Planta (Berl.) 111, 271--274 (1973) 9 by Springer-Verlag 1973
Short Communication
Glutamine Synthetase in the Chloroplasts of Viciafaba Anthony I t a y s t e a d Department of Biological Sciences, University of Dundee, Dundee, DD 1 4 HN, U.K. Received February 12, 1973
Summary. A glutamine synthetase has been localised in the chloroplasts of Vicia/aba. The enzyme has requirements for Mg2+ and ATP in the biosynthetic reaction and in addition will catalyse a 7-glutamyl transferase reaction in the presence of Mn~+ and arsenate. The enzyme is inhibited by AMP, CTP, glycine and alanine. These results are discussed in relation to the possible chloroplastic synthesis of nucleotide bases. Estimations of glutamine amide-2-oxoglutarate amino transferase (oxido-reductase) have demonstrated only low levels of activity in the chloroplast extracts. This enzyme is generally active in organisms where GS has an assimilary role. It is concluded that glutamine synthetase has a biosynthetic and not an assimilatory role in the chloroplast. A number of authors have demonstrated t h a t the enzyme L-glutam a t e dehydrogenase (GDH) is present in the chloroplasts of higher plants (Leech and Kirk, 1968; Santorius and Stocking, 1969; Tscnova, 1972) and K i r k and Leech (1972) in a study of amino acid biosynthesis and steady state pools in the chloroplasts of Vicia/aba have concluded t h a t the primary reaction in ammonia assimilation is the reductive amination of 2-oxoglutarate catalyscd by G D H (see also Givan and Leech, 1971). Santorius and Stocking (1969), however, have demonstrated a rapid amidation of glutamate to form glutamine b y intact isolated chloroplasts of Spinacia oleracea. I n contrast Givan et al. (1970) using intact chloroplasts of Vicia/aba were unable to show glutamine synthesis from added 2-oxoglutarate. I n addition to reports of glutamate and gintamine synthesis, the activity of alanine and aspartate dehydrogenase, both enzymes catalysing reactions in which ammonia is assimilated, has been reported in the chloroplasts of higher plants (Santorius and Stocking, 1969; Tsukamoto, 1970). Glutamine has been shown to play a central role in the metabolism of both eukaryotie and prokaryotic cells both as a primary substrate in the synthesis of purines, pyrimidines and a number of amino acids (see
272
A. Haystead: Table 1. Activity of glutamine synthetase in chloroplasts of Vicia/aba Reaction
Activity
y-glutamyl transferase Pi release from ATP Glutamine synthesis
0.98 ~moles mg protein-1 rain-1 0.68 ttmoles mg protein-1 min-1 0.07 txmoles mg protein-1 rain-1
?-glutamyl transferase and ATP hydrolysis were assayed essentially as described in Shapiro and Stadtman (1970) and glutamine synthesis by the method of Wood et al. (1972). All assays were performed in 0.025 M K. HEPES at pH 7.2. Table 2. Substrate requirements for glutamine synthetase activity (Pi release) in extracts of Vicia/aba chloroplasts Assay mixture
Specific activity
Complete --ATP --Mg ~+ --glutamate --NH4+
0.68 0.00 0.02 0.00 0.00
Glutamine synthetase activity is expressed as izmoles Pi released mg protein-1 rain-1.
Shapiro and Stadtman, 1970) and as an early product of ammonia assimilation (Sims and Folkes, 1964; Holzer et al., 1967; Tempest et al., 1967; Nagatani etal., 1972; Dharmawardene etal., 1972). This communication presents evidence t h a t glutamlne m a y be important in the metabolism of the chloroplast and t h a t glutamine synthetase (L-glutam a t e : a m m o n i a ligase (ADP), EC 6.3.1.2.) m a y play an important role in the chloroplast as a biosynthetic enzyme. Table 1 shows t h a t extracts prepared from isolated intact chloroplasts of Vicia ]aba prepared as described in Givan et al. (1970) and disrupted by high pressure extrusion in a French press possess glutamine synthetase ( G S ) a n d ~-glutamyl transferase activity. The chloroplastic origin of the GS activity was confirmed b y the extremely low levels of associated succinic dehydrogenase activity ( < 5 % of whole cell activity). Table 2 shows t h a t the requirements for this activity are identical with those of the microbial enzyme (Hubbard and Stadtman, 1967). The relatively low rates of glutamine synthesis compared with glutamate and ammonia dependent A T P hydrolysis suggested t h a t a glutaminase activity(L-glutamine aminohydrolase, EC 3.5.1.2.) m a y be present in the chloroplast extract. Assay of the ghitaminase activity using the method of Wood et al. (1972) gave maximal levels of glutamate production of 0.27 ~moles mg protein -1 min -1.
