Eur. J. Biochem. 84, 285-291 (1978)

N-Acetylglutamate Synthetase from Rat-Liver Mitochondria Partial Purification and Catalytic Properties Katsuya SHICJESADA and Masamiti TATTBANA Department of Biochemistry, Chiba University School of Medicine (Received September 20, 1977)

N-Acetyl-L-glutamate synthetase was purified approximately 300-fold from the extract of rat liver mitochondria. The enzyme shows a high substrate specificity for L-glutamate and acetyl-CoA. No or very low activity is observed with other amino acids and acyl compounds. The reaction velocity fits normal Michaelis-Menten kinetics with respect to both L-glutamate (K,, 3.0 mM) and acetylCoA (K,, 0.7 mM). Acetylglutamate, the reaction product, and some of its structural analogues, such as N-acetylaminoadipate, N-propionylglutamate, and N-acetylglutamine, significantly inhibited the enzyme reaction. The Ki for acetylglutamate was 0.07 mM. The enzyme activity is sensitive to thiol-blocking agents and is inhibited by divalent cations. The enzyme activity is markedly stimulated by arginine, presumably by an allosteric mechanism. Other intermediates of the urea cycle and various structural analogues of arginine were practically not effective. Arginine increases the maximal velocity with no influence on K, values for the substrates. The activation shows a sigmoidal dependence on arginine concentrations and reaches a halfmaximal level at 5 - 10 p M ~The enzyme behaves as a mixture of multiple forms upon gel filtration (the range of molecular weight, 30000 to 300000) and its mean value is considerably increased in the presence of arginine. The specific activity of the enzyme increases with higher enzyme concentrations in the assay. The effect is more marked in the presence of arginine than in its absence. These observations suggest that the enzyme undergoes association and dissociation with concomitant changes in catalytic properties.

Acetylglutamate serves as a specific and obligatory activator of mitochondria1 carbamoyl-phosphate synthetase I, the first enzyme of urea biosynthesis in ureotelic animals including mammals [l,21. However, the physiological significance of the activator function of acetylglutamate remains obscure. We reported observations [3,4] which support the proposal that varying intracellular levels of acetylglutamate control the activity of carbamoyl-phosphate synthetase I, thus effecting regulation of entry of NH, into the urea cycle [5].In line with these studies, we demonstrated enzyme activity in mitochondria of rat and mouse livers which catalyzes the acetyl-Co A-dependent acetylation of the amino group of glutamic acid [4]. A unique property of the enzyme is that it is activated specifically by L-arginine. Ahhreoicrtion. Acetylglutarnate, N-acetyl-r-glutamate. Enzymes. Acetylglutamate synthetase o r acetyl-CoA: L-glutamate N-acetyltransferase (EC 2.3.1.1); carbamoyl-phosphate synthetase 1 orcarbamoyl-phosphate synthetase (ammonia) (EC 2.7.2.5).

This paper deals with partial purification of acetylglutamate synthetase from rat liver and characterization of its kinetic and regulatory properties. A preliminary account of part of this work has already been documented [6].

MATERIALS A N D METHODS Chemicals, Enzymes, and Isotopes N-Acetylglutamate, N-acetyl-L-ornithine, L-argininic acid, and other derivatives of glutamate and arginine, dithiothreitol, 5,5'-dithio-bis(2-nitrogenzoic acid), N-ethylmaleimide andp-hydroxymercuribenzoic acid were purchased from Sigma Chemical Co. (St Louis, Mo., U.S.A.). D- and L-glutamate, D- and Larginine, L-homoarginine, L-canavanine, other naturally occurring amino acids, and CoA were obtained from Kyowa Hakko Kogyo Co. (Tokyo, Japan). Labeled and unlabeled acetyl-CoA and other acyl-

286

CoA's were prepared [7], and the purity was determined by the hydroxamic acid method [8]. DEAEcellulose (type DE32) was supplied by Whatman, Sephadex G-100 and G-200 were from Pharmacia Fine Chemicals (Uppsala, Sweden), and the Diaflo XM-100 membrane from Amicon Far East Ltd (Tokyo, Japan). The collodion bags were from Sartorius Membranfilter GmbH (Gottingen, F.R.G.). Carbamoyl-phosphate synthetase I was prepared from frog liver [9] and ornithine carbamoyltransferase from bovine liver [lo]. ~ -[U'~ C]Glu tamate (10 Ci/mol), [l-*4C]aceticanhydride ( 5 Ci/mol) and L-[guanidino14C]arginine (2 Ci/mol) were supplied by the Radiochemical Centre (Amersham, England). ['4C]Glutamate was purified by adsorption on and elution from a Dowex 50-X8 (H' form) column [3]. Animals

Male Wistar rats, weighing 200 - 300 g, were used throughout and were usually maintained on a commercial laboratory chow containing 24 % crude protein. In some cases, synthetic diets containing various amounts of casein were provided [l I]. Assay of Acetylglutamate Synthetase

Enzyme was added to the standard mixture at 37 "C containing, in a final volume of 100 pl, 1.O mM ~-['~C]glutarnic acid (5.0 Ci/mol), 0.5 mM acetyl-CoA, 1.0 mM EDTA, 50 mM Tris-HC1 (pH 8.2) and incubated for 30 min. Where indicated, ['4C]glutamate and acetyl-CoA were replaced by unlabeled glutamate and ['4C]acetyl-CoA. The reaction was stopped with 50 pl of 1 M HCOOH containing 5 pmol of acetylglutamate. A 100-p1 aliquot of the mixture was applied to a small column (0.2 x 1.0 cm) of Dowex 50-X8 (H' form, 200-400 mesh), which was then washed with 1 ml of 0.1 M HCOOH and the whole effluent containing the ['4C]acetylgl~tamate formed, but not unreacted ['4C]glutamate, was collected. In the assay of partially purified enzyme, the radioactivity of the whole effluent was directly counted, with 10 ml of Bray's solution [12], in a Beckmann LS-100 scintillation counter. In the assay of crude mitochondria1 extracts, the above fraction was further purified by chromatography on Whatman 3MM paper in ethylether/benzene/formic acidlwater (22191712, v/v/v/v) [3]. The spot of ['4C]acetylglutamate was cut out and counted by the direct immersion method [3]. When [14C]acetyl-CoA was the labeled substrate, the reaction was stopped by adding 2 mg of activated charcoal suspended in 50 p1 of 1 M HCl containing 50 (v/v) ethanol to adsorb unreacted ['4C]acetylCoA, followed by centrifugation of the mixture. ['4C]Acetylglutamate in the supernatant was further purified by paper chromatography and counted as

Acetylglutamate Synthetase

described above. One enzyme unit is defined as that activity of enzyme which catalyzes the formation of 1 nmol of acetylglutamate in 30 min under the standard conditions. In some cases, the determination of acetylglutamate formed was based on its activator function for carbamoyl-phosphate synthetase I [3]. Other Determinations

Arginase activity was assayed by incubating enzyme in a 100-pl mixture containing 20 nmol of [guanidino''C]arginine (2 Cilniol) and 50 mM Tris-HC1 (pH 8.2) at 37 "C. After 30 min, lop1 of 2 M HCOOH was added to the mixture and a 50-yl aliquot was subjected to electrophoresis on Whatman 3MM paper at pH 1.8 [13]. The spot of [I4C]urea was cut out and counted directly [3]. Protein was colorimetrically determined [14] with crystalline bovine serum albumin as a standard.

RESULTS Purijication of Acetylglutamate Synthetase Conditionqfor Enzyme Assay. Since acetylglutamate synthetase activity in the rat liver is so low as to catalyze the formation of only 50 nmol of acetylglutamate x (g tissue)-' x h-' under the standard conditions in the absence of added arginine [4], the radiochemical assay was used. Even by this method, the activity was not detectable in the whole tissue homogenate, presumably because [14C]glutamateand acetylCoA were consumed by other reactions. The existence became clear using extracts of isolated mitochondria as enzyme, together with subsequent purification of the reaction product. ['4C]Acetylglutamate thus formed was identified as described previously [3]. With enzyme preparation after the ammonium sulfate step, the activity was simply assayed by measuring acetylCoA-dependent incorporation of radioactivity from ['4C]glutamate into the acidic components unadsorbed on the Dowex 50 column. With preparations after the hydroxyapatite step, the activity was a linear function of time up to 30 min under the standard contitions. It is notable that the enzyme activity was not linear with the amount of protein as described below (Fig. 2). Several attempts including addition of bovine serum albumin to the assay system failed to produce linearity. Although the enzyme is inhibited by the product acetylglutamate, the amount of the accumulated product was far below its Ki value and thus was not the cause of the nonlinearity. It is possible that dissociation and association of the enzyme took place depending on its concentration, as is discussed later. The specific activity of each enzyme preparation was expressed by a reciprocal of that amount of protein in mg, which

