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. 1977 Mar;15(3):863–870. doi: 10.1128/iai.15.3.863-870.1977

Cholesteryl de-esterifying enzyme from Staphylococcus aureus: separation from alpha toxin, purification, and some properties.

N R Harvie
PMCID: PMC421453  PMID: 858644

Abstract

A cholesteryl de-esterifying enzyme found in partially purified preparations of alpha toxin produced by the Wood 46 strain on Staphylococcus aureus has been separated from other staphylococcal proteins and from alpha toxin by isoelectric focusing and gel filtration. Preparations of alpha toxin from Bi-Gel P-60 columns and of the cholesteryl esterase from Bio-Gel P-200 columns showed a high degree of purity, as determined by analytical ultracentrifugation, gel diffusion, immunoelectrophoresis, and polyacrylamide gel electrophoresis. The molecular weight of the cholesteryl esterase determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate was 25,500 and on Bio-Gel P-300 columns it was 175,000, indicating an associating system. The density of the enzyme was lower than expected for simple proteins (about 1.19 g/ml). Chloroform-methanol extracts showed the presence of a neutral lipid that did not contain cholesterol. This material, possibly a glycolipid, might play a role in the stabilization of the enzymatically active protomer. The isoelectric point of the esterase was 9.1. Cholesteryl esterase was labile and lost its activity easily. It could bind reversibly to agarose-containing gels. After elution, it was enzymatically inactive, with an isoelectric point of less than 6.2. The W46M mutant of the Wood 46 strain, which does not produce alpha toxin, also does not produce cholesteryl esterase.

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Selected References

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

  1. Arbuthnott J. P., Freer J. H., Bernheimer A. W. Physical states of staphylococcal alpha-toxin. J Bacteriol. 1967 Oct;94(4):1170–1177. doi: 10.1128/jb.94.4.1170-1177.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BERNHEIMER A. W., SCHWARTZ L. L. Isolation and composition of staphylococcal alpha toxin. J Gen Microbiol. 1963 Mar;30:455–468. doi: 10.1099/00221287-30-3-455. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Eckhardt A. E., Hayes C. E., Goldstein I. J. A sensitive fluorescent method for the detection of glycoproteins in polyacrylamide gels. Anal Biochem. 1976 May 21;73(1):192–197. doi: 10.1016/0003-2697(76)90154-8. [DOI] [PubMed] [Google Scholar]
  5. FOLCH J., ASCOLI I., LEES M., MEATH J. A., LeBARON N. Preparation of lipide extracts from brain tissue. J Biol Chem. 1951 Aug;191(2):833–841. [PubMed] [Google Scholar]
  6. Filipek-Wender H., Borgström B. Cholesterol esterase activity of rat pancreatic juice. Effect of pH and bile salt on the enzyme-catalysed equilibrium state. Acta Biochim Pol. 1971;18(1):1–10. [PubMed] [Google Scholar]
  7. Forlani L., Bernheimer A. W., Chiancone E. Ultracentrifugal analysis of staphylococcal alpha toxin. J Bacteriol. 1971 Apr;106(1):138–142. doi: 10.1128/jb.106.1.138-142.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Harvie N. R. De-esterification of cholesteryl esters in human plasma alpha-lipoprotein (HDL) by preparations of staphylococcal alpha toxin. Biochem Biophys Res Commun. 1974 Dec 23;61(4):1283–1288. doi: 10.1016/s0006-291x(74)80423-7. [DOI] [PubMed] [Google Scholar]
  9. Hyun J., Treadwell C. R., Vahouny G. V. Pancreatic juice cholesterol esterase. Studies on molecular weight and bile salt-induced polymerization. Arch Biochem Biophys. 1972 Sep;152(1):233–242. doi: 10.1016/0003-9861(72)90211-1. [DOI] [PubMed] [Google Scholar]
  10. Kapitany R. A., Zebrowski E. J. A high resolution PAS stain for polyacrylamide gel electrophoresis. Anal Biochem. 1973 Dec;56(2):361–369. doi: 10.1016/0003-2697(73)90202-9. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Lowry R. R. Ferric chloride spray detector for cholesterol and cholesteryl esters on thin-layer chromatograms. J Lipid Res. 1968 May;9(3):397–397. [PubMed] [Google Scholar]
  13. McNiven A. C., Owen P., Arbuthnott J. P. Multiple forms of staphylococcal alpha-toxin. J Med Microbiol. 1972 Feb;5(1):113–122. doi: 10.1099/00222615-5-1-113. [DOI] [PubMed] [Google Scholar]
  14. OUCHTERLONY O. Diffusion-in-gel methods for immunological analysis. II. Prog Allergy. 1962;6:30–154. doi: 10.1159/000313795. [DOI] [PubMed] [Google Scholar]
  15. REISFELD R. A., LEWIS U. J., WILLIAMS D. E. Disk electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature. 1962 Jul 21;195:281–283. doi: 10.1038/195281a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Wiseman G. M. The hemolysins of Staphylococcus aureus. Bacteriol Rev. 1975 Dec;39(4):317–344. doi: 10.1128/br.39.4.317-344.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]

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