Gtutamine Syathetase in Chloroplasts
273
Table 3. Effect of various metabolites on the activity of GS from the chloroplast
Vicia ]aba Metabolite (5 r a M )
Specific activity
% inhibition
Control AMP CTP glycine glucosamine-6-P alanine tryptophan histidine proline
0.68 0.35 0.51 0.51 0.68 0.56 0.68 0.68 0.68
0 49 25 24 0 18 0 0 0
Glutamine synthetase activity is expressed as ~tmoles Pi released mg prorein-1 min-L
A characteristic of microbial GS is its sensitivity to feedback inhibition by a number of metabolites synthesised via pathways involving glutamine (Shapiro and Stadtman, 1970; Hubbard and Stadman, 1968; Deuel and Stadtman, 1970). An investigation of the effects of a number of potential products of glutamine metabolism is presented in Table 3. The inhibitory effect of AMP and CTP on chloroplast GS activity may be of considerable significance in that it may indicate a possible autonomy in purine and pyrimidine biosynthesis. Joussaume and Bourdu (1966) have reported the incorporation of radioactive orotic acid into uridylic and cytidylic acids, a further indication that chloroplasts can synthesise the pyrimidine skeleton. The demonstration of inhibition by glycine is surprising in that this amino acid is generally considered to be a product of glycollate metabolism (see Goldsworthy, 1970). In those microorganisms in which ammonia is first assimilated into the amide group of glutamlne further metabolism of the newly assimilated nitrogen is mediated by the enzyme L-glutamine mide: 2-oxoglutarate amino transferase (oxido-reduetase) (GOGAT) which catalyses the production of glutamate from glutamine and 2-oxoglutarate. In the assay mixture of Brown and Stanley (1972) only very low levels of this activity ( ~ 0.5 nmoles N A D P H oxidised mg protein -1 min -1) could be detected in the chloroplast extracts, a level of activity which could be attributed to GDH acting on ammonia produced in the glutaminase reaction. I t may be that modification of the extraction or assay procedure used here may reveal higher levels of this activity. I t seems probable, however, that ammonia assimilation into glutamine may be o f secondary importance compared with that into glutamate via GDH in the biosynthesis of most protein amino acids.
274
A. Haystead: Glutamine Synthetase in Chloroplasts
I should like to thank Dr. J.A. Raven, Dr. R. Lyne, Dr. G. E. Codd, Mr. M. W. N. Dharmawardene and Prof. W. D. P. Stewart for their comments during the preparation of the manuscript and Imperial Chemical Industries for a postdoctoral fellowship. References Brown, C. M., Stanley, S. 0.: Environment mediated changes in the cellular content of the "pool" constituents and their associated changes in cell physiology. J. appl. Chem. Biotechnol. 22, 363-389 (1972). Deuel, R. F., Stadtman, E. R. : Some kinetic properties of Bacillus subtilis glutamine synthetase. J. biol. Chem. 245, 5206--5213 (1970). Dharmawardene, M. W. N., Stewart, W. D. P., Stanley, S. O. : Nitrogenase activity, amino acid pool patterns and amination in blue-green algae. Planta (Berl.) 108, 133-145 (1972). Givan, C.V., Givan, A.L., Leech, R.M.: Photoreduction of ~-keteglutarate to glutamate by Vicia faba chloroplasts. P1. Physiol. (Wash.) 45, 624-630 (1970). Givan, C.V., Leech, R.M.: Biochemical autonomy of higher plant chloroplasts and their synthesis of small molecules. Biol. Rev. 46, 409428 (1971). Goldsworthy, A.: Photorespiration. Bot. Rev. 86, 321-34i (1970). Holzer, H., Mecke, K., Wulff, K., Leiss, K., :Heilmeyer, L.: Metabolite induced enzymatic inactivation of glutamine synthetase in E. coli. Advanc. Enz. Reg. 5, 211-225 (1967). Hubbard, J. S., Stadtman, E. R. : Regulation of glutamine synthetase. II. Patterns of feedback inhibition in microorganisms. J. Bact. 93, 1045-1055 (1967). Joussaume, M., Bourdu, R.: Conversion of carbon-14-1abelled orotic acid into pyrimidine nucleotides by chloroplasts. Nature (Lond.) 210, 1363-1365 (1966). Kirk, P. R., Leech, R. M. : Amino acid biosynthesis by isolated chloroplasts during photosynthesis. P1. Physiol. (Wash.) 50, 228-234 (1972). Leech, R. M., Kirk, P. R. : An NADP-dependent L-glutamate dehydrogenase from chloroplasts of Vicia faba. Biochem. biophys. Res. Commun. 