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. 1983 Mar;80(6):1516–1520. doi: 10.1073/pnas.80.6.1516

Overproduction of a Mr 92,000 protomer of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in compactin-resistant C100 cells

Edna C Hardeman *, Hans-Stephan Jenke †, Robert D Simoni *,‡
PMCID: PMC393632  PMID: 6572913

Abstract

We describe a cell line, designated C100, that displays a 100-fold increase in the major regulatory enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase [HMG-CoA; mevalonate:NADP+ oxido-reductase (CoA-acylating), EC 1.1.1.34]. Immunoprecipitation of [35S]methionine-labeled enzyme from C100 microsomal membranes prepared in the presence of the protease inhibitors phenyl-methylsulfonyl fluoride and leupeptin revealed two up regulated proteins: a major band of Mr 92,000 and a minor band of Mr 63,000. We conclude that the Mr 92,000 protein is probably the intact form of HMG-CoA reductase protomer based on the following criteria. (i) It is a highly up regulated microsomal membrane protein that coincides with the increase in HMG-CoA reductase specific activity in this cell line. (ii) It is recognized by a specific HMG-CoA reductase antiserum under a variety of stringencies. (iii) Isolation and solubilization of [35S]methionine-labeled C100 microsomal membranes in the absence of protease inhibitors resulted in the disappearance of the Mr 92,000 protein and the appearance of two proteins of Mr 52,000 and 38,000. (iv) Analysis of cells labeled for 30 min with [35S]methionine, well under the half-life of HMG-CoA reductase, revealed only the Mr 92,000 protein to be present in total cell extract. (v) The previously reported single immunoprecipitation polypeptide for HMG-CoA reductase of Mr 62,000 [Chin, D. J., Luskey, K. L., Anderson, R. G. W., Faust, J. R., Goldstein, J. L. & Brown, M. S. (1982) Proc. Natl. Acad. Sci. USA 79, 1185-1189] can be isolated and appears to be the result of both proteolysis and sample preparation for NaDodSO4 gel electrophoresis. Analysis of C100 cells labeled with [35S]methionine for 24 hr indicates that the predominant steady-state form of the enzyme is the Mr 92,000, rather than the Mr 63,000, protein, further suggesting that the two proteins do not have a classical precursor-product relationship.

Keywords: undegraded protomer, regulation of cholesterol synthesis

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Selected References

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

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