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. 1979 Dec 1;183(3):481–494. doi: 10.1042/bj1830481

Inosine 5'-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5'-monophosphate.

H J Gilbert, C R Lowe, W T Drabble
PMCID: PMC1161627  PMID: 44191

Abstract

Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic 'finger-printing' demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  2. Barrio J. R., Secrist J. A., 3rd, Leonard N. J. Fluorescent adenosine and cytidine derivatives. Biochem Biophys Res Commun. 1972 Jan 31;46(2):597–604. doi: 10.1016/s0006-291x(72)80181-5. [DOI] [PubMed] [Google Scholar]
  3. Brox L. W., Hampton A. Inosine-5'-phosphate dehydrogenase of Aerobacter aerogenes. Studies of tertiary structure. Biochim Biophys Acta. 1970 May 13;206(2):215–223. doi: 10.1016/0005-2744(70)90105-1. [DOI] [PubMed] [Google Scholar]
  4. Buzzee D. H., Levin A. P. Demonstration of an effector site for the enzyme inosine 5'-phosphate dehydrogenase. Biochem Biophys Res Commun. 1968 Mar 27;30(6):673–677. doi: 10.1016/0006-291x(68)90565-2. [DOI] [PubMed] [Google Scholar]
  5. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  6. Hampton A., Nomura A. Inosine 5'-phosphate dehydrogenase. Site of inhibition by guanosine 5'-phosphate and of inactivation by 6-chloro- and 6-mercaptopurine ribonucleoside 5'-phosphates. Biochemistry. 1967 Mar;6(3):679–689. doi: 10.1021/bi00855a006. [DOI] [PubMed] [Google Scholar]
  7. Heyde E., Morrison J. F. Studies on inosine monophosphate dehydrogenase. An associating-dissociating system. Biochim Biophys Acta. 1976 May 13;429(3):635–644. doi: 10.1016/0005-2744(76)90313-2. [DOI] [PubMed] [Google Scholar]
  8. Heyde E., Nagabhushanam A., Vonarx M., Morrison J. F. Studies on inosine monophosphate dehydrogenase. Steady state kinetics. Biochim Biophys Acta. 1976 May 13;429(3):645–660. doi: 10.1016/0005-2744(76)90314-4. [DOI] [PubMed] [Google Scholar]
  9. Ikehara M., Uesugi S. Studies on nucleosides and nucleotides. 38. Synthesis of 8-bromoadenosine nucleotides. Chem Pharm Bull (Tokyo) 1969 Feb;17(2):348–354. doi: 10.1248/cpb.17.348. [DOI] [PubMed] [Google Scholar]
  10. Ishii K., Shiio I. Regulation of purine ribonucleotide synthesis by end product inhibition. I. Effect of purine nucleotides on inosine-5'-phosphate dehydrogenase, xanthosine-5'-phosphate aminase and adenylosuccinate lyase of Bacillus subtilis. J Biochem. 1968 May;63(5):661–669. doi: 10.1093/oxfordjournals.jbchem.a128826. [DOI] [PubMed] [Google Scholar]
  11. Krishnaiah K. V. Inosinic acid 5'-monophosphate dehydrogenase of Escherichia coli: purification by affinity chromatography and some properties. Arch Biochem Biophys. 1975 Oct;170(2):567–575. doi: 10.1016/0003-9861(75)90152-6. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Lambden P. R., Drabble W. T. Inosine 5'-monophosphate dehydrogenase of Escherichia coli K12: the nature of the inhibition by guanosine 5'-monophosphate. Biochem J. 1973 Jul;133(3):607–608. doi: 10.1042/bj1330607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lambden P. R., Drabble W. T. The gua operon of Escherichia coli K-12: evidence for polarity from guaB to guaA. J Bacteriol. 1973 Sep;115(3):992–1002. doi: 10.1128/jb.115.3.992-1002.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lowe C. R., Mosbach K. Biospecific affinity chromatography in aqueous-organic cosolvent mixtures. The effect of ethylene glycol on the binding of lactate dehydrogenase to an immobilised-AMP analogue. Eur J Biochem. 1975 Mar 3;52(1):99–105. doi: 10.1111/j.1432-1033.1975.tb03977.x. [DOI] [PubMed] [Google Scholar]