82, 685-690 (1968). Nagatanti, H., Schimizu, M., Valentine, R. C.: The mechanism of ammonia assimilation in nitrogen fixing bacteria. Arch. Microbiol. 79, 164-175 (1972). Santorius, K.A., Stocking, C. R. : Intra~ellular localisation of enzymes in leaves and chloroplast membrane permeability to compounds involved in amino acid synthesis. Z. Naturforsch. 246, 1170-1179 (1969). Shapiro, B. M., Stadtman, E. R. : Glutamine synthetase (Escherichia coli). Meth. Enzymol. 17A, 910-922 (1970). Sims, A. P., Folkes, B. F. : A kinetic study of the assimilation of (15N) ammonia and the synthesis of amino acids in an exponentially growing culture of Candida utilis. Proc. roy. Soc. B 159, 479-502 (1964). Tsenova, E. N. : Isolation and properties of L-glutamate dehydrogenase from pea chloroplasts. Enzymologia 48, 397-408 (1972). Tsukamoto, A. : Reductive carboxylation and amination of keto acids by spinach chloroplasts. Plant Cell Physiol. 11, 221-230 (1970). Wood, A. W., McCrea, M. E., Seegmiller, J. E. : A radiochemical assay for glutaminase and glutamic decarboxylase. Analyt. Biochem. 48, 581-587 (1972).
Short Communication
Glutamine Synthetase in the Chloroplasts of Viciafaba Anthony I t a y s t e a d Department of Biological Sciences, University of Dundee, Dundee, DD 1 4 HN, U.K. Received February 12, 1973
Summary. A glutamine synthetase has been localised in the chloroplasts of Vicia/aba. The enzyme has requirements for Mg2+ and ATP in the biosynthetic reaction and in addition will catalyse a 7-glutamyl transferase reaction in the presence of Mn~+ and arsenate. The enzyme is inhibited by AMP, CTP, glycine and alanine. These results are discussed in relation to the possible chloroplastic synthesis of nucleotide bases. Estimations of glutamine amide-2-oxoglutarate amino transferase (oxido-reductase) have demonstrated only low levels of activity in the chloroplast extracts. This enzyme is generally active in organisms where GS has an assimilary role. It is concluded that glutamine synthetase has a biosynthetic and not an assimilatory role in the chloroplast. A number of authors have demonstrated t h a t the enzyme L-glutam a t e dehydrogenase (GDH) is present in the chloroplasts of higher plants (Leech and Kirk, 1968; Santorius and Stocking, 1969; Tscnova, 1972) and K i r k and Leech (1972) in a study of amino acid biosynthesis and steady state pools in the chloroplasts of Vicia/aba have concluded t h a t the primary reaction in ammonia assimilation is the reductive amination of 2-oxoglutarate catalyscd by G D H (see also Givan and Leech, 1971). Santorius and Stocking (1969), however, have demonstrated a rapid amidation of glutamate to form glutamine b y intact isolated chloroplasts of Spinacia oleracea. I n contrast Givan et al. (1970) using intact chloroplasts of Vicia/aba were unable to show glutamine synthesis from added 2-oxoglutarate. I n addition to reports of glutamate and gintamine synthesis, the activity of alanine and aspartate dehydrogenase, both enzymes catalysing reactions in which ammonia is assimilated, has been reported in the chloroplasts of higher plants (Santorius and Stocking, 1969; Tsukamoto, 1970). Glutamine has been shown to play a central role in the metabolism of both eukaryotie and prokaryotic cells both as a primary substrate in the synthesis of purines, pyrimidines and a number of amino acids (see
272
A. Haystead: Table 1. Activity of glutamine synthetase in chloroplasts of Vicia/aba Reaction
Activity
y-glutamyl transferase Pi release from ATP Glutamine synthesis
0.98 ~moles mg protein-1 rain-1 0.68 ttmoles mg protein-1 min-1 0.07 txmoles mg protein-1 rain-1
?-glutamyl transferase and ATP hydrolysis were assayed essentially as described in Shapiro and Stadtman (1970) and glutamine synthesis by the method of Wood et al. (1972). All assays were performed in 0.025 M K. HEPES at pH 7.2. Table 2. Substrate requirements for glutamine synthetase activity (Pi release) in extracts of Vicia/aba chloroplasts Assay mixture
Specific activity
Complete --ATP --Mg ~+ --glutamate --NH4+
0.68 0.00 0.02 0.00 0.00
Glutamine synthetase activity is expressed as izmoles Pi released mg protein-1 rain-1.
