ,/-glutamyltransferase activity
Additions during preincubation
% activity of control
Additions during preincubation
% activity of control
None (extract alone) Mg 2 + ATP Mg 2+ + A T P L-glutamate NH4Cl L-glutamine Sodium arsenate ADP
28
None (extract alone) Mn 2+ ADP Mn 2+ + A D P L-glutamine Hydroxylamine Sodium arsenate
39
95 50 98 95 28 96 43 49
85 53 85 76 5 51
a period of 90 min. The results of heat treatment, in the presence of various substrates, on Mg 2+-dependent biosynthetic activity of the enzyme are presented in Table 5a. This activity was protected almost completely by Mg 2§ or glutamate and to a lesser extent by ATP whereas ammonium chloride was ineffective. Glutamine or ADP, which are the substrates for 7-glutamyltransferase activity, were as effective as glutamate and ATP respectively in stabilizing the biosynthetic activity against thermal denaturation. A similar response of the ?-glutamyltransferase activity to various substrates during heat treatment was
S.K. Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena
60I !4~ o~2
1 0 5 Incubation
1
~ 10 t i m e (rnin)
15
Fig. 8. Time course of inactivation of biosynthetic and y-glutamyltransferase activities by hydroxylamine. Desalted enzyme preparation (100 lxl) was preincubated with 2.0 Izmol hydroxylamine hydrochloride (neutralized with NaHCO3) for the indicated time. The biosynthetic (using 70 mmol 1- t Mg z§ ?-glutamyltransferase (using 2.5 mmol I ~ Mn 2 +) activities were determined as described in Table 2, except that the reaction mixture for the latter activity contained 8 gmol of hydroxylamine. The biosynthetic ( i m) and 7-glutamyltransferase (A A) activities of the untreated sample were 0.28 pmol Pi and 3.40 pmol of 7-glutamylhydroxamate, respectively, produced in 30 min
Table 6. Effect of various substrates on inactivation of biosynthetic
and 7-glutamyltransferase activities by hydroxylamine The experimental details were the same as in Table 5 but instead of heat treatment, the enzyme was preincubated at 37 ~ C for 5 min with 2 gmol hydroxylamine. The concentration of hydroxylamine for ),-glutamyltransferase activity was 10 Izmol. Activities of the control samples (without hydroxylamine for the biosynthetic activity) were 0.28 gmoI of Pi and 3.40 gmol y-glutamylhydroxamate produced respectively in 30 rain. The per cent biosynthetic activities for treatments using either L-glutamine, ADP or arsenate and the treatment involving Mg 2+ in ~-glutamyltransferase activity, were calculated from corresponding controls assayed in the presence of these compounds a) Biosynthetic Activity
b) ,/-glutamyltransferase activity
Additions during preincubation
% activity of control
Additions during preincubation
% activity of control
None Mg 2+ ATP Mg2 + + ATP L-glutamate NH4C1 L-glutamine ADP Sodium arsenate
38 81 84 87 84 46 60 64 78
None Mn 2 + ADP Mn z+ + A D P L-glutamine Sodium arsenate Mg 2 +
40 92 72 100 86 100 85
297
observed (Table 5 b). Thus Mn 2 § and glutamine were equally effective in protecting the enzyme whereas ADP was less so and hydroxylamine accelerated the inactivation. The likelihood that the decomposition of hydroxylamine at high temperatures would result in lower 7-glutamyltransferase activity was discounted because the addition of hydroxylamine pre-equilibrated at 50~ for 30 min produced similar activities to the controls. The effect of hydroxylamine on glutamine synthetase was further investigated. Results in Figure 8 show an almost comparable loss of both 7-glutamyltransferase and biosynthetic activities on incubating the enzyme with 2 Izmol hydroxylamine. More than 80% of the biosynthetic activity was retained when the enzyme was preincubated with hydroxylamine in the presence of either Mg 2+, ATP or glutamate. Each of the substrates of 7-glutamyltransferase activity viz. glutamine, ADP or sodium aresenate also provided a significant protection to the biosynthetic activity against hydroxylamine (Table 6a). Similarly, the loss of y-glutamyltransferase activity, on preincubating the enzyme with hydroxylamine was largely prevented by either Mn 2", Mg 2+, ADP, glutamine or sodium arsenate (Table 6b). Discussion
These investigations on glutamine synthetase from Anabaena cylindrica show that it resembles the enzyme from other organisms in several of its properties, viz. requirement for divalent cations, pH optimum and K m values for substrates. As with the enzyme from other sources (Hubbard and Stadtman, 1967b) the maximum biosynthetic activity was obtained with Mg 2". The results indicate that the lower activity with Co 2--, than with Mg 2+, was largely due to its relatively high Km for glutamate. The response of the algal enzyme to concentrations of ATP relative to those of the divalent cations and also the more acidic pH optimum for the Co 2 +- and Mn 2 +-dependent than the Mg 2 § activity, are consistent with the properties of the enzyme from other organisms (Deuel et al., 1969; Dharmawardene et al., 1973; O'Neal and Joy, 1973; McParland etal., 1976). Our results show that the pH optimum and also the K m values for hydroxylamine, glutamine and ADP were the same for the Mn 2+, Co 2+ and Mg 2+ dependent 7-glutamyltransferase activities. As found for the enzyme from bacteria (Hunt et al., 1975) and plants (Speck, 1949; Elliot, 1953; Kanamori and Matsumoto, 1972; McParland et al., 1976), there were, however, marked differences in the optimum concentrations of the divalent cation s required for the maximum 7-glutamyltransferase activity.
298
S.K. Sawhneyand D.J.D. Nicholas: Glutamine Synthetasefrom Anabaena
Significant amount of 7-glutamylhydroxamate was produced in the Mn 2 § reaction of the algal enzyme even in the absence of ADP. The requirement for Mn 2§ and arsenate, indicated that this reaction was catalyzed by 7-glutamyltransferase rather than by other possible contaminants like amidohydrolases (Hubbard and Stadtman, 1967b) or glutaminases (Meister, 1974). Synthesis of v-glutamylhydroxamate, in the absence of the nucleotide was observed only with Mn 2+ even though the activities with either Mn 2--, Mg 2§ or Co 2§ were saturated at the same concentration of ADP. Thus, these results do not support the suggestion (Lajtha et al., 1953; Meister, 1974) that this activity was due to enzyme bound adenine nucleotide. It is possible that these cations stabilize different conformational states of the enzyme and the one induced by Mn 2§ is partially active even in the absence of ADP. On the other hand, configurations attained by Co 2+ or Mg 2§ could require further modification by ADP for maximal enzyme activity. Such a role for ADP in v-glutamyltransferase reaction is possible in view of its requirement in catalytic amounts and secondly because it does not enter directly into the transferase reaction. In this work, glutamine synthetase has been shown to be much more stable in the presence of divalent cations. This effect is evident on comparing the extent of inactivation of both the biosynthetic and 7-glutamyltransferase activities, in the presence or the absence of divalent cations, on treatment with urea, hydroxylamine or high temperature. The partial stabilization of the desalted enzyme at 37~ by /%mercaptoethanol, suggests that its -SH groups become more exposed after removal of the cations. These observations are in agreement with the results for glutamine synthetase from E. coli (Kingdon et al., 1968; Shapiro and Stadtman, 1968) where the removal of divalent cation resulted in conversion of the enzyme to a 'relaxed' state. The biosynthetic activity of desalted enzyme preparation was protected against thermal denaturation by either Mg 2§ ATP, glutamate, glutamine or ADP. In this respect, this enzyme behaves differently from the sheep brain enzyme which was stabilized when both Mg 2+ and ATP were present together (Pamiljans et al., 1962). Protection of the enzyme by either glutamate or glutamine against heat denaturation as well as hydroxylamine implies that these substrates interact with the algal enzyme even in the absence of metal ions and adenine nucleotides. These observations thus support the proposed random (Wedler, 1974) rather than sequential order of binding of the substrates (Pamiljans et al., 1962). The failure of ammonia or hydroxylamine to provide such protection may be due either to their inability to bind to