287

K . Shigesada and M Tdtibana

Table 1. Purification of acetylglutamate syntherase Starting material was 67 g of rat liver. A unit of arginase activity is defined as that amount of activity which catalyzes liberation of 1 nmol urea in 30 min. The detailed conditions for the assay are described in thc text. The unit of acetylglutamate synthetase activity is defined in the text

-

~

~~

~

~~

Total protein

Purification step

Acetylglutamate synthetase

-

~~

~

~~

-

Arginase .~

~~

total activity

specific activity

activation by 1 mM arginine

mg

units

unitslmg

:4

unitslmg

1140 310 28 4.9 2.0

1070 2280 1410 870 670

0.94 7.4 50.4 196 335

210 690 720 740 710

1000 400 16 9 5

~

Sonic extracts of mitochondria Ammonium sulfate (0 -400;) Hydroxyapatite DEAE-cellulose column Scphadex G-I00 gel filtration

exhibits one unit of activity under the standard assay conditions. Preparation of Mitochondria1 Extracts. Subsequent operations were performed at 0 - 4 "C and buffer solutions used contained 0.1 mM dithiothreitol, unless otherwise stated. Generally 50 g of liver were used as the starting material. Mitochondria were isolated [15], suspended in 25 ml of 50 m M potassium phosphate (pH 7 3 , disrupted with a sonic disintegrator (Kaijo Denki, model 2310) at 20 kHz for 2 min, and centrifuged at 105000 x g for 60 min. Ammonium SulJhte Fractionation. The supernatant was brought to 400/:, saturation with solid ammonium sulfate (243 g/l) and, after 15 min, centrifuged at 15000 x g for 30 min. The pellet was dissolved in 20 ml of 0.01 M potassium phosphate (pH 7.0). Chromatography on Hydroxyapatite. The solution was applied to a hydroxyapatite column (2 x 10 cm) equilibrated with 0.01 M potassium phosphate (pH 7.0). Elution was carried out successively with 0.05, 0.075, and 0.20 M potassium phosphate (pH 7.0) in respective volumes of 40, 40, and 100 ml, and 20-ml fractions were collected. The enzyme activity was usually recovered in the 0.20 M eluates, which were pooled and concentrated to 5 ml or less in an Amicon ultrafiltration cell using an XM-100 membrane. Chromatography on DEAE-cellulose. The concentrated enzyme solution was desalted on a Sephadex (3-25 column (1.5 x 20 cm) equilibrated with 0.01 M potassium phosphate buffer, pH 7.5, and applied to a column (1 x 20 cm) of DEAE-cellulose (Whatman DE32) equilibrated with the same buffer. Elution was performed with a linear gradient (120 ml) of 0.01 -0.25 M potassium phosphate (pH 7.5). Active fractions were pooled and concentrated to 2 ml or less in a Sartorius collodion bag. Gel Filtration on Sephadex G-100. The enzyme solution was applied to a Sephadex G-100 column (1.5 x 40 cm) equilibrated with 0.1 M potassium phos-

