Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1973 Oct;116(1):88–97. doi: 10.1128/jb.116.1.88-97.1973

Evidence for Proteolytic Degradation of Histidinol Phosphate Phosphatase Specified by Nonsense Mutants of the hisB Gene of Salmonella typhimurium

L L Houston 1
PMCID: PMC246395  PMID: 4355487

Abstract

The gene products from hisB nonsense mutants having histidinol phosphate phosphatase activity were isolated from Salmonella typhimurium. The enzyme from strain TR691 (hisB278 hisT1529 aroD5) was isolated in the presence of diisopropylfluorophosphate. Three electrophoretically separable forms were demonstrated, and all were shown to have a mol wt of approximately 38,000 and to consist of a single polypeptide chain. Previously, two forms of the phosphatase enzyme from this strain were isolated without diisopropylfluorophosphate and shown to have a different subunit composition. Strain TA387 (hisB2133 his 01242) was shown to have two electrophoretically separable phosphatases with a mol wt of about 52,000 and consisted of 17,000- to 19,000-mol wt polypeptide chains as evidenced by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The results could be explained by proteolytic cleavage of the primary gene product which can partially be prevented by the protease inhibitor. Strain TA387 phosphatase lost all activity in 8 M urea but could be renatured by dialysis. Gel filtration showed that it also regained its original molecular weight. The values of Km of histidinol phosphate and the competition inhibition constant for histidinol were determined. The addition of MnCl2 to the assay was shown to shift the optimal pH value to a lower pH value.