the enzyme in the absence of the other substrates (Rhee et al., 1976) or simply to the nonconversion of the enzyme into a more stable form. The results of the comparative studies on the biosynthetic and 7-glutamyltransferase activities presented in this paper provide further evidence that they are associated with the same enzyme. It has also been shown that v-glutamyltransferase activity was inhibited by the substrates of the biosynthetic reaction. Thus both ammonia or glutamate inhibited the transferase activity competitively with glutamine and non-competitively with hydroxylamine. Gass and Meister (1970) have suggested that glutamine interacts with the enzyme in such a way that its NH2-group occupies the ammonia binding site while the 'oxygenbinding' site to which glutamate is normally bound, is required for the attachment of the corresponding oxygen group of glutamine. Thus the observed competitive effects of glutamate or ammonia with glutamine in the 7-glutamyltransferase reaction reported herein are consistent with their postulated model for the active site of the enzyme, but not with the suggestion that corresponding pairs of the substrates for biosynthetic and transferase reactions share common sites (Hubbard and Stadtman, 1967a). Our results also suggest that the interaction of glutamine with N H 2 binding site, would preclude the binding of hydroxylamine at this locus, A lack of competition between ammonia and hydroxylamine was further demonstrated by the inability of the former substrate to protect the algal enzyme against its inactivation by hydroxylamine. The 7-glutamyltransferase reaction was also inhibited by ATP, possibly competitively with ADP, since its effect was reversed by increasing concentrations of ADP. The results of these kinetic experiments suggest that during 7-glutamyltransferase reaction glutamine interacts with the enzyme at the sites where glutamate and ammonia bind in the biosynthetic reaction while the adenine nucleotide binding site could be occupied by either ATP or ADP. Further work is required to examine the effects of various substrates of the transferase reaction on the biosynthetic activity with a view to elucidating the pattern of binding sites for the substrates of these two reactions of glutamine synthetase. This work was supported by a generous grant from the Australian Research Grants Committee and the Australian Water Resources Council. The skilled technical assistance of Mr. Michael Byrne is gratefully acknowledged. References
Brown, C.M., MacDonald Brown, D.S., Meers, J.L. : Physiological aspects of microbial inorganic nitrogen metabolism, in Ad-
S.K. Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena vances in Microbial physiology, 11, pp I 52, Rose, A.H., Tempest, D.W. eds., London: Academic Press 1974 Brownell, P.F., Nicholas, D.J.D. : Some effects of sodium on nitrate assimilation and Nz fixation in Anabaena cylindrica. Plant Physiol. 42, 915 921 (1967) Dharmawardene, M.W.N., Stewart, W.D.P., Stanley, S.O.: Nitrogenase activity, amino acid pool patterns and amination in blue-green algae. Planta 108, 133-145 (1972) Dharmawardene, M.W.N., Haystead, A., Stewart, W.D.P. : Glutamine synthetase of nitrogen fixing alga, Anabaena cylindrica. Arch. Mikrobiol. 90, 281 295 (1973) Deuel, T.F., Stadtman, E.R.: Some kinetic properties of Bacillus subtilis glutamine synthetase. J. biol. Chem. 245, 5206-5213 (1970) Deuel, T.F., Shelton, E., Stadtman, E.R. : Structure and regulation of Bacillus subtilis glutamine synthetase. Fed. Proc. 28, 341 (1969) Elliot, W.H. : Isoiation of glutamine synthetase and gtutamotransferase from green peas. J. biol. Chem. 201, 661 672 (1953) Gass, J.D., Meister, A. : Computer analysis of active site of glutamine synthetase. Biochemistry 9, 1380-1389 (1970) Hachimori, A., Matsunaga, A., Shimizu, M., Samejima, T., Nosoh, Y.: Purification and properties of glutamine synthetase from Bacillus stearothermophilus. Biochim. Biophys. Acta 350, 461-474 (1974) Hubbard, J.S., Stadtman, E.R. : Regulation of glutamine synthetase VI. Interactions of inhibitors for Bacillus licheniformis glutamine synthetase. J. Bacteriol. 94, 1016-1024 (1967a) Hubbard, J.S., Stadtman, E.R. : Regulation of glutamine synthetase II. Patterns of feedback inhibition in microorganisms. J. Bacteriol. 93, 1045-1055 (1967b) Hunt, J.B., Smyrniots, P.Z., Ginsburg, A., Stadtman, E.R.: Metal ion requirement by glutamine synthetase of Escherichia coli in catalysis of 7-glutamyl transfer. Arch. Biochem. Biophys. 166, 102-124 (1975) Kanamori, T., Matsumoto, H.: Glutamine synthetase from rice plant roots. Arch. Biochem. Biophys. 152, 404-412 (1972) Kingdon, H.S., Hubbard, J.S., Stadtman, E.R. : Regulation of glutamine synthetase XI. The nature and implications of lag phase in the Escherichia coli glutamine synthctase reaction. Biochemistry 7, 2136-2142 (1968) Lajtha, A., Mela, P., Waelsch, H. : Manganese-dependent glutamotransferase. J. biol. Chem. 205, 553-564 (1953) Levintow, L., Meister, A., Hogeboom, G.H., Kuff, E.L.: Studies on the relationship between the enzymatic synthesis of glutamine and glutamyl transfer reaction. J. Amer. Chem. Soc. 77, 5304-5308 (1955) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 262-275 (1951) McParland, R.H., Guevara, J.G., Becker, R.R., Evans, H.J. : The purification and properties of glutamine synthetase from cytosol of soy-bean root nodules. Biochem. J. 153, 597-606 (1976) Meister, A.: Glutamine synthetase of mammals, in The Enzymes Vol X, pp 699-754, 3rd ed., Boyer, P.D. ed, New York and London: Academic Press 1974 Miflin, B.J., Lea, P.J.: The pathway of nitrogen assimilation in plants. Phytochemistry 15, 873-885 (1976) Nagatani, H., Shimuzu, M., Valentine, R.C.: The mechanism of ammonia assimilation in nitrogen fixing bacteria. Arch. Mikrobiol. 79, 164-175 (1971)
299
O'Neal, D., Joy, K.W.: Glutamine synthetase of pea leaves: 1. Purification stabiiization and pH optima. Arch. Biochem. Biophys. 159, 1I3-122 (1973) Pamiljans, V., Krishnaswamy, P.R., Dumville, G., Meister, A.: Studies on the mechanism of glutamine synthesis; Isolation and properties of the enzyme from sheep brain. Biochemistry 1, 153-158 (1962) Rhee, S.G., Chock, P.B., Stadtman, E.R.: Mechanistic studies of glutamine synthetase from Escherichia coli. An integrated mechanism for biosynthesis, transferase and ATPase reaction. Biochemistry 58, 35-49 (1976) Ronzio, R,A., Rowe, W.B., Wilk, S., Meister, A.: Preparation and studies on the characterisation of sheep brain glutamine synthetase. Biochemistry 8, 2670-2674 (1969) Seyama, S., Kuroda, Y., Katunuma, N. : Purification and comparison of glutamine synthetase from rat and chick livers. J. Biochem. 72, 1017-1027 (1972) Shapiro, B.M., Stadtman, E.R.: Regulation of glutamine synthetase IX. Reactivity of the sulphydryl groups of the enzyme from Escherichia coli. J. biol. Chem. 242, 5069 5079 (1968) Shapiro, B.M., Stadtman, E.R. : Glutamine synthetase (Escherichia coli), in Methods in Enzymology, Vol. 17A, pp 910-922, Tabor, H., Tabor, C.W., eds., New York and London: Academic Press 1970 Speck, J.F. : The enzymatic synthesis of glutamine, a reaction utilising adenosine triphosphate. J. biol. Chem. 179, 1405 1426 (1949) Stadtman, E.R., Giusburg, A. : Glutamine synthetase of Escheriehia coli, in The Enzymes, Vol. X, pp 755 807, 3rd ed., Boyer, P.D. ed,, New York and London: Academic Press 1974 Stewart, G.R., Rhodes, D. : Evidence for the assimilation of ammonia via glutamine pathway in nitrate-grown Lemna minor. FEBS Lett. 64, 296-299 (1976) Stewart, W.D.P., Rowell, P. : Effects of L-methioninc-DL-sulphoximine on assimilation of newly fixed NH3, acetylene reduction and heterocyst production in Anabaena cylindrica. Biochem. Biophys. Res. Commun. 65, 846-856 (1975) Tate, S.S., Meister, A.: Regulation of rat liver glutamine synthetase: Activation by c~-ketoglutarate and inhibition by glycine, alanine and carbamyI phosphate. Proc. Natl. Acad. Sci., USA. 68, 781-785 (1971) Tare, S.S., Leu, F.Y., Meister, A. : Rat liver glutamine synthetase. Preparation, properties and mechanism of inhibition by carbamyl phosphate. J. biol. Chem. 247, 5312 5321 (1972) Thomas, J., Meeks, J.C., Wolk, C.P., Shaffer, P.W., Austin, S.M., Chien, W.S.: Formation of glutamine from [a3N] ammonia, [13N] dinitrogen and [14C] glutamate by heterocysts isolated from Anabaena cylindrica. J. Bacteriol. 129, I545-1555 (1977) Wedler, F.C.: Mechanisms of substrate binding with glutamine synthetase. Equilibrium isotope exchanges with ovine brain, pea seed and Escherichia coli enzymes. J. biol. Chem. 249, 5080-5087 (1974) Wolk, C.P., Thomas, J., Shaffer, P.W., Austin, S.M., Galonsky, A.: Pathway of nitrogen metabolism after fixation of 13Nlabeled nitrogen gas by the cyanobacterium, Anabaena cylindrica. J. biol. Chem. 251, 5027-5034 (1976)
Received 6 December; accepted 22 December 1977
P l a n t a 9 by Springer-Verlag 1978
Some Properties of Giutamine Synthetase from Anabaena cylindrica S.K. Sawhney and D.J.D. Nicholas Department of Agricultural Biochemistry, Waite Agricultural Research Institute, University of Adelaide, Glen Osmond, South Australia, 5064
Abstract. Some properties of the biosynthetic and 7-glutamyltransferase activities of glutamine synthetase (EC 6.3.1.2) from A n a b a e n a c y l i n d r i c a are described, including requirement for divalent cations, pH optimum and K m for substrates. The y-glutamyltransferase reaction was inhibited by L-glutamate, ammonia and ATP. The inhibition by L-glutamate and ammonia was competitive for L-glutamine and non-competitive for hydroxylamine. Both the biosynthetic and the 7-glutamyltransferase activities of the desalted enzyme were much more sensitive to inactivation by treatments such as urea, hydroxylamine and incubation at 50~ than the preparation which contained a divalent cation. The effects of some substrates of these reactions on protection against thermal denaturation and hydroxylamine were examined. An interpretation of these results in terms of the sequence of binding of substrates both in the biosynthetic and the 7-glutamyltransferase reactions are discussed. Key words: A n a b a e n a - Biosynthetic and transferase activities - Glutamine synthetase.