phate (pH 7 . 9 , and eluted with the same buffer at a flow rate of 10 ml/h. The enzyme appeared as a single peak immediately after the void volume. Active fractions were pooled and concentrated in a Sartorius collodion bag to about 2 ml and frozen rapidly in small portions (0.5 ml) and stored at - 80 "C. A typical result of purification is summarized in Table 1. Since the total activity usually increased by 50 - 100% after the ammonium sulfate fractionation, the indicated activity of the initial mitochondria1 extracts should be regarded as a minimum estimate. With this reservation, purification up to 300-fold with a final recovery of over 50% was achieved. However, in view of the specific activity of the final preparation (Table 1 ) and chromatographic patterns of enzyme purification, it appears that further extensive purification, possibly 100-fold or more, is necessary to obtain an homogeneous preparation. Preparations after the hydroxyapatite step contained only a trace of arginase activity (Table l), the presence of which can interfere with studies on the activation of the enzyme by arginine. The extent of activation by arginine did not change after the ammonium sulfate fractionation (Table l), although it varied with different lots of preparation. From the elution pattern of the enzyme from the Sephadex G-100 column, its mean molecular weight was estimated to be more than 150000, although its apparent molecular size was variable with experimental conditions as is discussed later.

Stability of Enzynze The purified enzyme lost activity by about 10% after 24 h at 4 "C in 0.1 M potassium phosphate buffer (pH 7.5). When stored frozen at - 80 "C without intermittent thawing and freezing, about 5004 of the activity remained after 6 months.

288

Dietary Variations in the Enzyme Leoel The enzyme level varied in proportion to protein contents of given diets. For example, the activity as measured with the ammonium sulfate fraction was 46 nmol acetylglutamate formed x (g liver)-' x h-' in animals fed a 5 % casein diet for a weak and 76 nmol x (g liver)-' x h-' in those fed a 6004 casein diet. In these experiments, animals were provided food ad lihiturn and used for the preparation of enzyme at 9 a.m. Substrate Specijicity The enzyme showed a relatively high substrate specificity toward L-glutamate and acetyl-CoA. With 0.5 mM ['4C]acetyl-CoA as acyl donor, glycine, DLa-aminoadipate, and D-glutamine at 1 mM showed a low activity as acyl acceptors (8,6, and 2 % of L-glutamate, respectively). L-Glutamine and L-aspartate were totally inactive. The minute activity with D-glutamate might be due to its possible contamination with Lglutamate. With 1 mM ~-['~C]gIutamate as acyl acceptor, 0.5 mM propionyl-CoA could substitute for acetyl-CoA to a limited extent (7 %). Butyryl-CoA and benzoyl-CoA showed only a very low activity (1 :d of acetyl-CoA). Acetylphosphate, acetyl carnitine, and N-acetyl-L-ornithine were totally ineffective as acyl donors. Efect of Metal Ions The enzymic formation of acetylglutamate did not require added metal ions. The reaction was little inhibited by 10 mM EDTA. Rather, the activity was inhibited by a low concentration of various polyvalent metal ions: 30-500/, inhibition was observed with MgCl,, CaCl,, and BaCI, at 1 mM, and with FeSO,, FeCl, , CoCl,, MnCl, , and Ni(NO,), at 0.1 mM. The inhibition by these salts was completely abolished by EDTA added in excess. p H Dependence The enzyme activity showed a broad pH-dependence with an optimum at pH 8 in the absence of arginine. In the presence of arginine, the optimum shifted to pH 8.5 and the peak of the pH/velocity curve became much steeper than in its absence. The activities in 50 mM possium phosphate buffer in the presence and absence of arginine were about 40% and 70% of those in 50 mM Tris-HC1, respectively. The activity in 50 mM diethanolamine-HC1 buffer (pH 8.5) was slightly lower than in Tris buffer. Egects of Sulfhydryl Reagents and Ionic Strength The enzyme was strongly inhibited by sulfhydrylblocking agents such as p-hydroxymercuribenzoate,

Acetylglutamate Synthetase

5,5'-dithio-bis(2-nitrobenzoate)and heavy metal salts such as AgNO, , Hg(CH,COO), , Pb(NO,), , CuSO,, and ZnC1,. These agents caused 70 - 90 inhibition at 0.1 mM. The enzyme activity was inhibited by a high concentration of KCI; the extent was 3004 at 100 mM and 5004 at 200 mM, Potassium phosphate and K,S04 were even more inhibitory, Therefore, care was taken in the enzyme assay to keep the total salt concentration as low as possible. Inhibition by Product and Its Analogues The enzyme reaction was strongly inhibited by its product, acetylglutamate (88% at 2 mM). Some structural analogues of acetylglutamate that effected inhibition and the extents of inhibition are as follows: N-acetyl-DL-a-aminoadipate(78 'd), N-propionyl-LN-acetyl-L-glutamine (46 Nglutamate (63 carbamoyl-L-glutamate (31 %), N-acetyl-o-glutamate (30%), N-benzoyl-L-glutamate (29 N-acetyl-L-aspartate (25 'd), N-butyryl-L-glutamate (19%), L-aacetoxylglutarate (1 7 %), N-acetylglycine and succinate (21 %). Free CoA, the other reaction product, exerted a 20"/, inhibition at 1 mM.

