PURIFICATION AND CHARACTERIZATION HMG-COENZYME A REDUCTASE PHOSPHATASES

WILLIS EDMUND BROWN, Purdue University

Abstract

Activation of HMG-CoA reductase (reductase, E.C. 1.1.1.34) inactivated by MgATP is mediated by dephosphorylation. Activation can be achieved by treatment with acid phosphatase, alkaline phosphatase or acetyl-CoA carboxylase phosphatase. Reductase phosphatase undergoes a large decrease in molecular weight and a large increase in activity following solvent treatment. The properties of reductase phosphatases treated with solvents (low MW reductase phosphatase) or not treated with solvents (native reductase phosphatase) were studied. Native reductase phosphatase, detected in all vertebrate livers examined, is primarily a cytosolic enzyme. Heat-stable inhibitor II inhibits the activity of low MW reductase phosphatase, but not that of native reductase phosphatase. Purification of native reductase phosphatase reveals two peaks of activity designated native reductase phosphatase I and II. Native reductase phosphatase I and II can be distinguished on DE-52, gel filtration or hydroxylapatite chromatography. Native reductase phosphatase II was purified 800-fold and has an apparent molecular weight of 130,000. Native reductase phosphatase I, purified 50-fold, has an apparent molecular weight of 190,000. Low MW reductase phosphatase and native reductase phosphatases I and II dephosphorylate ('32)P-labeled reductase with an accompanying increase in reductase activity. For all three phosphatase preparations, no further activation is observed after approximately 50% of the ('32)P has been released. Approximately one mole of phosphate is released per mole of reductase subunit activated. Release of additional ('32)P did not result in any change in reductase activity. These results suggest that reductase is phosphorylated at more than one site in vitro. Native reductase phosphatases I and II also dephosphorylate phosphorylase a, acetyl-CoA carboxylase and reductase kinase. Substrate specificity toward reductase and reductase kinase was observed. Neither native reductase phosphatase hydrolyzes ATP, p-nitrophenylphosphate or 4-methylumbelliferyl phosphate. Pyrophosphate is a potent inhibitor of both low MW and of native reductase phosphatase activity. Half maximal inhibition is observed at < 1 mM. Fluoride, a potent inhibitor of low MW reductase phosphatase, is a less effective inhibitor of native reductase phosphatase I or II.

Degree

Ph.D.

Subject Area

Biochemistry

COinS