Introduction
Glutamine synthetase (L-glutamate:ammonia tigase, ADP, EC 6.3.1.2) plays an important role in nitrogen metabolism of bacteria (Nagatani et al., 1971 ; Brown et al., 1974), algae (Dharmawardene et al., 1972; Stewart and Rowell, 1975 ; Wolk et al., 1976 ; Thomas et al., 1977) and plants (Stewart and Rhodes, 1976; Miflin and Lea, 1976) since glutamine formed in the reaction is utilized for a variety of nitrogenous metabolites of a cell. In addition to catalyzing biosynthetic reaction (reaction I), highly purified preparations of glutamine synthetase from various organisms exhibit
several other activities (Meister, 1974; Stadtman and Ginsburg, 1974). Among these, 7-glutamyltransferase activity (reaction II) has been used extensively during purification of the enzyme by various workers : L-Glutamate + ATP + NH 3 -
Mez+
L-Glutamine + ADP + Pi
(i)
L-Glutamine + NH2OH Me2+'ADP, arsenate or Pi
7-Glutamylhydroxamate+ NH 3
0I)
The relationship between these two activities of glutamine synthetase is not known. Investigations by Levintow et al. (1955) discounted the possibility that 7glutamyltransferase reaction is the reversal of biosynthetic activity in which hydroxylamine substitutes for ammonia. It has been stated that y-glutamyltransferase activity might represent a simple uncoupling of the reverse biosynthetic reaction (Hunt et al., 1975). Hubbard and Stadtman (1967b) mentioned that the substrate-binding sites for these two activities are probably shared by a corresponding pair of substrates like glutamate and glutamine, ammonia and hydroxylamine and ATP and ADP. There is, however, no experimental evidence for these common sites. Although glutamine synthetase from bacteria (Hubbard and Stadtman, 1967a; Deuel and Stadtman, 1970; Stadtman and Ginsburg, 1974; Hachimori et al., 1974), plants (Elliot, 1953; Kanamori and Matsumoto, 1972; O'Neal and Joy, 1973) and animal tissues (Ronzio et al., 1969; Tate et al., 1972; Seyama et al., 1972; Tare and Meister, 1971) has been extensively characterized, little information (Dharmawardene et al., 1973) is available about this enzyme in algae. In this paper the properties as well as the relation between the biosynthetic and 7-glutamyl-
290
S.K. Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena
transferase activities of glutamine synthetase are described for a blue-green algae, Anabaena cylindrica.
1"0
1.0 .~
0"8
5.6
Materials and Methods ~0.6
Materials
i\!.i'.,. ....:.....--(_J.!._._if:':-.-:
ve
?
A culture of the blue-green alga, Anabaena eylindriea (Lemmerman strain 1403/2A) was obtained from the Culture Collection of Algae and Protozoa, The Botany School, Cambridge, U.K. L-glutamate, L-glutamine, 7-glutamylhydroxamate and ATP were purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A. /7mercaptoethanol was from B.D.H., Poole, England, Hydroxylamine hydrochloride was bought from Ajax Chemical Ltd., Sydney. All the other chemicals used in these investigations were of analytical grade.
Methods Preparation of Cell-Free Extract. Anabaena cylindrica was grown, in a medium containing 5 mmol 1- ~ KNO3, as described by Brownell and Nicholas (1967). All the operations during harvesting and preparation of cell-free extracts were done at 4~C. The sevendays old cells were collected by centrifuging at ),000 x g for 10 rain. The cells were broken by passing them through an Aminco French pressure cell at 16,000 p.s.i. The homogenate was centrifuged at 6,000 x g for 20 min and the supernatant fraction ($6) obtained was used for the purification of glutamine synthetase.
DesaIting Procedure. In some of the experiments the purified enzyme preparation was used after desalting. About 5 ml of the enzyme was passed through a sephadex G-10 column (2.5 • 20 cm) which had been previously equilibrated with 0.1 mol 1 1 Tris-HC1 pH 7.5. The enzyme was eluted from the column with the same buffer. The desalted enzyme was always examined for both the biosynthetic and the y-glutamyltransferase activities, in the absence of divalent cation, to ascertain complete removal of MgC12 from the preparation. Enzyme Assays. Both the biosynthetic and 7-glutamyltransferase activities of glutamine synthetase were determined by the procedures described by Shapiro and Stadtman (1970). The reaction mixture for ~/-glutamyltransferase activity, in a final volume of 1 ml contained, in ~tmol: Imidazole-HC1 buffer (pH 7.0), 40; L-glutamine, 30; hydroxylamine hydrochloride (neutralized with NaOH), 30; MnCI2, 3.0; sodium arsenate, 2.0; ADP, 0.4 and an appropriate amount of the enzyme. Control tubes from which L-glutamine was omitted were always included. The amount of 7-glutamylhydroxamate produced after 15 rain at 37~ was determined colorimetrically using a reference curve prepared from authentic ,/-glutamylhydroxamate (Sigma Chemical Co~, USA). For the biosynthetic acti+ity, the assay mixture in a final volume of 0.3 ml contained in lamol: Imidazole-HC1 buffer (pH 7.0), 10; .Lglutamate, 10; MgC12, 20; NH4C1, 10; ATP, 3,0 and the enzyme preparation. From the control tubes L-glutamate was omitted. The amount of Pi produced after 30 rain was determined as described by Shapiro and Stadtman (1970). For every batch of purified glutamine synthetase, the amount of enzyme for the biosynthetic and y-glutamyltransferase activities was determined so that, under the conditions of the assay, the activity was linear over a period of 45 min.
Protein Content. Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
0"4
0'2
2-B
-g
1"4 :~
30
40
50
Fraction
60
70
80
90
100
number
Fig. 1. Elution profile for glutamine synthetase from a Sepharose 6B column. Experimental details are given in the text. Enzyme activity was determined by measuring the rate of 7-glutamylhydroxamate (v-GH) production. Reaction mixture in a final volume of 1 ml contained, in gmol: Imidazole-HC1 buffer (pH 7.0) 40; L-glutamine, 30; hydroxylamine, 30; ADP, 0.4; sodium arsenate, 2; MnC12, 3 and 0.2 ml of enzyme. Reaction carried out at 37 ~ C for 15 rain. Absorbance at 280 nm, e - - e ; Enzyme activity, i - - i
Enzyme Purification Glutamine synthetase from Anabaena cylindrica was purified by the procedure described below. Unless otherwise stated all the operations were carried out at 4~
Heat Treatment. Cell-free extract (S 6 fraction) was kept in a water bath at 50~ for 20 min. After cooling, the precipitated proteins were removed by centrifuging at 30,000 x g for 30 min.
p H Precipitation. The pH of the supernatant ($30 heated) from the preceding step was adjusted to 5.2 with M-acetic acid. After standing it on ice for 30 min, it was centrifuged at 30,000 x g for 30 min. The pellet was discarded and pH of the supernatant fraction was brought to 4.0 by further addition of M-acetic acid. After 30 rain, the preparation was centrifuged at 30,000 x g for 30 min and the pellet dissolved in 17 ml of 0.1 mol 1-1 Tris-HC1 buffer (pH 7.5) containing 10 mmol 1-1 MgC12.