x),

x),

x),

(Ox),

Kinetic Studies The velocity of the synthetase reaction versus concentrations of glutamate with a fixed concentration of acetyl-CoA showed Michaelis-Menten kinetics. Similar hyperbolic kinetics were obtained for acetylCoA concentration/velocity relationship. Double-reciprocal plots of these data gave K , values for glutamate and acetyl-CoA of 3.0 and 0.7 mM, respectively (Fig. 1). These values are considerably higher than those reported previously (1.8 mM for glutamate and 0.3 mM for acetyl-CoA) [4]. The discrepancy is probably due in part to different buffers used for the enzyme assay: potassium phosphate (pH 7.5) was used formerly and Tris-HC1 (pH 8.2) was used in the present experiments. The inhibitory effects of 0.2 mM acetylglutamate with variable concentrations of either glutamate or acetyl-CoA are also shown in Fig. 3 . The kinetics are consistent with competitive inhibition. From these data, the intrinsic value of Ki for acetylglutamate was calculated to be 0.07 mM. Actiuation by Arginine The enzyme is subject to activation by arginine [4]. [~uuni~ino-14C]Arginine added to the assay system

was recovered almost quantitatively (more than 95 %) after the reaction, with liberation of only a minute quantity of [14C]urea due to contaminating arginase activity. An allosteric mechanism for the activation was also supported by the occurrence of the partly desensitized enzyme as described hereafter.

K. Shigesada and M. Tatibana

289

D /'

I

1 / [Glutamate] (mM-')

1 2 3 4 l/[Acetyl-CoA] (rnM-')

I

5

Fig. 1. Double-reciproccd plot of variation of velocity against varying concentrutions of substrates. Standard assay conditions were used except for the variations in substrate concentrations. In some assays, 0.1 niM, arginine (-- ) or 0.2 mM acetylglutamate (-- - - ) was added. Enzyme: DEAE-cellulose fraction, 2 pg protein. (A) Glutamate concentrations were varied as indicated. Acetyl-CoA concentrations were 0.20 mM (O), 0.35 mM (Uj, 0.50 mM (A) and 0.80 mM (O), respectively. (B) Acetyl-CoA concentrations were varied as indicated. Glutamate concentrations were 1.0 m M (0). 1.75 m M (U). 2.5 mM (A) and 4.0 mM (O), respectively. Initial velocity, v, was measured as nmol acetylglutamate produced in 30 min

Variable Sensitivity of DifSerent Preparation to Activation by Arginine. The extent of activation by arginine varied with different enzyme preparations, in a range from 200 % to 800 11/0. About 400 % activation was observed most frequently. It appeared that the enzyme underwent some sort of desensitization during the early steps of the purification procedure, as is suggested by the following experiments. A batch of liver homogenate was divided into two equal parts; one was immediately subjected to purification by the step of ammonium sulfate fractionation, and the other was kept at 4 "C for 5 h and then purified in the same way. The extent of arginine activation was 450% in the former preparation while only 50% in the latter one. Those preparations showing a higher extent of activation (more than 400%) and at the DEAEcellulose step were generally used in studies presented in this paper. Specijicity of Arginine as Activator. The specificity of arginine as activator is quite strict. Other natural amino acids, intermediates of the urea cycle and synthetic derivatives of arginine tested were without effect, with the exception of slight stimulation (+ 30 %) by L-argininic acid [ 6 ] .Some of ineffective compounds tested were D-arginine, L-homoarginine, L-canavanine, N-acetyl-L-arginine, L-arginine methyl ester, L-arginine ethyl ester, L-arginine hydroxamate, agmatine, Lornithine, N'-acetyl-L-ornithine, L-lysine, L-citrulline and spermidine. EfSect of Enzyme Concentration andpH on Arginine Activation. The extent of activation by arginine varied greatly with enzyme concentrations and pH values. As shown in Fig. 2, the specific activity of the enzyme in the presence of arginine (1 mM) became greater with increased enzyme concentrations, and the rela-

