Abstract
Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn2+-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.
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Abbreviations
- GS:
-
glutamine synthetase
- MOPS:
-
2-(N-morpholine) propane sulfonate
- MSX:
-
l-methionine-d,l-sulfoximine
References
Alef K, Zumft WG (1981) Regulatory properties of glutamine synthetase from the nitrogen-fixing phototrophic bacterium Rhodopseudomonas palustris. Z Naturforsch 36c:784–789
Alef K, Burkhardt HJ, Horstmann HJ, Zumft WG (1981) Molecular characterization of glutamine synthetase from the nitrogenfixing phototrophic bacterium Rhodopseudomonas palustris. Z Naturforsch 36c:246–254
Arp DJ, Zumft WG (1983) l-Methionine-SR-sulfoximine as probe for the role of glutamine synthetase in nitrogenase switch-off by ammonia and glutamine in Rhodopseudomonas palustris. Arch Microbiol 134:17–22
Ayling PD (1981) Methionine sulfoxide is transported by highaffinity methionine and glutamine transport system in Salmonella typhimurium. J Bacteriol 148:514–520
Bender RA, Janssen KA, Resnick AD, Blumenberg M, Foor F, Magasanik B (1977) Biochemical parameters of glutamine synthetase from Klebsiella aerogenes. J Bacteriol 129:1001–1009
Caballero FJ, Cejudo FJ, Florencio FJ, Cárdenas J, Castillo F (1985) Molecular and regulatory properties of glutamine synthetase from the phototrophic bacterium Rhodopseudomonas capsulata E1F1. J Bacteriol 162:804–809
Cárdenas J, Mortenson LE (1975) Role of molybdenum in dinitrogen fixation by Clostridium pasteurianum. J Bacteriol 123:978–984
Engelhardt H, Klemme JH (1981) In vivo control of glutamine synthetase in the facultative phototrophic bacterium Rhodopseudomonas sphaeroides. Z Naturforsch 36c:407–410
Hernández G, Sánchez-Pescador R, Palacios R, Mora J (1983) Nitrogen source regulates glutamine dehydrogenase NADP synthesis in Neurospora crassa. J Bacteriol 154:524–528
Johansson BC, Gest H (1976) Inorganic nitrogen assimilation by the photosynthetic bacterium Rhodopseudomonas capsulata. J Bacteriol 128:683–688
Johansson BC, Gest H (1977) Adenylylation/deadenylylation control of the glutamine synthetase of Rhodopseudomonas capsulata. Eur J Biochem 81:365–371
Jones BL, Monty KJ (1979) Glutamine as a feedback inhibitor of the Rhodopseudomonas sphaeroides nitrogenase system. J Bacteriol 139:1007–1013
Kleiner D, Alef K, Hartmann A (1983) Uptake of methionine sulfoximine by some N2-fixing bacteria, and its effect on ammonium transport. FEBS Lett 164:121–123
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Michalski WP, Nicholas DJD (1984) Regulation of N2 fixation and ammonia assimilation in Rhodopseudomonas sphaeroides f. sp. denitrificans. Role of glutamine. J Gen Microbiol 130:1069–1077
Moreno-Vivián C, Cejudo FJ, Cárdenas J, Castillo F (1983) Ammonia assimilation pathways in Rhodopseudomonas capsulata E1F1. Arch Microbiol 136:147–151
Nagatani H, Shimizu M, Valentine RC (1971) The mechanism of ammonia assimilation in nitrogen fixing bacteria. Arch Mikrobiol 79:164–175
Norklund S, Kanemoto RH, Murrel SA, Ludden PW (1985) Properties and regulation of glutamine synthetase from Rhodospirillum rubrum. J Bacteriol 161:13–17
Orr J, Haselkorn R (1982) Regulation of glutamine synthetase activity and synthesis in free-living and symbiotic Anabaena spp. J Bacteriol 152:626–635
Ouchterlony O, Nilsson LA (1973) Immunodiffusion and immunoelectrophoresis. In: Weir DM (ed) Handbook of experimental immunology. Blackwell Scientific Publications, Oxford, pp 19.1–19.34
Scolnik PA, Virosco J, Haselkorn R (1983) The wild-type gene for glutamine synthetase restores ammonia control of nitrogen fixation to Gln- (glnA) mutants of Rhodopseudomonas capsulata. J Bacteriol 155:180–185
Shapiro BM, Stadtman ER (1970) Glutamine synthetase (Escherichia coli). In: Colowick SP, Kaplan NO (eds) Methods in enzymology, vol 17 A. Academic Press, New York, pp 910–922
Stadtman ER, Ginsburg A, Ciardi JE, Yeh J, Hennig SB, Shapiro BM (1970) Multiple molecular forms of glutamine synthetase produced by enzyme catalyzed adenylylation and deadenylylation reactions. Adv Enzym Reg 8:99–118
Weaver PF, Wall JD, Gest H (1975) Characterization of Rhodopseudomonas capsulata. Arch Microbiol 105:207–216
Weeke B (1973) Rocket immunoelectrophoresis. Crossed immunoelectrophoresis. In: Axelsen NH, Kroll J, Week B (eds) Quantitative immunoelectrophoresis. Scan J Immunol, vol 2, suppl 1. Universitetsforlaget, Oslo, pp 37–56
Yoch DC, Cantu M, Zhang ZM (1983) Evidence for a glutamine synthetase association in the phototroph Rhodospirillum rubrum: purification, properties and regulation of the enzyme. J Bacteriol 154:632–639
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Romero, F., Caballero, F.J., Castillo, F. et al. Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine. Arch. Microbiol. 143, 111–116 (1985). https://doi.org/10.1007/BF00411032
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DOI: https://doi.org/10.1007/BF00411032