Identification of a cholesterol-regulated 180-kDa microsomal protein in rat hepatocytes.
The immunoprecipitation by antibodies to 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of extracts of [35S]methionine-pulse-labelled isolated hepatocytes, followed by electrophoresis and fluorography, showed the presence not only of 97-kDa HMG-CoA reductase, but also of another protein of 180 kDa. Boiling the immunoprecipitates both in the presence and in the absence of 2-mercaptoethanol, followed by SDS/polyacrylamide gel electrophoresis both in the presence and in the absence of 8 M urea, was not found to change the ratio of 180-kDa/97-kDa proteins. These facts suggest that the 180-kDa protein is not an aggregated form of HMG-CoA reductase. A different batch of antibodies obtained from a newly purified HMG-CoA reductase fully titrated the reductase activity, but did not immunoprecipitate the 180-kDa protein, showing that there is no cross-reactivity between these proteins. The 180-kDa polypeptide is a glycoprotein of N-linked high-mannose oligosaccharide chains, which is not processed on the Golgi system. The apparent molecular mass of the carbohydrate is 16 kDa. The incubation of rat hepatocytes with sterols produces, on the one hand, a decrease in the rate of synthesis, and on the other hand, an acceleration in the turnover rate of the 180-kDa protein. In addition, mevalonate is known to decrease its rate of synthesis. The carbohydrate-free 164-kDa protein was found to degrade only a tenth as fast as the glycoprotein and, furthermore, the degradation was no longer accelerated by sterols. These results support the notion that the 180-kDa protein is not a modified form of 97-kDa reductase, but probably a different protein related to cholesterol metabolism, and also that the N-linked, high-mannose chains, which are bound to the glycoprotein, are required for rapid and controlled degradation of the protein.
Haro
D
D
Unit of Biochemistry, School of Pharmacy, University of Barcelona, Spain.
Marrero
P F
PF
Ayté
J
J
Hegardt
F G
FG
D000818:Animals; D000936:Antigen-Antibody Complex / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D006596:Hexosaminidases; D006903:Hydroxymethylglutaryl CoA Reductases / Q000276:immunology / Q000378:metabolism; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000378:metabolism; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009500:Neutralization Tests; D009844:Oligosaccharides / Q000032:analysis; D011233:Precipitin Tests; D051381:Rats; D014415:Tunicamycin / Q000494:pharmacology; D014508:Urea
D000936:Antigen-Antibody Complex; D006023:Glycoproteins; D009844:Oligosaccharides; D014415:Tunicamycin; D014508:Urea; D002784:Cholesterol; D006903:Hydroxymethylglutaryl CoA Reductases; D006904:Hydroxymethylglutaryl-CoA Synthase; D006596:Hexosaminidases; D008358:Mannose