Shapiro and Stadtman, 1970) and as an early product of ammonia assimilation (Sims and Folkes, 1964; Holzer et al., 1967; Tempest et al., 1967; Nagatani etal., 1972; Dharmawardene etal., 1972). This communication presents evidence t h a t glutamlne m a y be important in the metabolism of the chloroplast and t h a t glutamine synthetase (L-glutam a t e : a m m o n i a ligase (ADP), EC 6.3.1.2.) m a y play an important role in the chloroplast as a biosynthetic enzyme. Table 1 shows t h a t extracts prepared from isolated intact chloroplasts of Vicia ]aba prepared as described in Givan et al. (1970) and disrupted by high pressure extrusion in a French press possess glutamine synthetase ( G S ) a n d ~-glutamyl transferase activity. The chloroplastic origin of the GS activity was confirmed b y the extremely low levels of associated succinic dehydrogenase activity ( < 5 % of whole cell activity). Table 2 shows t h a t the requirements for this activity are identical with those of the microbial enzyme (Hubbard and Stadtman, 1967). The relatively low rates of glutamine synthesis compared with glutamate and ammonia dependent A T P hydrolysis suggested t h a t a glutaminase activity(L-glutamine aminohydrolase, EC 3.5.1.2.) m a y be present in the chloroplast extract. Assay of the ghitaminase activity using the method of Wood et al. (1972) gave maximal levels of glutamate production of 0.27 ~moles mg protein -1 min -1.
Gtutamine Syathetase in Chloroplasts
273
Table 3. Effect of various metabolites on the activity of GS from the chloroplast
Vicia ]aba Metabolite (5 r a M )
Specific activity
% inhibition
Control AMP CTP glycine glucosamine-6-P alanine tryptophan histidine proline
0.68 0.35 0.51 0.51 0.68 0.56 0.68 0.68 0.68
0 49 25 24 0 18 0 0 0
Glutamine synthetase activity is expressed as ~tmoles Pi released mg prorein-1 min-L
A characteristic of microbial GS is its sensitivity to feedback inhibition by a number of metabolites synthesised via pathways involving glutamine (Shapiro and Stadtman, 1970; Hubbard and Stadman, 1968; Deuel and Stadtman, 1970). An investigation of the effects of a number of potential products of glutamine metabolism is presented in Table 3. The inhibitory effect of AMP and CTP on chloroplast GS activity may be of considerable significance in that it may indicate a possible autonomy in purine and pyrimidine biosynthesis. Joussaume and Bourdu (1966) have reported the incorporation of radioactive orotic acid into uridylic and cytidylic acids, a further indication that chloroplasts can synthesise the pyrimidine skeleton. The demonstration of inhibition by glycine is surprising in that this amino acid is generally considered to be a product of glycollate metabolism (see Goldsworthy, 1970). In those microorganisms in which ammonia is first assimilated into the amide group of glutamlne further metabolism of the newly assimilated nitrogen is mediated by the enzyme L-glutamine mide: 2-oxoglutarate amino transferase (oxido-reduetase) (GOGAT) which catalyses the production of glutamate from glutamine and 2-oxoglutarate. In the assay mixture of Brown and Stanley (1972) only very low levels of this activity ( ~ 0.5 nmoles N A D P H oxidised mg protein -1 min -1) could be detected in the chloroplast extracts, a level of activity which could be attributed to GDH acting on ammonia produced in the glutaminase reaction. I t may be that modification of the extraction or assay procedure used here may reveal higher levels of this activity. I t seems probable, however, that ammonia assimilation into glutamine may be o f secondary importance compared with that into glutamate via GDH in the biosynthesis of most protein amino acids.