Ammonium Sulphate Precipitation. Solid ammonium sulphate was added gradually, to the above preparation, to obtain 30% saturation. The precipitated proteins were removed after 20 rain by centrifuging at 30,000 x g for 30 min. The ammonium sulphate concentration was then raised to 60% saturation. The pellet, after centrifuging at 30,000 x g for 30 rain, was dissolved in 6 ml of extraction buffer and dialyzed overnight against the same buffer. Gel Filtration, The dialyzed enzyme was then loaded onto a Sepharose 6B column (3 x 70 cm) which had been previously equilibrated with 0.1 mol 1 1 Tris-HCl (pH 7.5) containing 10 mmol 1-1 MgC12. The sample was eluted with the same buffer at a flow rate of 12 ml/h and 5 ml fractions were collected. The elution profiles for protein and glutamine synthetase are shown in Figure 1. The enzyme was recovered in the fractions just prior to the bluishcoloured second protein peak. The pooled fractions from the column had 20-25-fold higher activity than the $6 preparation and about 13% of the original activity was recovered by this procedure (Table 1). This partially purified enzyme was stable for several weeks at 0~
S.K. Sawhney a n d D.J.D. Nicholas: Glutamine Synthetase from Anabaena
291
Table 1. Purification of glutamine synthetase from Anabaena cylindrica Experimental details given in the text. Enzyme activity was determined as described in Figure 1. One unit of enzyme activity represents 1 ~tmol of 7-glutamylhydroxamate produced per min Purification step
Total volume (ml)
Total activity (units)
Total protein (rag)
% recovery
$6 Supernatant of broken cells centrifuged at 6000 x g for 10 min
75
307.5
517.5
100
0.6
1
S30(heated) S 6 heated to 50~ for 20 rain and centrifuged at 30000 x g for 30 min
71
305.3
216.5
99
1.4
2
Pellet pH 4.0
20
243.0
61.0
79
4.0
7
6
125.0
26.1
41
4.8
8
88
39.8
13
15.1
26
30 60% a m m o n i u m sulphate fraction Pooled Sepharose 6B fractions
2.64
Specific activity units/mg protein
Purification (fold)
Results
Table 2. Biosynthetic and y-glutamyltransferase activities with various divalent cations
Properties of Biosynthetic and y-Glutamyhransferase Activities
Biosynthetic and ~/-glutamyltransferase activities were determined from the rates of formation of Pi and y-glutamylhydroxamate, respectively. Purified enzyme preparation was used after desalting through a Sephadex G-10 column (2.5 x 20 cm). For the biosynthetic activity, the reaction mixture in a final volume of 0.3 ml contained, in Ixmol: hnidazole-HCl buffer (pH 7.0), 10; L-glutamate, 20; a m m o n i u m chloride, 10; ATP, 3; enzyme preparation 0.25 ml (I0 ~tg protein) and the specified concentrations of various cations. The a m o u n t of Pi produced after 45 min at 37~ was determined as in Methods. The assay mixture for q/-glutamyltransferase activity was as described in Figure 1 except that M n 2 § was replaced by other cations as indicated in the table
Both the biosynthetic and y-glutamyltransferase reactions were catalyzed by the 25-fold purified glutamine synthetase preparation. The properties of these two activities of the enzyme were compared.
Divalent Cation Specificity. The relative rates of biosynthetic activity, at p H 7.0, in the presence of various divalent cations are given in Table 2. At 70 mmol 1- t, m a x i m u m activity was obtained with Mg 2 +. The observed order of effectiveness of the different cations was Mg 2+ > Co 2+ > Cu 2+ > Fe z+ > Mn 2+ = Z n 2+ . The lower activity at 14 mmol 1-1 Mg 2+ than with Co 2+ was due to a relatively higher concentration of Mg 2+ required to saturate the enzyme. This effect is illustrated in Figure 2A; thus at lower concentrations of either Mg z+ or Co z+, enzyme activity with the latter was higher than with Mg 2+. However, at saturating concentrations of the metal ions, activity with Mg 2+ was significantly greater than with Co 2+. For 7-glutamyltransferase activity, Mn z+ was the most effective cation followed by Mg 2+ and Co 2+ (Table 2). A relatively small activity was obtained with Cu 2+ and the other cations Zn z+, Fe 2+, Ba 2+, Ca 2+ were ineffective. Marked differences were observed in the concentrations of Mn 2+, Co 2+ and Mg 2+ required for m a x i m u m enzyme activity (Figs. 2B, C, D). As found for the biosynthetic reaction, relatively higher amounts of Mg 2+ than Co 2+ were required to saturate the enzyme.
Effect ofpH. The p H optimum for the biosynthetic reaction was influenced by the type of activating metal ion used. Thus m a x i m u m activities with Mg 2+
Metal ion
Mg 2+ M n 2+ Co 2+ Cu 2+ Fe 2+ Zn 2+ Ni 2+ Ca 2+ a b
Biosynthetic activity a Metal ion (mmol i- 1)
7-Glutamyltransferase activity u Metal ion (mmol 1 1)
1.4
14
70
0.5
5.0
50.0
20 20 20 20 20 20 7 7
160 73 184 20 80 50 20 11
280 80 157 123 107 80 34 34
0.24 1.60 0.22 0.07 0 0 0 0
0.35 1.43 0.29 0.16 0 0 0 0
0.43 0.60 0.21 0.07 0 0 0 0
nmol Pi produced in 45 min gmol ~/-glutamylhydroxamate produced in 30 rain
( 1 0 0 m m o l l - 1 ) , Co 2+ ( 3 5 m m o l l -~) and Mn 2+ (20 m m o l l 1) w e r e obtained at pH 7.3, 6.9 and 6.0 respectively. However, an identical p H o p t i m u m of 8.0 was observed for the y-glutamyltransferase reaction with all three cations (concentrations used were 10 m m o l 1-1 of either Mg 2+ or Co 2+ and 3 m m o l 1-a Mn2+). At optimal conditions of p H and metal ion concentration, the biosynthetic activities with Co 2+ and Mn 2+ were 50 and 25% respectively of that with
292
S.K. S a w h n e y a n d D . J . D . N i c h o l a s : G l u t a m i n e S y n t h e t a s e f r o m A n a b a e n a
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I 0.5
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Fig. 2 A - D , Biosynthetic a n d 7 - g l u t a m y l t r a n s f e r a s e activities in the presence o f v a r i o u s c o n c e n t r a t i o n s of divalent cations. E x p e r i m e n tal details as given in T a b l e 2. A B i o s y n t h e t i c activity with M g 2+, 9 9 OF C o 2 +, A - I k . B, C, D. y-glutamyltransferase activity with M n 2 +, Co 2 + a n d M g z+, respectively. ( ? - G H = y - g t u t a m y l h y d r oxamate)
Fig. 3. Effect of v a r y i n g c o n c e n t r a t i o n s of L - g l u t a m a t e on the M g 2+ a n d C o Z + - d e p e n d e n t b i o s y n t h e t i c activity. D e t a i l s of the r e a c t i o n m i x t u r e as given in T a b l e 2 except t h a t the c o n c e n t r a t i o n of L - g l u t a m a t e was v a r i e d as i n d i c a t e d a n d the p H of the assay m i x t u r e s for d e t e r m i n i n g the activities w i t h M g 2+ a n d C o 2+ were 7.5 a n d 6.5 respectively. Purified e n z y m e p r e p a r a t i o n was used after d e s a l t i n g t h r o u g h a S e p h a d e x G-10 column. C o n c e n t r a t i o n s of d i v a l e n t cations u s e d were: M g 1+, 70 m m o l l - 1 9 9 C o 2+ 30mmoll • 9 9
Mg 2+ and for the 7-glutamyltransferase reaction Mg 2+ and Co 2+ gave 35 and 30% of the activity obtained with Mn 2+.
catalyzed by Mn 2 + did not have an absolute requirement for A D P but was stimulated by about 35% on adding this nucleotide. M a x i m u m activities with all the three cations were obtained with 0.05 m m o l 1ADP.