A

pH70

B pH82

C pH95

Enzyme concentration (wg protein/tube)

Fig. 2. Dependence of speci$c activiry of the synthefase on enzyme concentration. The activity was measured in the presence ( 0 ) and of 1 m M arginine under the standard assay conditions absence (0) except for the variations in pH and buffer. (A) p H 7.0 (50 mM Tris-HC1); (Bj p H 8.2 (50 mM Tris-HCI); (C) p H 9.5 (50 mM diethanolamine-HC1)

tionship was more pronounced at pH 8.2 and 9.5 than at pH 7.0. A similar relationship between the specific activity and the enzyme concentration was observed even in the absence of arginine, though to a lesser extent. It is assumed that the enzyme underwent molecular association depending on its concentration with a concomitant increase in catalytic activity and that the enzyme association was promoted by binding with arginine. Possible Association of Enzyme Promoted by Arginine. The possible effect of arginine on the molecular size of enzyme was studied by gel filtration chromato-

Acetylglutamate Synthetase

290

DISCUSSION

i

Fig. 3. Effect of arginine on association of'fhe enzyme. Enzyme preparation after the Sephadex G-100 step (0.3 mg protein in 0.1 M potassium phosphate, pH 7.5) was applied to a Sephadex G-200 column (1 .5 x 40 cm) equilibrated with 0.1 M potassium phosphate (pH 7.5) containing 0.5 mM arginine and eluted with the same buffer at a Row rate of 10 ml/h. Fractions of 1 ml each were collected. A control run of chromatography was performed in the absence of arginine. The enzyme activity was measured in the presence of 1 mM and the absence arginine. Elution profiles in the presence (0 ).(0-0) of arginine. The arrows indicate the void volume and elution volumes of marker proteins : V,, , void volume; (1) apoferritin ( M , 480000); (2) human immunoglobulin ( M , 160000); (3) bovine serum albumin ( M , 67000); (4) ovalbumin ( M , 45000); ( 5 ) beef pancreas chymotrypsinogen ( M , 25000); (6) horse heart cytochrome c ( M , 12400)

graphy on Sephadex G-200. As shown in Fig. 3, in the absence of arginine the enzyme activity exhibited a broad elution profile with a main peak corresponding to a molecular weight of about 100000. In the presence of arginine, the mean molecular weight increased to 200000. These results strongly suggest that the enzyme underwent various degrees of association. From the figure, the molecular weights of the smallest and the largest enzyme forms were roughly estimated to be 30 000 and 300 000, respectively. EfSect of Arginine Concentrations. Activation of the synthetase at variable concentrations of arginine yielded a sigmoidal curve as reported previously [ 6 ] . The extent of activation became maximal at about 0.1 mM and declined at concentrations higher than 1 mM. A half maximal activation was reached at 5 - 10 pM. The extent of maximal activation varied with pH, as discussed above (Fig. 2), and with the concentration of enzyme. As shown in Fig. 1, arginine affected only the maximal velocity of the enzyme without any effect on K,,, values either for glutamate or acetyl-CoA. Conversely, the apparent affinity of the enzyme for arginine was not affected by changes in the substrate concentrations (data not shown).

