Cholesterol gallstone disease afflicts 16 to 20 million people in the United States, with one million new cases developing each year. Epidemiological studies have shown that up to 75% of certain populations have clinically apparent gallstones. Available evidence points to decreased bile acid output as the major contributing factor in the production of lithogenic bile, yet the metabolic defect remains unknown. A great body of physiological data points to cholesterol 7alpha- hydroxylase as the rate-limiting enzyme in bile acid biosynthesis. However, the basic physiology of this system has been explored only at the whole animal level. Contemporary molecular techniques which have been successfully applied to the characterization of other membrane-bound enzymes have yet to be applied to enzymes in the bile acid biosynthetic pathway. We will focus on the key enzyme in bile acid production, cholesterol 7alpha-hydroxylase, as the primary avenue toward understanding normal and pathological features of this pathway. 7alpha-Hydroxylase has been partially purified from liver microsomes of cholestyramine-fed rats. The primary objective of this proposal is to purify 7alpha-hydroxylase to homogeneity and raise antibodies against the protein. Production of monoclonal antibodies will begin immediately using the partially purified protein as antigen. Polyclonal antibody production will begin when homogeneous 7alpha-hydroxylase is available. In future studies, immunological tools and catalytic measurements will be used to ask how bile acid biosynthesis is regulated. Purification and characterization of 7alpha-hydroxylase and its regulation are the first steps towards development of necessary molecular probes to study genetic polymorphisms in populations at risk for cholesterol gallstone disease.
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