Requirements for Enzyme Activity. The biosynthetic activity, with either Mg 2+, Co 2+ or Mn 2+ was dependent on the presence of L-glutamate, a m m o n i u m chloride and ATP. Negligible amounts of P~ were liberated when any of these components were omitted from the reaction mixture. When hydroxylamine substituted for a m m o n i u m chloride, then only 30% of the activity was recorded. All the components of the standard 7-glutamyltransferase reaction mixture were required for the Mg 2+- and Co2+-dependent activities. Less than 5% 7-glutamylhydroxamate was produced in the absence of any of the following constituents: L-glutamine, hydroxylamine, metal ion, A D P or arsenate. Similar results were obtained with the Mn2+-activated transferase reaction except that activity in the absence of A D P was about 65% of that of the complete assay mixture. Thus, unlike the Mg 2+- and Co2+-dependent activities, the reaction
Effect of Concentration of Substrates. Because the biosynthetic activity with Mn 2+ was small, only Mg 2+and Co2+-dependent activities were studied in the subsequent experiments. The rate of reaction increased proportionally with increasing amounts of a m m o n i u m chloride up to 1 m m o l 1- 1 and an identical K m value of 0.7 m m o l 1 - 1 for the a m m o n i u m ion was obtained for both the Mg 2+- and Co2+-depen dent activities. M a x i m u m Mg2+-dependent activity occurred at 6.6 mmol 1-1 L-glutamate and concentrations exceeding 25 mmol 1-~ were inhibitory. In contrast, the Co 2 +-dependent activity increased progressively up to 50 m m o l 1-1 L_glutamate and it was still increasing at the highest concentration used, viz. 100 m m o l 1- 1 L-glutamate (Fig. 3 inset.) It should be noted that under these conditions the Co2+-depen -
S.K. Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena
I 10
30
50
70
B
I
10
I
30
mM A T P
293
5
I
70
10
30
mM ATP
50
70
mM A T P
Fig. 4A-C. Effect of varying concentrations of A T P on the biosynthetic activity in presence of various a m o u n t s of divalent cations. Experimental details as described in Table 2 except that the concentration of A T P was varied as indicated and the pH of the assay mixtures for Mg 2+, Co z+ and M n 2+ were 7.5, 6.5 and 6.0, respectively. The concentrations of divalent cations used were (mmol 1 1). AMg2+:33.3, 9149 50, J, A; 66.6, 9 =; 100, o o ; B Co2+: 16.6, 9 9 33.3, s - - A ; 66.6, 9 C Mn2+: 6.6, 9 9 16.6, A A; 33.3, . - - ; 66.6, 9 1 6 9
,o!./
.
I
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.
~
.
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.
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I
.
I 0.5
,0L.
I 0.5
I 1.0
I 1.5
I 2.0
! 2.5
1
mM H y d r o x y l a m i n e
Fig. 5A and B. ?-Glutamyltransferase activity with different concentrations of L-glutamine and hydroxylamine in the presence of various divalent cations. Reaction mixture for assaying 7-glutamyltransferase activity was as given in Table 2 except that the a m o u n t s of L-glutamine A and hydroxylamine B were varied as shown. Purified enzyme preparation, desalted by passing through a Sephadex G-10 column was used. The concentrations of divalent cations in the assay mixture were : M n 2 + 3 mmol l - 1, 9 9 ; Mg 2 + 50 mmol l - 1 9 9; Co 2+ 5 mmol 1 1 A - - A . -/-GH=)~-glutamylhydroxamate and l / V represents the reciprocal of gmol of y - G H produced in 30 min
dent activity was about 80% of the m a x i m u m activity with Mg z+. The K m values for L-glutamate for the Mg 2+ and Co 2+ catalyzed activites were 2.3 and 23.2 m m o l 1-1 respectively (Fig. 3). The results in Figures 4A, B, C show that the o p t i m u m amount of A T P required varied with the divalent cation used. Thus with Mg 2 + (Fig. 4A) the amount of Pi produced
increased progressively up to 33 m m o l 1-1 A T P but above this concentration, irrespective of the level of Mg 2+, A T P inhibited the enzyme. In contrast, the response of Co 2 + = (Fig. 4 B) and Mn 2 + = (Fig. 4 C) dependent reactions to varying amounts of ATP, was determined by the concentration of the cation. The enzyme activity increased with higher amounts of
294
S.K, Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena
51" 60
/
.//
=/I/!
A
I I 10 20 rnM NH4C1
B
I 30
/
0.25
1
0.5
0.75
1.0
mM G l u ~ a m l n e
0.1
0.2
l
0.3
0.4
0.5
mM H y d r o x y l a m l n e
Fig. 6A and B. Inhibition of y-glutamyltransferase activity by a m m o n i u m chloride. Assay procedure, using a desalted enzyme preparation, for determining ~?-glutamyltransferase activity (with 3 mmol 1 1 Mn2+) as described in Table 2 was used with the following modifications: A (inset): normal reaction mixture was supplemented with various a m o u n t s of a m m o n i u m chloride. Concentration of glutamine was varied as shown and different a m o u n t s of a m m o n i u m chloride used were: without a m m o n i u m chloride, e - - - e ; 5 mmol I 1, . . _ _ . . ; l0 m m o l I 1, 9 A. B concentration of hydroxylamine was varied as shown and the following a m o u n t s of a m m o n i u m chloride were included in the assay mixture: Without a m m o n i u m chloride, 9 e; 5 mmol1-1, 9 ..; 1 0 m m o l l -~, 9 9 15mmol1-1, [] D. 1IV represents reciprocal of gmol of y-glutamylhydroxamate formed in 30 min
ATP until the concentration of this nucleotide was approximately equivalent to that of the cation. Relatively higher concentrations of ATP to that of the cations inhibited the activity. The effects of various divalent cations (Figs. 2 B, C, D) and ADP on 7-glutamyltransferase activity have been presented earlier in this paper. Enzyme activities with Mn 2+, Co 2+ and Mg a+ respectively, with varying concentrations of L-glutamine are shown in Figure 5A (inset). The production of 7-glutamylhydroxamate with either of the three cations increased only slightly beyond 20 mmol 1- 1 glutamine. Double-reciprocal plots (Fig. 5A) gave an identical Km value of 5 mmol 1-1 glutamine with all three cations. The effects of varying the concentration of hydroxylamine on 7-glutamyltransferase activity are presented in Figure 5B (inset). With either Mn 2+ or Mg 2+, the enzyme was saturated with 12 mmol 1- 1 hydroxylamine whereas with Co 2+, no further increase in the activity occurred beyond 6 mmol 1-1 hydroxylamine. The activity with Mn 2§ or Mg 2+ was inhibited by 38 and 10% respectively at 60 mmol 1 - 1 hydroxylamine while that for Co 2+ was not affected. The Km values for hydroxylamine with Mg 2+ and Mn 2§ were 5.88 and 6.25 mmol 1-1 respectively and a lower value of
2.70 mmol 1-1 was obtained for the CoZ+-dependent activity (Fig. 5 B).
Effects of Substrates of the Biosynthetic Reaction on 7-Glutamyltransferase Activity. The effects of various substrates of the biosynthetic reaction on the Mn 2+dependent y-glutamyltransferase activity were examined. 7-glutamyltransferase activity was inhibited progressively in the presence of increasing amounts of ammonium chloride (Fig. 6A inset), and its Ki value was 23.0 mmol 1 - 1 Kinetic experiments on the inhibition of enzyme activity by ammonium ions with varying concentrations of glutamine (Fig. 6A) and hydroxylamine (Fig. 6B) indicated that it is a competitive inhibitor for glutamine and is non-competitive for hydroxylamine. The inhibitor effects on 7-glutamyltransferase activity of L-glutamate, in the presence of three concentrations of glutamine, are illustrated in Figure 7A (inset). Double-reciprocal plots of the inhibition by Lglutamate indicated that it was a competitive inhibitor of L-glutamine (Fig. 7A) and non-competitive with hydroxylamine (Fig. 7 B). 7-Glutamyltransferase activity was also inhibited by ATP and this effect was reversed with higher
S.K. Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena
2.0
3o1"
/~x
9
295
1.2
1.5
mM G l u t a m a t e , ,
=~
~
f
1.0
11.4 0.5
I
I
0.1
0.2
I
I
0.3
0.4
I
0.5
1
mM G l u t a m i n e
I
I
0.1
0.2
I
!
0.3
0.4
!