Full text

PDF
88

Selected References

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

  1. Ames B. N., Garry B. COORDINATE REPRESSION OF THE SYNTHESIS OF FOUR HISTIDINE BIOSYNTHETIC ENZYMES BY HISTIDINE. Proc Natl Acad Sci U S A. 1959 Oct;45(10):1453–1461. doi: 10.1073/pnas.45.10.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anfinsen C. B. The formation and stabilization of protein structure. Biochem J. 1972 Jul;128(4):737–749. doi: 10.1042/bj1280737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brady D. R., Houston L. L. New assay for histidinol phosphate phosphatase using a coupled reaction. Anal Biochem. 1972 Aug;48(2):480–482. doi: 10.1016/0003-2697(72)90101-7. [DOI] [PubMed] [Google Scholar]
  4. Brady D. R., Houston L. L. Some properties of the catalytic sites of imidazoleglycerol phosphate dehydratase-histidinol phosphate phosphatase, a bifunctional enzyme from Salmonella typhimurium. J Biol Chem. 1973 Apr 10;248(7):2588–2592. [PubMed] [Google Scholar]
  5. Chaiken I. M. Chemical studies of structural features in staphylococcal nuclease-T'. J Biol Chem. 1972 Apr 10;247(7):1999–2007. [PubMed] [Google Scholar]
  6. Chang G. W., Roth J. R., Ames B. N. Histidine regulation in Salmonella typhimurium. 8. Mutations of the hisT gene. J Bacteriol. 1971 Oct;108(1):410–414. doi: 10.1128/jb.108.1.410-414.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clark J. F., Jakoby W. B. Yeast aldehyde dehydrogenase. 3. Preparation of three homogeneous species. J Biol Chem. 1970 Nov 25;245(22):6065–6071. [PubMed] [Google Scholar]
  8. Fink G. R., Klopotowski T., Ames B. N. Histidine regulatory mutants in Salmonella typhimurium. IV. A positive selection for polar histidine-requiring mutants from histidine operator constitutive mutants. J Mol Biol. 1967 Nov 28;30(1):81–95. doi: 10.1016/0022-2836(67)90245-8. [DOI] [PubMed] [Google Scholar]
  9. Fink G. R., Roth J. R. Histidine regulatory mutants in Salmonella typhiumium. VI. Dominance studies. J Mol Biol. 1968 May 14;33(3):547–557. doi: 10.1016/0022-2836(68)90305-7. [DOI] [PubMed] [Google Scholar]
  10. Fowden L., Lewis D., Tristram H. Toxic amino acids: their action as antimetabolites. Adv Enzymol Relat Areas Mol Biol. 1967;29:89–163. doi: 10.1002/9780470122747.ch3. [DOI] [PubMed] [Google Scholar]
  11. Goldberg A. L. Degradation of abnormal proteins in Escherichia coli (protein breakdown-protein structure-mistranslation-amino acid analogs-puromycin). Proc Natl Acad Sci U S A. 1972 Feb;69(2):422–426. doi: 10.1073/pnas.69.2.422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldschmidt R. In vivo degradation of nonsense fragments in E. coli. Nature. 1970 Dec 19;228(5277):1151–1154. doi: 10.1038/2281151a0. [DOI] [PubMed] [Google Scholar]
  13. HARTMAN P. E., HARTMAN Z., SERMAN D. Complementation mapping by abortive transduction of histidine requiring Salmonella mutants. J Gen Microbiol. 1960 Apr;22:354–368. doi: 10.1099/00221287-22-2-354. [DOI] [PubMed] [Google Scholar]
  14. HJERTEN S. "Molecular sieve" chromatography on polyacrylamide gels, prepared according to a simplified method. Arch Biochem Biophys. 1962 Sep;Suppl 1:147–151. [PubMed] [Google Scholar]
  15. Hartman P. E., Hartman Z., Stahl R. C. Classification and mapping of spontaneous and induced mutations in the histidine operon of Salmonella. Adv Genet. 1971;16:1–34. doi: 10.1016/s0065-2660(08)60352-1. [DOI] [PubMed] [Google Scholar]
  16. Houston L. L. Specialized subregions of the bifunctional hisB gene of Salmonella typhimurium. J Bacteriol. 1973 Jan;113(1):82–87. doi: 10.1128/jb.113.1.82-87.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. LOPER J. C. Enzyme complementation in mixed extracts of mutants from the Salmonella histidine B locus. Proc Natl Acad Sci U S A. 1961 Sep 15;47:1440–1450. doi: 10.1073/pnas.47.9.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. LOPER J. C., GRABNAR M., STAHL R. C., HARTMAN Z., HARTMAN P. E. GENES AND PROTEINS INVOLVED IN HISTIDINE BIOSYNTHESIS IN SALMONELLA. Brookhaven Symp Biol. 1964 Dec;17:15–52. [PubMed] [Google Scholar]
  19. 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]
  20. MANDELSTAM J. The intracellular turnover of protein and nucleic acids and its role in biochemical differentiation. Bacteriol Rev. 1960 Sep;24(3):289–308. doi: 10.1128/br.24.3.289-308.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nath K., Koch A. L. Protein degradation in Escherichia coli. I. Measurement of rapidly and slowly decaying components. J Biol Chem. 1970 Jun 10;245(11):2889–2900. [PubMed] [Google Scholar]
  22. Pine M. J. Steady-state measurement of the turnover of amino acid in the cellular proteins of growing Escherichia coli: existence of two kinetically distinct reactions. J Bacteriol. 1970 Jul;103(1):207–215. doi: 10.1128/jb.103.1.207-215.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Roth J. R., Silbert D. F., Fink G. R., Voll M. J., Antón D., Hartman P. E., Ames B. N. Transfer RNA and the control of the histidine operon. Cold Spring Harb Symp Quant Biol. 1966;31:383–392. doi: 10.1101/sqb.1966.031.01.050. [DOI] [PubMed] [Google Scholar]
  24. Roth J. R. UGA nonsense mutations in Salmonella typhimurium. J Bacteriol. 1970 May;102(2):467–475. doi: 10.1128/jb.102.2.467-475.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schulze I. T., Colowick S. P. The modification of yeast hexokinases by proteases and its relationship to the dissociation of hexokinase into subunits. J Biol Chem. 1969 May 10;244(9):2306–2316. [PubMed] [Google Scholar]
  26. Shaffer B., Edelstein S., Fink G. R. His4 a gene complex of Saccharomyces cerevisiae. Brookhaven Symp Biol. 1972;23:250–270. [PubMed] [Google Scholar]
  27. 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]
  28. Vasington F. D., LeBeau P. Sedimentation properties of the enzymes of the histidine B gene. Biochem Biophys Res Commun. 1967 Jan 23;26(2):153–161. doi: 10.1016/0006-291x(67)90227-6. [DOI] [PubMed] [Google Scholar]
  29. Villarejo M., Zamenhof P. J., Zabin I. Beta-galactosidase. In vivo -complementation. J Biol Chem. 1972 Apr 10;247(7):2212–2216. [PubMed] [Google Scholar]
  30. WHITFIELD H. J., Jr, SMITH D. W., MARTIN R. G. SEDIMENTATION PROPERTIES OF THE ENZYMES OF THE HISTIDINE OPERON. J Biol Chem. 1964 Oct;239:3288–3291. [PubMed] [Google Scholar]
  31. 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]

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

RESOURCES