274
A. Haystead: Glutamine Synthetase in Chloroplasts
I should like to thank Dr. J.A. Raven, Dr. R. Lyne, Dr. G. E. Codd, Mr. M. W. N. Dharmawardene and Prof. W. D. P. Stewart for their comments during the preparation of the manuscript and Imperial Chemical Industries for a postdoctoral fellowship. References Brown, C. M., Stanley, S. 0.: Environment mediated changes in the cellular content of the "pool" constituents and their associated changes in cell physiology. J. appl. Chem. Biotechnol. 22, 363-389 (1972). Deuel, R. F., Stadtman, E. R. : Some kinetic properties of Bacillus subtilis glutamine synthetase. J. biol. Chem. 245, 5206--5213 (1970). Dharmawardene, M. W. N., Stewart, W. D. P., Stanley, S. O. : Nitrogenase activity, amino acid pool patterns and amination in blue-green algae. Planta (Berl.) 108, 133-145 (1972). Givan, C.V., Givan, A.L., Leech, R.M.: Photoreduction of ~-keteglutarate to glutamate by Vicia faba chloroplasts. P1. Physiol. (Wash.) 45, 624-630 (1970). Givan, C.V., Leech, R.M.: Biochemical autonomy of higher plant chloroplasts and their synthesis of small molecules. Biol. Rev. 46, 409428 (1971). Goldsworthy, A.: Photorespiration. Bot. Rev. 86, 321-34i (1970). Holzer, H., Mecke, K., Wulff, K., Leiss, K., :Heilmeyer, L.: Metabolite induced enzymatic inactivation of glutamine synthetase in E. coli. Advanc. Enz. Reg. 5, 211-225 (1967). Hubbard, J. S., Stadtman, E. R. : Regulation of glutamine synthetase. II. Patterns of feedback inhibition in microorganisms. J. Bact. 93, 1045-1055 (1967). Joussaume, M., Bourdu, R.: Conversion of carbon-14-1abelled orotic acid into pyrimidine nucleotides by chloroplasts. Nature (Lond.) 210, 1363-1365 (1966). Kirk, P. R., Leech, R. M. : Amino acid biosynthesis by isolated chloroplasts during photosynthesis. P1. Physiol. (Wash.) 50, 228-234 (1972). Leech, R. M., Kirk, P. R. : An NADP-dependent L-glutamate dehydrogenase from chloroplasts of Vicia faba. Biochem. biophys. Res. Commun. 82, 685-690 (1968). Nagatanti, H., Schimizu, M., Valentine, R. C.: The mechanism of ammonia assimilation in nitrogen fixing bacteria. Arch. Microbiol. 79, 164-175 (1972). Santorius, K.A., Stocking, C. R. : Intra~ellular localisation of enzymes in leaves and chloroplast membrane permeability to compounds involved in amino acid synthesis. Z. Naturforsch. 246, 1170-1179 (1969). Shapiro, B. M., Stadtman, E. R. : Glutamine synthetase (Escherichia coli). Meth. Enzymol. 17A, 910-922 (1970). Sims, A. P., Folkes, B. F. : A kinetic study of the assimilation of (15N) ammonia and the synthesis of amino acids in an exponentially growing culture of Candida utilis. Proc. roy. Soc. B 159, 479-502 (1964). Tsenova, E. N. : Isolation and properties of L-glutamate dehydrogenase from pea chloroplasts. Enzymologia 48, 397-408 (1972). Tsukamoto, A. : Reductive carboxylation and amination of keto acids by spinach chloroplasts. Plant Cell Physiol. 11, 221-230 (1970). Wood, A. W., McCrea, M. E., Seegmiller, J. E. : A radiochemical assay for glutaminase and glutamic decarboxylase. Analyt. Biochem. 48, 581-587 (1972).