0.5
I
mM H y d r o x y l a m i n e
Fig. 7A and B. Effect of L-glutamate on ,/-glutamyltransferase activity in the presence of varying concentrations of L-glutamine and hydroxylamine. 7-glutamyltransferase activity with 3 mmol 1 1 Mn2+, using a desalted enzyme preparation, was determined as in Table 2 with the following modifications: A Inset : effect of L-glutamate was examined in presence of three concentrations of glutamine: 5 mmol 1- t, 9 9 10 mmol 1 1, 9 9 20 mmol 1-~, 9 9 Concentration of L-glutamine was varied as shown and the reaction mixture contained the following amounts of glutamate: without glutamate, 9 9 ; 10 mmol 1-1, 9 9 ; 20 mmol 1-1 9 9 ; 30 mmol 1- a, z x - - z x . B Reaction mixture contained 5 mmol 1 1 L-glutamine, various amounts of hydroxylamine and the following concentrations of L-glutamate: without glutamate, 9 9 ; 20 mmol 1-1, 9 9 ; 30 mmol 1-1, 9 9 1/Vrepresents reciprocal of gmol of ?-glutamylhydroxamate produced in 30 rain
concentrations of ADP. Thus in the presence of 0.05 mmol 1- ~ ADP, a 40% inhibition of 7-glutamyltransferase activity by 2 m m o l 1- a ATP, was reduced to 30 and 15% on increasing A D P to 0.1 and 0.4 mmol 1 1 respectively. As mentioned earlier, a substantial ?-glutamyltransferase activity with Mn 2 + was obtained even in the absence of ADP. This apparently ADP-independent activity was also inhibited by A T P but this effect did not exceed 20% even at saturating concentration of ATP. This should be compared with a 40% inhibition of the activity in the presence of 0.05 m m o l 1- ~ ADP.
Effects of Various Denaturing Treatments on the Biosynthetic and 7-Glutamyltransferase Activities. Results in Table 3 demonstrate that treatment of the enzyme with urea caused a relatively greater loss of 7-glutamyltransferase than the biosynthetic activity. Thus 2 tool 1-~ urea reduced the biosynthetic activity by 17%, whereas 7-glutamyltransferase activity was inhibited by 35%. The denaturation of the enzyme by urea was significantly suppressed by adding metal ions (Table 3). Thus a loss of 48% of the biosynthetic activity with 3 tool 1 1 urea was reduced to 5% when
the enzyme was incubated with urea in the presence of Mg 2§ In similar experiments (Table 3) Mn 2§ lowered the extent of inhibition of 7-glutamyltransferas 9 activity by 3 mol 1-1 urea from 59 to 18%. The data in Table 4 indicate that both the biosynthetic and v-glutamyltransferase activities were lost on incubating the desalted enzyme preparation at 50~ for 1 h. This thermal denaturation was prevented significantly and to the same extent, by including ]~-mercaptoethanol in the incubation mixture during the heat treatment. However, both enzyme activities were fairly stable to the heat treatment when the preparation was used without desalting, when the enzyme preparation contained 1 0 m m o l 1 - 1 MgC12. Under these conditions the addition of /~-mercaptoethanol induced loss of enzyme activity. The biosynthetic activity was affected to a greater extent since with 0.05 m m o l 1 - 1 fi-mercaptoethanol its activity was reduced by 24% whereas the corresponding loss of 7-glutamyltransferase activity was only 7%. A preliminary experiment showed that Mn 2§ Mg 2§ and Co 2 + dependent ?-glutamyltransferase and Mg 2+ dependent biosynthetic activities were lost concomitantly on incubating the enzyme at 50~ over
296
S.K. Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena
Table 3. Effect of urea on biosynthetic and ,/-glutamyltransferase activities
Table 5. Effect of various substrates on thermal stability of biosynthetic and y-glutamyltransferase activities
Desalted enzyme preparation was preincubated either in the presence or absence of divalent cation (70 m m o l l - 1 Mg 2+ for the biosynthetic and 2.5 mmol 1-1 Mn2+ for 7-glutamyltransferase activities, respectively) with various concentrations of urea for 15 min at 37 ~C. Both the biosynthetic and 7-glutamyltransferase reactions were started by adding the corresponding assay mixtures, as described in Table2, except that 23 p,mol of Mg 2§ and 2.50 gmol of Mn 2+ were used in the reaction, mixtures for the biosynthetic and 7-glutamyltransferase activities respectively. Cation-free reaction mixtures were added to the tubes which already contained the metal ions during preincubation. The biosynthetic and 7-glutamyltransferase activities of the controls were 0.26 and 2.75 gmol of Pi and 7-glutamylhydroxamate, respectively, produced in 30 min
For biosynthetic activity (Table5a), 125 ~tl of desalted enzyme was preincubated at 50~ for 30 min along with the following concentrations of the compounds indicated in the table (in ~tmol): ATP, 3; L-glutamate, 10; ammonium chloride, 10; Mg 2+, 20; L-glutamine, 10; ADP, 0.5; sodium arsenate, 10. After preincubation the tubes were cooled and 10 gmol of imidazole-HC1 buffer (pH 7.0) added to each tube. Components of the biosynthetic assay mixture other than the ones already present during preincubation, were added, the final volume made to 0.3 ml and the enzyme activity determined as described in Methods. Controls, containing each component individually, were run simultaneously to determine their heat stability and also to correct for any formation of Pi from ATP or A D P at higher temperatures. The results are expressed as per cent of the biosynthetic activity of the enzyme without heat treatment except where either L-glutamine, A D P or arsenate were included. In the latter case, per cent activities were calculated from corresponding control samples assayed in the presence of these compounds. The conditions for preincubating the enzyme for ),-glutamyltransferase (Table 5b) were the same as for the biosynthetic activity, except that the concentrations of various compounds used were, in lamol; glutamine, 20; sodium arsenate, 10; ADP, 2.0; hydroxylamine, 15; and M n z+ 1.5. After preincubation the enzyme activity was determined by adding the remaining components of ?~-glutamylhydroxamate assay mixture, at the concentrations specified above, in a final volume of 0.3 ml. The biosynthetic and 7-glutamylhydroxamate activities of the controls (without heat treatment) were 0.25 gmol of P~ and 2.89 gmol of 7-glutamylhydroxamate produced respectively in 30 min
Urea
Biosynthetic
mol 1 1
Preincubation at 37~ Without Mg 2+
y-glutamyttransferase for 15 min
With Mg 2+
Without Mn 2+
With Mn 2+
100 92 65 41 33 24 11
100 87 84 82 68 52 37
%activity of control 0 t 2 3 4 5 6
100 i00 83 52 22 8 3
100 103 100 95 86 69 59
Table 4. Biosynthetic and 7-glutamyltransferase activities after preincubating enzyme with fi-mercaptoethanol Purified enzyme without desalting (containing 10 mmol l - 1 MgC12) or after desalting was incubated with various concentrations of fl-mercaptoethanol at 50 ~C for 1 h. After cooling, the biosynthetic activity (with 70 mmol 1-~ Mg 2+) and y-glutamyltransferase activity (with 2.5 mmol 1-1 Mn 2+) were started by adding the corresponding assay mixtures as given in Table 2 Activities of the control sample (without heat treatment and in absence of fi-ercaptoethanol) without desalting were 0.25 p,mol Pi and 3.30 gmol of 7-glutamylhydroxamate and those after desalting were 0.20 btmol Pi and 2.45 gmol ~/-glutamylhydroxamate produced in 30 min for the biosynthetic and ~,-glutamyltransferase activities respectively fi-mercaptoethanol (Ixmol I- t)
After desalting
Without desalting
Bioy-glutamylsynthetic transferase
Biosynthetic
~-glutamyitransferase
100 94 81 76 68
100 100 100 93 83
%activity of control 0 10 25 50 100
21 32 41 69 57
14 38 48 72 53
a) Biosynthetic activity
b) ,/-glutamyltransferase activity
Additions during preincubation
% activity of control
Additions during preincubation
% activity of control
None (extract alone) Mg 2 + ATP Mg 2+ + A T P L-glutamate NH4Cl L-glutamine Sodium arsenate ADP
28
None (extract alone) Mn 2+ ADP Mn 2+ + A D P L-glutamine Hydroxylamine Sodium arsenate
39
95 50 98 95 28 96 43 49
85 53 85 76 5 51
a period of 90 min. The results of heat treatment, in the presence of various substrates, on Mg 2+-dependent biosynthetic activity of the enzyme are presented in Table 5a. This activity was protected almost completely by Mg 2§ or glutamate and to a lesser extent by ATP whereas ammonium chloride was ineffective. Glutamine or ADP, which are the substrates for 7-glutamyltransferase activity, were as effective as glutamate and ATP respectively in stabilizing the biosynthetic activity against thermal denaturation. A similar response of the ?-glutamyltransferase activity to various substrates during heat treatment was