Acetylglutamate synthetase has a high specificity for arginine as activator [4,6] (and this paper). The concentration of arginine required to give a half maximal activation varied in the range of 5 - 10 pM. The intracellular content of arginine in mouse liver was found to be 26- 170 pmol x (g liver)-' 1161 and therefore appears to be adequate for the enzyme activation, although it is not known whether the intramitochondria1 concentration is proportional to the mean concentration in the whole cellular space. It was suggested that the enzyme undergoes concentration-dependent association and dissociation with concomitant changes in its catalytic and regulatory capacity (Fig.2 and 3). Furthermore, it is likely that arginine promotes the association of enzyme and that the stimulatory effect of arginine increases with higher degrees of association of the enzyme. Such effects of arginine may be of physiological importance since the concentration of the enzyme in the mitochondria must be much higher than that in the assay system in vitro and thus the enzyme is presumably more sensitive to arginine activation in vivo. In fact, [14C]acetylglutamate synthesis was shown to be remarkably stimulated by arginine in the isolated mouse liver mitochondria (more than 10-fold 141). The enzyme is subject to a fairly strong product inhibition by acetylglutamate in a manner competitive with glutamate and acetyl-CoA. Since the intramitochondrial concentration of acetylglutamate, 0.1 - 0.5 mM [3,16], is well above Ki,its inhibitory effect might be of physiological significance in the control of acetylglutamate synthesis in vivo. Recently acetylglutamate synthetase of Escherichia coli has been purified and characterized [17]. In microorganisms, acetylglutamate serves as the first intermediate for synthesis of ornithine and arginine ; the role is distinct from that in mammalian liver. In spite of the distinction, a considerable resemblance exists or is suggested between the two synthetases from E. coli [I71 and rat liver [4,6] (and this paper) with regard to the kinetic and molecular properties. These include the following : concentration-dependent association and dissociation of the oligomeric protein accompanied by changes in enzyme activity ;sensitivity to regulation by arginine, though the direction of the effects on the respective enzymes is opposite (inhibition for the E. coli enzyme and stimulation for the rat liver enzyme) ; the inhibitory effects of divalent cations, thiol blocking agents, and ionic strength. This investigation was supported in part by research grants from the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan (B748077 and D867023) and the Tanabe Amino Acid Research Foundation. The authors wish to thank M. Ohara for help in preparation of the manuscript.

29 1

K . Shigesada and M. Tatibana

REFERENCES 1. Grisolia, S. & Cohen, P. P. (1953) J . Biol. Chem. 204,753-757. 2. Hall, L. M., Metzenberg, R. L. & Cohen, P. P. (1958) J . Biol. Chern. 230, 1013-1021. 3. Shigesada, K. &Tatibana, M. (1971) J . Biol. Chem.246,55885595. 4. Shigesada, K. & Tatibana, M. (1971) Biochem. Biophys. Res. Commun. 44, 1117-1124. 5. Krebs, H. A., Hems, R. & Lund, P. (1973) Adu. Enzyme Regul. 11, 361 -377. 6. Tatibana, M., Shigesada, K. & Mori, M. (1976) in The Urea Cycle (Grisolia, S., Baguena, R. & Mayor, F., eds) pp. 95105, John Wiley & Sons, New York. 7. Simon, E. J. & Shemin, D. (1953) J . Am. Chem. Soc. 75, 2520. 8. Lipmann, F. & Tuttle, L. C. (1945) J . Biol. Chern. 159, 21 -28.

9. Marshall, M., Metzenberg, R. L. & Cohen, P. P. (1958) J . B i d . Chem. 233, 102 - 105. 10. Marshall, M. & Cohen, P. P. (1972) J . Biol. Chem. 247, 1641 1653. 11. Harper, A. E. (1959) J . Nutr. 68, 405-418. 12. Bray, G. A. (1960) Anal. Biochem. I , 279-285. 13. Rothman, F. & Higa, A. (1962) Anal. Biochem. 3,173- 177. 14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 15. Hogeboom, G. H., Schneider, W. C. & Palade, G. E. (1948) J . Biol. Chem. 172, 619-635. 16. Tatibana, M. & Shigesada, K. (1976) in The Urecr Cycle (Grisolia, S., Baguena, R. & Mayor, F., eds) pp. 301 -313, John Wiley & Sons, New York. 17. Marvil, D. K. & Leisinger, T. (1977) J . B i d . Chem. 252, 32953303.

K. Shigesada, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan 606

M. Tatibana*, Department of Biochemistry, Chiba University School of Medicine, Inohana, Chiba, Japan 280

*

To whom correspondence should be addressed.

N-Acetylglutamate synthetase from rat-liver mitochondria. Partial purification and catalytic properties.

Eur. J. Biochem. 84, 285-291 (1978) N-Acetylglutamate Synthetase from Rat-Liver Mitochondria Partial Purification and Catalytic Properties Katsuya SH...
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