  16. Lowe C. R. The synthesis of several 8-substituted derivatives of adenosine 5'-monophosphate to study the effect of the nature of the spacer arm in affinity chromatography. Eur J Biochem. 1977 Feb 15;73(1):265–274. doi: 10.1111/j.1432-1033.1977.tb11316.x. [DOI] [PubMed] [Google Scholar]
  17. MAGASANIK B., MOYED H. S., GEHRING L. B. Enzymes essential for the biosynthesis of nucleic acid guanine; inosine 5'-phosphate dehydrogenase of Aerobacter aerogenes. J Biol Chem. 1957 May;226(1):339–350. [PubMed] [Google Scholar]
  18. Michal G., Mühlegger K., Nelboeck M., Thiessen C., Weimann G. Cyclophosphates VI. Cyclophosphates as substrates and effectors of phosphodiesterase. Pharmacol Res Commun. 1974 Jun;6(3):203–252. doi: 10.1016/s0031-6989(74)80043-3. [DOI] [PubMed] [Google Scholar]
  19. Mosbach K., Larsson P. O., Lowe C. Immobilized coenzymes. Methods Enzymol. 1976;44:859–887. doi: 10.1016/s0076-6879(76)44063-6. [DOI] [PubMed] [Google Scholar]
  20. Neville D. M., Jr, Glossmann H. Plasma membrane protein subunit composition. A comparative study by discontinuous electrophoresis in sodium dodecyl sulfate. J Biol Chem. 1971 Oct 25;246(20):6335–6338. [PubMed] [Google Scholar]
  21. Nijkamp H. J. Regulatory role of adenine nucleotides in the biosynthesis of guanosine 5'-monophosphate. J Bacteriol. 1969 Nov;100(2):585–593. doi: 10.1128/jb.100.2.585-593.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ofengand J. The function of pseudouridylic acid in transfer ribonucleic acid. I. The specific cyanoethylation of pseudouridine, inosine, and 4-thiouridine by acrylonitrile. J Biol Chem. 1967 Nov 10;242(21):5034–5045. [PubMed] [Google Scholar]
  23. Powell G. F. Subunit structure of inosinic acid dehydrogenase from Escherichia coli. Biochemistry. 1973 Apr 10;12(8):1592–1595. doi: 10.1021/bi00732a020. [DOI] [PubMed] [Google Scholar]
  24. Powell G. Rajagopalan KV, Handler P: Purification and properties of inosinic acid dehydrogenase from Escherichia coli. J Biol Chem. 1969 Sep 10;244(17):4793–4797. [PubMed] [Google Scholar]
  25. Trayer I. P., Trayer H. R., Small D. P., Bottomley R. C. Preparation of adenosine nucleotide derivatives suitable for affinity chromatography. Biochem J. 1974 Jun;139(3):609–623. doi: 10.1042/bj1390609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  27. Vaitukaitis J., Robbins J. B., Nieschlag E., Ross G. T. A method for producing specific antisera with small doses of immunogen. J Clin Endocrinol Metab. 1971 Dec;33(6):988–991. doi: 10.1210/jcem-33-6-988. [DOI] [PubMed] [Google Scholar]
  28. Weiner A. M., Platt T., Weber K. Amino-terminal sequence analysis of proteins purified on a nanomole scale by gel electrophoresis. J Biol Chem. 1972 May 25;247(10):3242–3251. [PubMed] [Google Scholar]
  29. Wu T. W., Scrimgeour K. G. Properties of inosinic acid dehydrogenase from Bacillus subtilis. I. Purification and physical properties. Can J Biochem. 1973 Oct;51(10):1380–1390. doi: 10.1139/o73-181. [DOI] [PubMed] [Google Scholar]
  30. Wu T. W., Scrimgeour K. G. Properties of inosinic acid dehydrogenase from Bacillus subtilis. II. Kinetic properties. Can J Biochem. 1973 Oct;51(10):1391–1398. doi: 10.1139/o73-182. [DOI] [PubMed] [Google Scholar]
  31. Yokosawa H., Tobita T., Yamada T. Studies on inosine-5-phosphate dehydrogenase of Bacillus subtilis. Purification and general properties. Biochim Biophys Acta. 1971 Mar 10;227(3):538–553. doi: 10.1016/0005-2744(71)90005-2. [DOI] [PubMed] [Google Scholar]

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