S.K. Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena
60I !4~ o~2
1 0 5 Incubation
1
~ 10 t i m e (rnin)
15
Fig. 8. Time course of inactivation of biosynthetic and y-glutamyltransferase activities by hydroxylamine. Desalted enzyme preparation (100 lxl) was preincubated with 2.0 Izmol hydroxylamine hydrochloride (neutralized with NaHCO3) for the indicated time. The biosynthetic (using 70 mmol 1- t Mg z§ ?-glutamyltransferase (using 2.5 mmol I ~ Mn 2 +) activities were determined as described in Table 2, except that the reaction mixture for the latter activity contained 8 gmol of hydroxylamine. The biosynthetic ( i m) and 7-glutamyltransferase (A A) activities of the untreated sample were 0.28 pmol Pi and 3.40 pmol of 7-glutamylhydroxamate, respectively, produced in 30 min
Table 6. Effect of various substrates on inactivation of biosynthetic
and 7-glutamyltransferase activities by hydroxylamine The experimental details were the same as in Table 5 but instead of heat treatment, the enzyme was preincubated at 37 ~ C for 5 min with 2 gmol hydroxylamine. The concentration of hydroxylamine for ),-glutamyltransferase activity was 10 Izmol. Activities of the control samples (without hydroxylamine for the biosynthetic activity) were 0.28 gmoI of Pi and 3.40 gmol y-glutamylhydroxamate produced respectively in 30 rain. The per cent biosynthetic activities for treatments using either L-glutamine, ADP or arsenate and the treatment involving Mg 2+ in ~-glutamyltransferase activity, were calculated from corresponding controls assayed in the presence of these compounds a) Biosynthetic Activity
b) ,/-glutamyltransferase activity
Additions during preincubation
% activity of control
Additions during preincubation
% activity of control
None Mg 2+ ATP Mg2 + + ATP L-glutamate NH4C1 L-glutamine ADP Sodium arsenate
38 81 84 87 84 46 60 64 78
None Mn 2 + ADP Mn z+ + A D P L-glutamine Sodium arsenate Mg 2 +
40 92 72 100 86 100 85
297
observed (Table 5 b). Thus Mn 2 § and glutamine were equally effective in protecting the enzyme whereas ADP was less so and hydroxylamine accelerated the inactivation. The likelihood that the decomposition of hydroxylamine at high temperatures would result in lower 7-glutamyltransferase activity was discounted because the addition of hydroxylamine pre-equilibrated at 50~ for 30 min produced similar activities to the controls. The effect of hydroxylamine on glutamine synthetase was further investigated. Results in Figure 8 show an almost comparable loss of both 7-glutamyltransferase and biosynthetic activities on incubating the enzyme with 2 Izmol hydroxylamine. More than 80% of the biosynthetic activity was retained when the enzyme was preincubated with hydroxylamine in the presence of either Mg 2+, ATP or glutamate. Each of the substrates of 7-glutamyltransferase activity viz. glutamine, ADP or sodium aresenate also provided a significant protection to the biosynthetic activity against hydroxylamine (Table 6a). Similarly, the loss of y-glutamyltransferase activity, on preincubating the enzyme with hydroxylamine was largely prevented by either Mn 2", Mg 2+, ADP, glutamine or sodium arsenate (Table 6b). Discussion
These investigations on glutamine synthetase from Anabaena cylindrica show that it resembles the enzyme from other organisms in several of its properties, viz. requirement for divalent cations, pH optimum and K m values for substrates. As with the enzyme from other sources (Hubbard and Stadtman, 1967b) the maximum biosynthetic activity was obtained with Mg 2". The results indicate that the lower activity with Co 2--, than with Mg 2+, was largely due to its relatively high Km for glutamate. The response of the algal enzyme to concentrations of ATP relative to those of the divalent cations and also the more acidic pH optimum for the Co 2 +- and Mn 2 +-dependent than the Mg 2 § activity, are consistent with the properties of the enzyme from other organisms (Deuel et al., 1969; Dharmawardene et al., 1973; O'Neal and Joy, 1973; McParland etal., 1976). Our results show that the pH optimum and also the K m values for hydroxylamine, glutamine and ADP were the same for the Mn 2+, Co 2+ and Mg 2+ dependent 7-glutamyltransferase activities. As found for the enzyme from bacteria (Hunt et al., 1975) and plants (Speck, 1949; Elliot, 1953; Kanamori and Matsumoto, 1972; McParland et al., 1976), there were, however, marked differences in the optimum concentrations of the divalent cation s required for the maximum 7-glutamyltransferase activity.
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Significant amount of 7-glutamylhydroxamate was produced in the Mn 2 § reaction of the algal enzyme even in the absence of ADP. The requirement for Mn 2§ and arsenate, indicated that this reaction was catalyzed by 7-glutamyltransferase rather than by other possible contaminants like amidohydrolases (Hubbard and Stadtman, 1967b) or glutaminases (Meister, 1974). Synthesis of v-glutamylhydroxamate, in the absence of the nucleotide was observed only with Mn 2+ even though the activities with either Mn 2--, Mg 2§ or Co 2§ were saturated at the same concentration of ADP. Thus, these results do not support the suggestion (Lajtha et al., 1953; Meister, 1974) that this activity was due to enzyme bound adenine nucleotide. It is possible that these cations stabilize different conformational states of the enzyme and the one induced by Mn 2§ is partially active even in the absence of ADP. On the other hand, configurations attained by Co 2+ or Mg 2§ could require further modification by ADP for maximal enzyme activity. Such a role for ADP in v-glutamyltransferase reaction is possible in view of its requirement in catalytic amounts and secondly because it does not enter directly into the transferase reaction. In this work, glutamine synthetase has been shown to be much more stable in the presence of divalent cations. This effect is evident on comparing the extent of inactivation of both the biosynthetic and 7-glutamyltransferase activities, in the presence or the absence of divalent cations, on treatment with urea, hydroxylamine or high temperature. The partial stabilization of the desalted enzyme at 37~ by /%mercaptoethanol, suggests that its -SH groups become more exposed after removal of the cations. These observations are in agreement with the results for glutamine synthetase from E. coli (Kingdon et al., 1968; Shapiro and Stadtman, 1968) where the removal of divalent cation resulted in conversion of the enzyme to a 'relaxed' state. The biosynthetic activity of desalted enzyme preparation was protected against thermal denaturation by either Mg 2§ ATP, glutamate, glutamine or ADP. In this respect, this enzyme behaves differently from the sheep brain enzyme which was stabilized when both Mg 2+ and ATP were present together (Pamiljans et al., 1962). Protection of the enzyme by either glutamate or glutamine against heat denaturation as well as hydroxylamine implies that these substrates interact with the algal enzyme even in the absence of metal ions and adenine nucleotides. These observations thus support the proposed random (Wedler, 1974) rather than sequential order of binding of the substrates (Pamiljans et al., 1962). The failure of ammonia or hydroxylamine to provide such protection may be due either to their inability to bind to
