RAT LIVER 3-HYDROXY-3-METHYLGLUTARYL COENZYME A REDUCTASE: REGULATION BY PHOSPHORYLATION - DEPHOSPHORYLATION AND SOLUBILIZATION AND PROPERTIES OF THE NATIVE ENZYME (KINASE)
Abstract
The objectives of this thesis were to (a) solubilize native HMG-CoA reductase, (b) examine its size and stability properties, (c) determine whether it catalyzed the reverse reaction (mevalonate to HMG-CoA) or either of the half-reactions (mevaldate to HMG-CoA or mevaldate to mevalonate), and (d) determine whether HMG-CoA reductase was inactivated by covalent phosphorylation. Substantial progress was achieved on all of these objectives. Native HMG-CoA reductase was solubilized and purified over 200-fold. It closely resembled the intact, microsomally-bound enzyme in its subunit size, heat-lability, and facility of inactivation. It was not associated with detergent micelles in solution, had a pH optimum for stability of 6.6, and had a subunit molecular weight of 88,800. Its molecular weight and sedimentation coefficient as determined by sucrose density gradient ultracentrifugation were 87,500 and 6.0, respectively. Both native and freeze-thaw solubilized HMG-CoA reductase catalyzed the reverse reaction and both half-reactions, as determined by spectrophotometric assays, inhibition studies, and identification of reaction products. The second half-reaction (mevaldate to mevalonate) was stimulated by CoASH. Native HMG-CoA reductase was reversibly inactivated with a facility approaching that observed for the microsomally-bound enzyme. Inactivation was observed whether HMG-CoA reductase activity was measured by radioisotopic (mevalonate production) or spectrophotometric (NADPH oxidation) means. Inactivation blocked catalysis of the forward reaction and both half-reactions. Freeze-thaw solubilized HMG-CoA reductase from microsomes treated with (gamma)-('32)P ATP contained covalently bound ('32)P as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing gels. Analysis by isoelectric focusing under denaturing conditions resolved three ('32)P-labelled species. These may reflect multiple phosphorylation of HMG-CoA reductase subunits, although other interpretations cannot as yet be ruled out.
Degree
Ph.D.
Subject Area
Biochemistry
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