IN VITRO PHOSPHORYLATION OF HYDROXYMETHYLGLUTARYL COENZYME A REDUCTASE: STRUCTURAL ANALYSIS OF THE LABELED ENZYME
Abstract
Microsomal HMG-CoA reductase {hydroxymethylglutaryl-CoA reductase (NADPH), EC 1.1.1.34} (reductase) is inactivated in vitro by the addition of MgATP. In preparation for an attempt to provide proof that reductase is phosphorylated during inactivation, methods were developed to inactivate reductase with (gamma)('32)P-ATP and Mg('+2) so that the labeled reductase could be solubilized and purified. Assays which determined the activity of active reductase (R(,a)) and th total activity of inactive and active reductase (R(,t)) during purification were also developed. NaF was present in purification buffers to prevent loss of ('32)P by phosphatases. In collaboration with D. Rogers and H. Rudney (Univ. of Cincinnati), labeled, inactivated reductase was purified to homogeneity with significant incorporation of ('32)P. A single ('32)P band, which comigrated with reductase monomer, was present on SDS polyacrylamide gels. Reductase activity and protein comigrated with ('32)P on nondenaturing polyacrylamide gels. Antibody raised against homogeneous reductase immunoprecipitated ('32)P. Reductase is therefore phosphorylated. Phosphoreductase (phosphorylated, ('32)P-labeled reductase) was subsequently purified to a form which, although not homogeneous with respect to protein, had only one ('32)P band on SDS gel electrophoresis. Immunoprecipitation with antibody made to homogeneous reductase quantitatively precipitated ('32)P. This is strong evidence that reductase is the only ('32)P-labeled protein present. The phosphate-reductase bond was acid-stable and base-labile, consistent with its being a phosphoester. Following high voltage electrophoresis, label comigrated only with phosphoserine. Tryptic digestion in 2M urea produces 2 peaks of ('32)P-labeled phosphopeptide (present in unequal amounts) when analyzed by reverse phase high performance liquid chromatography. Each high performance liquid chromatography peak is resolved into 3 peaks by high voltage electrophoresis at pH 3.6 or 6.4 Reductase is phosphorylated during inactivation in microsomes at seryl-residues and appears to contain at least six nonidentical phosphorylation sites.
Degree
Ph.D.
Subject Area
Biochemistry
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