Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1981 Apr;146(1):24–32. doi: 10.1128/jb.146.1.24-32.1981

Purification and properties of 5'-nucleotidase from the membrane of Vibrio costicola, a moderately halophilic bacterium.

C Bengis-Garber, D J Kushner
PMCID: PMC217047  PMID: 6163762

Abstract

Two different Mg2+-dependent adenosine 5'-triphosphate-hydrolyzing activities were detected in membranes of Vibrio costicola, a novel 5'-nucleotidase and an N,N'-dicyclohexylcarbodiimide-sensitive adenosine triphosphatase. The former and the latter had different requirements for Mg2+ and were selectively assayed in the membranes by using, respectively, 20 and 2 mM Mg2+. The two enzymes were extracted with a combination of Triton X-100 and octylglucoside, separated on a diethylaminoethyl cellulose column, and purified on glycerol gradients. The purified 5'-nucleotidase consisted of one major polypeptide of 70,000 daltons when analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate. The purified 5'-nucleotidase was similar in substrate specificities, divalent cation specificities, and pH profiles to the membrane-bound N,N'-dicyclohexylcarbodiimide-insensitive nucleotide-phosphohydrolyzing activity. The enzyme hydrolyzed nucleoside 5'-tri, 5'-di, and 5'-monophosphates at comparable rates. Inorganic pyrophosphate, p-nitrophenyl phosphate, glucose 6-phosphate, beta-glycerophosphate, adenosine 5'-diphosphate glucose, adenosine 3'-monophosphate, and cyclic adenosine 3',5'-monophosphate were not hydrolyzed, either im membranes or by the purified 5'-nucleotides. Actions of NaCl and KCl on the activity of the 5'-nucleotidase were studied. The enzyme was activated by both NaCl and KCl; the activation profiles however, were different for the membrane-bound and purified 5'-nucleotidase. The purified enzyme, unlike the membrane-bound enzyme, was markedly stimulated by high concentrations of NaCl (up to 3 M).

Full text

PDF
24

Images in this article

27Image
on p.27

Selected References

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

  1. Ahlers J., Günther T., Peter H. W. Phospholipid composition of plasma membranes and kinetic properties of membrane-bound nucleotidase from marine bacteria. Int J Biochem. 1978;9(8):573–578. doi: 10.1016/0020-711x(78)90117-9. [DOI] [PubMed] [Google Scholar]
  2. Baron C., Thompson T. E. Solubilization of bacterial membrane proteins using alkyl glucosides and dioctanoyl phosphatidylcholine. Biochim Biophys Acta. 1975 Mar 25;382(3):276–285. doi: 10.1016/0005-2736(75)90270-9. [DOI] [PubMed] [Google Scholar]
  3. Bengis-Garber C., Gromet-Elhanan Z. Purification of the energy-transducing adenosine triphosphatase complex from Rhodospirillum rubrum. Biochemistry. 1979 Aug 7;18(16):3577–3581. doi: 10.1021/bi00583a022. [DOI] [PubMed] [Google Scholar]
  4. CHRISTIAN J. H., WALTHO J. A. Solute concentrations within cells of halophilic and non-halophilic bacteria. Biochim Biophys Acta. 1962 Dec 17;65:506–508. doi: 10.1016/0006-3002(62)90453-5. [DOI] [PubMed] [Google Scholar]
  5. DRAPEAU G. R., MACLEOD R. A. NUTRITION AND METABOLISM OF MARINE BACTERIA. XII. ION ACTIVATION OF ADENOSINE TRIPHOSPHATASE IN MEMBRANES OF MARINE BACTERIAL CELLS. J Bacteriol. 1963 Jun;85:1413–1419. doi: 10.1128/jb.85.6.1413-1419.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. HERMAN E. C., Jr, WRIGHT B. E. At 5'-nucleotidase activated by ferrous iron. J Biol Chem. 1959 Jan;234(1):122–125. [PubMed] [Google Scholar]
  7. Hayashi M., Unemoto T., Kozuka Y., Hayashi M. Anion-activated 5'-nucleotidase in cell envelopes of a slightly halophilic Vibrio alginolyticus. Biochim Biophys Acta. 1970 Nov 11;220(2):244–255. doi: 10.1016/0005-2744(70)90010-0. [DOI] [PubMed] [Google Scholar]
  8. KOHN J., REIS J. L. BACTERIAL NUCLEOTIDASES. J Bacteriol. 1963 Oct;86:713–716. doi: 10.1128/jb.86.4.713-716.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Lanyi J. K. Salt-dependent properties of proteins from extremely halophilic bacteria. Bacteriol Rev. 1974 Sep;38(3):272–290. doi: 10.1128/br.38.3.272-290.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Neu H. C. The 5'-nucleotidase of Escherichia coli. I. Purification and properties. J Biol Chem. 1967 Sep 10;242(17):3896–3904. [PubMed] [Google Scholar]
  12. Neu H. C. The 5'-nucleotidase of Escherichia coli. II. Surface localization and purification of the Escherichia coli 5'-nucleotidase inhibitor. J Biol Chem. 1967 Sep 10;242(17):3905–3911. [PubMed] [Google Scholar]
  13. Shindler D. B., Wydro R. M., Kushner D. J. Cell-bound cations of the moderately halophilic bacterium Vibrio costicola. J Bacteriol. 1977 May;130(2):698–703. doi: 10.1128/jb.130.2.698-703.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. TAUSSKY H. H., SHORR E. A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem. 1953 Jun;202(2):675–685. [PubMed] [Google Scholar]
  15. Thompson J., Green M. L., Happold F. C. Cation-activated nucleotidase in cell envelopes of a marine bacterium. J Bacteriol. 1969 Sep;99(3):834–841. doi: 10.1128/jb.99.3.834-841.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. WANG T. P. Specific 5'-nucleotidase from a soil bacterium. J Bacteriol. 1954 Jul;68(1):128–128. doi: 10.1128/jb.68.1.128-128.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  18. Yagil E., Beacham I. R. Uptake of adenosine 5'-monophosphate by Escherichia coli. J Bacteriol. 1975 Feb;121(2):401–405. doi: 10.1128/jb.121.2.401-405.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Yayashi M., Uchida R. A cation activated adenosinetriphosphatase in cell membranes of halophilic Vibrio parahaemolyticus. Biochim Biophys Acta. 1965 Oct 25;110(1):207–209. doi: 10.1016/s0926-6593(65)80113-8. [DOI] [PubMed] [Google Scholar]
  20. de Médicis E., Rossignol B. Pyruvate kinase from the moderate halophile, Vibrio costicola. Can J Biochem. 1977 Aug;55(8):825–833. doi: 10.1139/o77-122. [DOI] [PubMed] [Google Scholar]

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

RESOURCES