The long range objective of the proposed research is to understand the regulation of cholesterol biosynthesis and in particular to determine the mechanism involved in the hormonal and dietary regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the enzyme which catalyzes the rate-limiting reaction of cholesterol biosynthesis. The rate of cholesterol biosynthesis is one of several factors contributing to the regulation serum cholesterol levels. Elevated serum cholesterol is a significant risk factor in the development of vascular diseases. In this proposal, we seek to determine the extent to which changes in 1) enzyme quantity 2) rates of enzyme synthesis and degradation and 3) mRNA levels for HMG-CoA reductase contribute to various hormonal and dietary changes in HMG-CoA reductase activity. Of particular interest are the changes due to thyroid hormones and insulin. The methodology to be used will include immunotitration, immunoblotting, in vitro translation using a rabbit reticulocyte lysate system, immunoprecipitation and enzyme purification. We also plan to investigate certain properties of native HMG-CoA reductase. These include intracellular location of the reductase by immunoelectron microscopy, isolation of native reductase by immuno affinity chromatography and determination of the subunit and overall molecular weight, amino acid composition, whether it is synthesized as a proenzyme, whether it is glycoslated, its Kms for substrates etc.
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