SPECIFIC AIMS: 1. To purify the native, undegraded form of rat liver HMG-CoA reductase to homogeneity, determine its molecular weight and subunit composition, and determine whether it is a glyco-or lipoprotein. 2. To establish the number of structurally distinct phosphorylation sites on native HMG-CoA reductase. 3. To determine which of the anticipated structurally distinct phosphorylation sites regulate HMG-CoA reductase activity. 4. To determine which of the sites in (2) regulate reductase activity. 5. To investigate the nature of the physical changes induced by reductase phosphorylation which render it catalytically inactive. METHODOLOGIES: Enzyme purification on affinity supports. Determination of physiochemical parameters. 32P-Labelling of phosphorylation sites and peptide mapping. Determination of site specificity of converter enzyme. Kinetics of 32p incorporation and release and correlation with changes in reductase activity. Covalent modification of active site residues. Use of immunological and protease probes to detect conformational changes. LONG-TERM OBJECTIVES: 1. To determine the catalytic mechanism for HMG-CoA reductase and the physical mechanism by which phosphorylation inactivates reductase activity. 2. To determine the number and structure of the phosphorylation sites on mammalian HMG-CoA reductase, their spatial relationship to the active site, and the functions of each (eg: Regulate activity? Regulate degradation rate?). 3. To identify the converter proteins that act on mammalian HMG-CoA reductase, their site specificities, and the regulatory signals to which they respond. 4. To determine the three-dimensional structure of mammalian HMG-CoA reductase, especially that of its catalytic, ligand binding, and phosphorylation domains. SCIENTIFIC DISCIPLINES INVOLVED: Enzymology, protein chemistry. HEALTH RELATEDNESS: HMG-CoA reductase main regulatory point in cholesterogenesis. Disease relatedness: hypercholesterolemias.
| Sherban, D G; Kennelly, P J; Brandt, K G et al. (1985) Rat liver 3-hydroxy-3-methylglutaryl-CoA reductase. Catalysis of the reverse reaction and two half-reactions. J Biol Chem 260:12579-85 |
| Kennelly, P J; Rodwell, V W (1985) Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by reversible phosphorylation-dephosphorylation. J Lipid Res 26:903-14 |
