Abstract
We have developed two procedures which allow the very rapid purification of glutamine synthetase (GS) from a diverse variety of bacteria. The first procedure, based upon differential sedimentation, depends upon the association of GS with deoxyribonucleic acid in cell extracts. The second procedure, derived from the method of C. Gross et al (J. Bacteriol. 128:382-389, 1976) for purifying ribonucleic acid polymerase by polyethylene glycol (PEG) precipitation, enabled us to obtain high yields of GS from either small or large quantities of cells. We used the PEG procedure to purify GS from Klebsiella aerogenes, K. pneumoniae, Escherichia coli, Salmonella typhimurium, Rhizobium sp. strain 32H1, R. meliloti, Azotobacter vinelandii, Pseudomonas putida, Caulobacter crescentus, and Rhodopseudomonas capsulata. The purity of the GS obtained, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was high, and in many instances only a single protein band was detected.
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- Anderson R. P., Roth J. R. Tandem genetic duplications in phage and bacteria. Annu Rev Microbiol. 1977;31:473–505. doi: 10.1146/annurev.mi.31.100177.002353. [DOI] [PubMed] [Google Scholar]
- Bender R. A., Janssen K. A., Resnick A. D., Blumenberg M., Foor F., Magasanik B. Biochemical parameters of glutamine synthetase from Klebsiella aerogenes. J Bacteriol. 1977 Feb;129(2):1001–1009. doi: 10.1128/jb.129.2.1001-1009.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bender R. A., Streicher S. L. Glutamine synthetase regulation, adenylylation state, and strain specificity analyzed by polyacrylamide gel electrophoresis. J Bacteriol. 1979 Feb;137(2):1000–1007. doi: 10.1128/jb.137.2.1000-1007.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bloom F. R., Streicher S. L., Tyler B. Regulation of enzyme synthesis by the glutamine synthetase of Salmonella typhimurium: a factor in addition to glutamine synthetase is required for activation of enzyme formation. J Bacteriol. 1977 Jun;130(3):983–990. doi: 10.1128/jb.130.3.983-990.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brenchley J. E., Prival M. J., Magasanik B. Regulation of the synthesis of enzymes responsible for glutamate formation in Klebsiella aerogenes. J Biol Chem. 1973 Sep 10;248(17):6122–6128. [PubMed] [Google Scholar]
- Deleo A. B., Magasanik B. Identification of the structural gene for glutamine synthetase in Klebsiella aerogenes. J Bacteriol. 1975 Jan;121(1):313–319. doi: 10.1128/jb.121.1.313-319.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ely B., Amarasinghe A. B., Bender R. A. Ammonia assimilation and glutamate formation in Caulobacter crescentus. J Bacteriol. 1978 Jan;133(1):225–230. doi: 10.1128/jb.133.1.225-230.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Frey T. G., Eisenberg D., Eiserling F. A. Glutamine synthetase forms three- and seven-stranded helical cables. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3402–3406. doi: 10.1073/pnas.72.9.3402. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gaillardin C. M., Magasanik B. Involvement of the product of the glnF gene in the autogenous regulation of glutamine synthetase formation in Klebsiella aerogenes. J Bacteriol. 1978 Mar;133(3):1329–1338. doi: 10.1128/jb.133.3.1329-1338.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garcia E., Bancroft S., Rhee S. G., Kustu S. The product of a newly identified gene, gInF, is required for synthesis of glutamine synthetase in Salmonella. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1662–1666. doi: 10.1073/pnas.74.4.1662. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gross C., Engbaek F., Flammang T., Burgess R. Rapid micromethod for the purification of Escherichia coli ribonucleic acid polymerase and the preparation of bacterial extracts active in ribonucleic acid synthesis. J Bacteriol. 1976 Oct;128(1):382–389. doi: 10.1128/jb.128.1.382-389.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hagen D. C., Magasanik B. Deoxyribonucleic acid-binding studies on the hut repressor and mutant forms of the hut repressor of Salmonella typhimurium. J Bacteriol. 1976 Aug;127(2):837–847. doi: 10.1128/jb.127.2.837-847.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kao-Huang Y., Revzin A., Butler A. P., O'Conner P., Noble D. W., von Hippel P. H. Nonspecific DNA binding of genome-regulating proteins as a biological control mechanism: measurement of DNA-bound Escherichia coli lac repressor in vivo. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4228–4232. doi: 10.1073/pnas.74.10.4228. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kustu S. G., McFarland N. C., Hui S. P., Esmon B., Ames G. F. Nitrogen control of Salmonella typhimurium: co-regulation of synthesis of glutamine synthetase and amino acid transport systems. J Bacteriol. 1979 Apr;138(1):218–234. doi: 10.1128/jb.138.1.218-234.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kustu S., Burton D., Garcia E., McCarter L., McFarland N. Nitrogen control in Salmonella: regulation by the glnR and glnF gene products. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4576–4580. doi: 10.1073/pnas.76.9.4576. [DOI] [PMC free article] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Ludwig R. A. Control of ammonium assimilation in Rhizobium 32H1. J Bacteriol. 1978 Jul;135(1):114–123. doi: 10.1128/jb.135.1.114-123.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Magasanik B. Classical and postclassical modes of regulation of the synthesis of degradative bacterial enzymes. Prog Nucleic Acid Res Mol Biol. 1976;17:99–115. doi: 10.1016/s0079-6603(08)60067-7. [DOI] [PubMed] [Google Scholar]
- Miller R. E., Shelton E., Stadtman E. R. Zinc-induced paracrystalline aggregation of glutamine synthetase. Arch Biochem Biophys. 1974 Jul;163(1):155–171. doi: 10.1016/0003-9861(74)90465-2. [DOI] [PubMed] [Google Scholar]
- Miller R. E., Stadtman E. R. Glutamate synthase from Escherichia coli. An iron-sulfide flavoprotein. J Biol Chem. 1972 Nov 25;247(22):7407–7419. [PubMed] [Google Scholar]
- Mäntsälä P., Zalkin H. Glutamate synthase. Properties of the glutamine-dependent activity. J Biol Chem. 1976 Jun 10;251(11):3294–3299. [PubMed] [Google Scholar]
- Nissley P., Anderson W. B., Gallo M., Pastan I., Perlman R. L. The binding of cyclic adenosine monophosphate receptor to deoxyribonucleic acid. J Biol Chem. 1972 Jul 10;247(13):4264–4269. [PubMed] [Google Scholar]
- Pahel G., Tyler B. A new glnA-linked regulatory gene for glutamine synthetase in Escherichia coli. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4544–4548. doi: 10.1073/pnas.76.9.4544. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pahel G., Zelenetz A. D., Tyler B. M. gltB gene and regulation of nitrogen metabolism by glutamine synthetase in Escherichia coli. J Bacteriol. 1978 Jan;133(1):139–148. doi: 10.1128/jb.133.1.139-148.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Riggs A. D., Reiness G., Zubay G. Purification and DNA-binding properties of the catabolite gene activator protein. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1222–1225. doi: 10.1073/pnas.68.6.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Siedel J., Shelton E. Purification and properties of Azotobacter vinelandii glutamine synthetase. Arch Biochem Biophys. 1979 Jan;192(1):214–224. doi: 10.1016/0003-9861(79)90086-9. [DOI] [PubMed] [Google Scholar]
- Streicher S. L., Bender R. A., Magasanik B. Genetic control of glutamine synthetase in Klebiella aerogenes. J Bacteriol. 1975 Jan;121(1):320–331. doi: 10.1128/jb.121.1.320-331.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Streicher S. L., Deleo A. B., Magasanik B. Regulation of enzyme formation in Klebsiella aerogenes by episomal glutamine synthetase of Escherichia coli. J Bacteriol. 1976 Jul;127(1):184–192. doi: 10.1128/jb.127.1.184-192.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tyler B. M., Goldberg R. B. Transduction of chromosomal genes between enteric bacteria by bacteriophage P1. J Bacteriol. 1976 Mar;125(3):1105–1111. doi: 10.1128/jb.125.3.1105-1111.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tyler B., Deleo A. B., Magasanik B. Activation of transcription of hut DNA by glutamine synthetase. Proc Natl Acad Sci U S A. 1974 Jan;71(1):225–229. doi: 10.1073/pnas.71.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tyler B. Regulation of the assimilation of nitrogen compounds. Annu Rev Biochem. 1978;47:1127–1162. doi: 10.1146/annurev.bi.47.070178.005403. [DOI] [PubMed] [Google Scholar]
- Weglenski P., Tyler B. Regulation of glnA messinger ribonucleic acid synthesis in Klebsiella aerogenes. J Bacteriol. 1977 Feb;129(2):880–887. doi: 10.1128/jb.129.2.880-887.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Woolfolk C. A., Shapiro B., Stadtman E. R. Regulation of glutamine synthetase. I. Purification and properties of glutamine synthetase from Escherichia coli. Arch Biochem Biophys. 1966 Sep 26;116(1):177–192. doi: 10.1016/0003-9861(66)90026-9. [DOI] [PubMed] [Google Scholar]
- von Hippel P. H., Revzin A., Gross C. A., Wang A. C. Non-specific DNA binding of genome regulating proteins as a biological control mechanism: I. The lac operon: equilibrium aspects. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4808–4812. doi: 10.1073/pnas.71.12.4808. [DOI] [PMC free article] [PubMed] [Google Scholar]