the enzyme in the absence of the other substrates (Rhee et al., 1976) or simply to the nonconversion of the enzyme into a more stable form. The results of the comparative studies on the biosynthetic and 7-glutamyltransferase activities presented in this paper provide further evidence that they are associated with the same enzyme. It has also been shown that v-glutamyltransferase activity was inhibited by the substrates of the biosynthetic reaction. Thus both ammonia or glutamate inhibited the transferase activity competitively with glutamine and non-competitively with hydroxylamine. Gass and Meister (1970) have suggested that glutamine interacts with the enzyme in such a way that its NH2-group occupies the ammonia binding site while the 'oxygenbinding' site to which glutamate is normally bound, is required for the attachment of the corresponding oxygen group of glutamine. Thus the observed competitive effects of glutamate or ammonia with glutamine in the 7-glutamyltransferase reaction reported herein are consistent with their postulated model for the active site of the enzyme, but not with the suggestion that corresponding pairs of the substrates for biosynthetic and transferase reactions share common sites (Hubbard and Stadtman, 1967a). Our results also suggest that the interaction of glutamine with N H 2 binding site, would preclude the binding of hydroxylamine at this locus, A lack of competition between ammonia and hydroxylamine was further demonstrated by the inability of the former substrate to protect the algal enzyme against its inactivation by hydroxylamine. The 7-glutamyltransferase reaction was also inhibited by ATP, possibly competitively with ADP, since its effect was reversed by increasing concentrations of ADP. The results of these kinetic experiments suggest that during 7-glutamyltransferase reaction glutamine interacts with the enzyme at the sites where glutamate and ammonia bind in the biosynthetic reaction while the adenine nucleotide binding site could be occupied by either ATP or ADP. Further work is required to examine the effects of various substrates of the transferase reaction on the biosynthetic activity with a view to elucidating the pattern of binding sites for the substrates of these two reactions of glutamine synthetase. This work was supported by a generous grant from the Australian Research Grants Committee and the Australian Water Resources Council. The skilled technical assistance of Mr. Michael Byrne is gratefully acknowledged. References
Brown, C.M., MacDonald Brown, D.S., Meers, J.L. : Physiological aspects of microbial inorganic nitrogen metabolism, in Ad-
S.K. Sawhney and D.J.D. Nicholas: Glutamine Synthetase from Anabaena vances in Microbial physiology, 11, pp I 52, Rose, A.H., Tempest, D.W. eds., London: Academic Press 1974 Brownell, P.F., Nicholas, D.J.D. : Some effects of sodium on nitrate assimilation and Nz fixation in Anabaena cylindrica. Plant Physiol. 42, 915 921 (1967) Dharmawardene, M.W.N., Stewart, W.D.P., Stanley, S.O.: Nitrogenase activity, amino acid pool patterns and amination in blue-green algae. Planta 108, 133-145 (1972) Dharmawardene, M.W.N., Haystead, A., Stewart, W.D.P. : Glutamine synthetase of nitrogen fixing alga, Anabaena cylindrica. Arch. Mikrobiol. 90, 281 295 (1973) Deuel, T.F., Stadtman, E.R.: Some kinetic properties of Bacillus subtilis glutamine synthetase. J. biol. Chem. 245, 5206-5213 (1970) Deuel, T.F., Shelton, E., Stadtman, E.R. : Structure and regulation of Bacillus subtilis glutamine synthetase. Fed. Proc. 28, 341 (1969) Elliot, W.H. : Isoiation of glutamine synthetase and gtutamotransferase from green peas. J. biol. Chem. 201, 661 672 (1953) Gass, J.D., Meister, A. : Computer analysis of active site of glutamine synthetase. Biochemistry 9, 1380-1389 (1970) Hachimori, A., Matsunaga, A., Shimizu, M., Samejima, T., Nosoh, Y.: Purification and properties of glutamine synthetase from Bacillus stearothermophilus. Biochim. Biophys. Acta 350, 461-474 (1974) Hubbard, J.S., Stadtman, E.R. : Regulation of glutamine synthetase VI. Interactions of inhibitors for Bacillus licheniformis glutamine synthetase. J. Bacteriol. 94, 1016-1024 (1967a) Hubbard, J.S., Stadtman, E.R. : Regulation of glutamine synthetase II. Patterns of feedback inhibition in microorganisms. J. Bacteriol. 93, 1045-1055 (1967b) Hunt, J.B., Smyrniots, P.Z., Ginsburg, A., Stadtman, E.R.: Metal ion requirement by glutamine synthetase of Escherichia coli in catalysis of 7-glutamyl transfer. Arch. Biochem. Biophys. 166, 102-124 (1975) Kanamori, T., Matsumoto, H.: Glutamine synthetase from rice plant roots. Arch. Biochem. Biophys. 152, 404-412 (1972) Kingdon, H.S., Hubbard, J.S., Stadtman, E.R. : Regulation of glutamine synthetase XI. The nature and implications of lag phase in the Escherichia coli glutamine synthctase reaction. Biochemistry 7, 2136-2142 (1968) Lajtha, A., Mela, P., Waelsch, H. : Manganese-dependent glutamotransferase. J. biol. Chem. 205, 553-564 (1953) Levintow, L., Meister, A., Hogeboom, G.H., Kuff, E.L.: Studies on the relationship between the enzymatic synthesis of glutamine and glutamyl transfer reaction. J. Amer. Chem. Soc. 77, 5304-5308 (1955) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 262-275 (1951) McParland, R.H., Guevara, J.G., Becker, R.R., Evans, H.J. : The purification and properties of glutamine synthetase from cytosol of soy-bean root nodules. Biochem. J. 153, 597-606 (1976) Meister, A.: Glutamine synthetase of mammals, in The Enzymes Vol X, pp 699-754, 3rd ed., Boyer, P.D. ed, New York and London: Academic Press 1974 Miflin, B.J., Lea, P.J.: The pathway of nitrogen assimilation in plants. Phytochemistry 15, 873-885 (1976) Nagatani, H., Shimuzu, M., Valentine, R.C.: The mechanism of ammonia assimilation in nitrogen fixing bacteria. Arch. Mikrobiol. 79, 164-175 (1971)
299
O'Neal, D., Joy, K.W.: Glutamine synthetase of pea leaves: 1. Purification stabiiization and pH optima. Arch. Biochem. Biophys. 159, 1I3-122 (1973) Pamiljans, V., Krishnaswamy, P.R., Dumville, G., Meister, A.: Studies on the mechanism of glutamine synthesis; Isolation and properties of the enzyme from sheep brain. Biochemistry 1, 153-158 (1962) Rhee, S.G., Chock, P.B., Stadtman, E.R.: Mechanistic studies of glutamine synthetase from Escherichia coli. An integrated mechanism for biosynthesis, transferase and ATPase reaction. Biochemistry 58, 35-49 (1976) Ronzio, R,A., Rowe, W.B., Wilk, S., Meister, A.: Preparation and studies on the characterisation of sheep brain glutamine synthetase. Biochemistry 8, 2670-2674 (1969) Seyama, S., Kuroda, Y., Katunuma, N. : Purification and comparison of glutamine synthetase from rat and chick livers. J. Biochem. 72, 1017-1027 (1972) Shapiro, B.M., Stadtman, E.R.: Regulation of glutamine synthetase IX. Reactivity of the sulphydryl groups of the enzyme from Escherichia coli. J. biol. Chem. 242, 5069 5079 (1968) Shapiro, B.M., Stadtman, E.R. : Glutamine synthetase (Escherichia coli), in Methods in Enzymology, Vol. 17A, pp 910-922, Tabor, H., Tabor, C.W., eds., New York and London: Academic Press 1970 Speck, J.F. : The enzymatic synthesis of glutamine, a reaction utilising adenosine triphosphate. J. biol. Chem. 179, 1405 1426 (1949) Stadtman, E.R., Giusburg, A. : Glutamine synthetase of Escheriehia coli, in The Enzymes, Vol. X, pp 755 807, 3rd ed., Boyer, P.D. ed,, New York and London: Academic Press 1974 Stewart, G.R., Rhodes, D. : Evidence for the assimilation of ammonia via glutamine pathway in nitrate-grown Lemna minor. FEBS Lett. 64, 296-299 (1976) Stewart, W.D.P., Rowell, P. : Effects of L-methioninc-DL-sulphoximine on assimilation of newly fixed NH3, acetylene reduction and heterocyst production in Anabaena cylindrica. Biochem. Biophys. Res. Commun. 65, 846-856 (1975) Tate, S.S., Meister, A.: Regulation of rat liver glutamine synthetase: Activation by c~-ketoglutarate and inhibition by glycine, alanine and carbamyI phosphate. Proc. Natl. Acad. Sci., USA. 68, 781-785 (1971) Tare, S.S., Leu, F.Y., Meister, A. : Rat liver glutamine synthetase. Preparation, properties and mechanism of inhibition by carbamyl phosphate. J. biol. Chem. 247, 5312 5321 (1972) Thomas, J., Meeks, J.C., Wolk, C.P., Shaffer, P.W., Austin, S.M., Chien, W.S.: Formation of glutamine from [a3N] ammonia, [13N] dinitrogen and [14C] glutamate by heterocysts isolated from Anabaena cylindrica. J. Bacteriol. 129, I545-1555 (1977) Wedler, F.C.: Mechanisms of substrate binding with glutamine synthetase. Equilibrium isotope exchanges with ovine brain, pea seed and Escherichia coli enzymes. J. biol. Chem. 249, 5080-5087 (1974) Wolk, C.P., Thomas, J., Shaffer, P.W., Austin, S.M., Galonsky, A.: Pathway of nitrogen metabolism after fixation of 13Nlabeled nitrogen gas by the cyanobacterium, Anabaena cylindrica. J. biol. Chem. 251, 5027-5034 (1976)
Received 6 December; accepted 22 December 1977
