Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1990 Oct;172(10):5750–5757. doi: 10.1128/jb.172.10.5750-5757.1990

Purification and characterization of two forms of hydrogenase isoenzyme 1 from Escherichia coli.

K Francis 1, P Patel 1, J C Wendt 1, K T Shanmugam 1
PMCID: PMC526891  PMID: 2211509

Abstract

A hydrogenase associated with dihydrogen uptake (HUP hydrogenase) was purified from an Escherichia coli mutant (strain SE1100) defective in utilization of molybdate and thus fermentative dihydrogen production. This protein had two subunits with apparent molecular weights of 59,000 and 28,000 (form 1). An immunologically cross-reactive hydrogenase was also purified from E. coli K10 grown in glucose-minimal medium and harvested at the mid-exponential phase of growth. Upon purification to homogeneity, this hydrogenase contained only one subunit with an apparent molecular weight of 59,000 (form 2). The two forms of the HUP hydrogenase exhibited similar kinetic characteristics. The electrophoretic properties of the enzyme and its response to pH suggest that this HUP hydrogenase is the HYD1 isoenzyme. The HYD1 isoenzyme was the only hydrogenase detectable during the stationary phase of growth in E. coli grown in Mo-deficient medium.

Full text

PDF
5751

Images in this article

5753Image
on p.5753

Selected References

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

  1. Adams M. W., Hall D. O. Purification of the membrane-bound hydrogenase of Escherichia coli. Biochem J. 1979 Oct 1;183(1):11–22. doi: 10.1042/bj1830011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adams M. W., Mortenson L. E., Chen J. S. Hydrogenase. Biochim Biophys Acta. 1980 Dec;594(2-3):105–176. doi: 10.1016/0304-4173(80)90007-5. [DOI] [PubMed] [Google Scholar]
  3. Ballantine S. P., Boxer D. H. Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli. Eur J Biochem. 1986 Apr 15;156(2):277–284. doi: 10.1111/j.1432-1033.1986.tb09578.x. [DOI] [PubMed] [Google Scholar]
  4. Ballantine S. P., Boxer D. H. Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12. J Bacteriol. 1985 Aug;163(2):454–459. doi: 10.1128/jb.163.2.454-459.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Ellis K. J., Morrison J. F. Buffers of constant ionic strength for studying pH-dependent processes. Methods Enzymol. 1982;87:405–426. doi: 10.1016/s0076-6879(82)87025-0. [DOI] [PubMed] [Google Scholar]
  9. FERGUSON K. A. STARCH-GEL ELECTROPHORESIS--APPLICATION TO THE CLASSIFICATION OF PITUITARY PROTEINS AND POLYPEPTIDES. Metabolism. 1964 Oct;13:SUPPL–SUPPL1002. doi: 10.1016/s0026-0495(64)80018-4. [DOI] [PubMed] [Google Scholar]
  10. Furth A. J. Removing unbound detergent from hydrophobic proteins. Anal Biochem. 1980 Dec;109(2):207–215. doi: 10.1016/0003-2697(80)90638-7. [DOI] [PubMed] [Google Scholar]
  11. Graham A., Boxer D. H., Haddock B. A., Mandrand-Berthelot A. M., Jones R. W. Immunochemical analysis of the membrane-bound hydrogenase of Escherichia coli. FEBS Lett. 1980 May 5;113(2):167–172. doi: 10.1016/0014-5793(80)80584-9. [DOI] [PubMed] [Google Scholar]
  12. Graham A. The organization of hydrogenase in the cytoplasmic membrane of Escherichia coli. Biochem J. 1981 Aug 1;197(2):283–291. doi: 10.1042/bj1970283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hedrick J. L., Smith A. J. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys. 1968 Jul;126(1):155–164. doi: 10.1016/0003-9861(68)90569-9. [DOI] [PubMed] [Google Scholar]
  14. Jones R. W. The role of the membrane-bound hydrogenase in the energy-conserving oxidation of molecular hydrogen by Escherichia coli. Biochem J. 1980 May 15;188(2):345–350. doi: 10.1042/bj1880345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. LOVENBERG W., BUCHANAN B. B., RABINOWITZ J. C. STUDIES ON THE CHEMICAL NATURE OF CLOSTRIDIAL FERREDOXIN. J Biol Chem. 1963 Dec;238:3899–3913. [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lambden P. R., Guest J. R. Mutants of Escherichia coli K12 unable to use fumarate as an anaerobic electron acceptor. J Gen Microbiol. 1976 Dec;97(2):145–160. doi: 10.1099/00221287-97-2-145. [DOI] [PubMed] [Google Scholar]
  18. Lee J. H., Patel P., Sankar P., Shanmugam K. T. Isolation and characterization of mutant strains of Escherichia coli altered in H2 metabolism. J Bacteriol. 1985 Apr;162(1):344–352. doi: 10.1128/jb.162.1.344-352.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee J. H., Wendt J. C., Shanmugam K. T. Identification of a new gene, molR, essential for utilization of molybdate by Escherichia coli. J Bacteriol. 1990 Apr;172(4):2079–2087. doi: 10.1128/jb.172.4.2079-2087.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Menon N. K., Robbins J., Peck H. D., Jr, Chatelus C. Y., Choi E. S., Przybyla A. E. Cloning and sequencing of a putative Escherichia coli [NiFe] hydrogenase-1 operon containing six open reading frames. J Bacteriol. 1990 Apr;172(4):1969–1977. doi: 10.1128/jb.172.4.1969-1977.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Moriyama R., Nakashima H., Makino S., Koga S. A study on the separation of reconstituted proteoliposomes and unincorporated membrane proteins by use of hydrophobic affinity gels, with special reference to band 3 from bovine erythrocyte membranes. Anal Biochem. 1984 Jun;139(2):292–297. doi: 10.1016/0003-2697(84)90005-8. [DOI] [PubMed] [Google Scholar]
  22. Robinson N. C., Wiginton D., Talbert L. Phenyl-Sepharose-mediated detergent-exchange chromatography: its application to exchange of detergents bound to membrane proteins. Biochemistry. 1984 Dec 4;23(25):6121–6126. doi: 10.1021/bi00320a034. [DOI] [PubMed] [Google Scholar]
  23. Sawers R. G., Ballantine S. P., Boxer D. H. Differential expression of hydrogenase isoenzymes in Escherichia coli K-12: evidence for a third isoenzyme. J Bacteriol. 1985 Dec;164(3):1324–1331. doi: 10.1128/jb.164.3.1324-1331.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sawers R. G., Boxer D. H. Purification and properties of membrane-bound hydrogenase isoenzyme 1 from anaerobically grown Escherichia coli K12. Eur J Biochem. 1986 Apr 15;156(2):265–275. doi: 10.1111/j.1432-1033.1986.tb09577.x. [DOI] [PubMed] [Google Scholar]
  25. Sawers R. G., Jamieson D. J., Higgins C. F., Boxer D. H. Characterization and physiological roles of membrane-bound hydrogenase isoenzymes from Salmonella typhimurium. J Bacteriol. 1986 Oct;168(1):398–404. doi: 10.1128/jb.168.1.398-404.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  27. Stoker K., Oltmann L. F., Stouthamer A. H. Partial characterization of an electrophoretically labile hydrogenase activity of Escherichia coli K-12. J Bacteriol. 1988 Mar;170(3):1220–1226. doi: 10.1128/jb.170.3.1220-1226.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES