SQUALENE EPOXIDASE AND OXIDOSQUALENE CYCLASE FROM LIVER

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Squalene epoxidase (SE) and oxidosqualene cyclase (OSC) are the two key enzymes required for the production of lanosterol from an acyclic polyene precursor. Squalene epoxidase requires a supernatant protein factor (SPF) for activity. Classical purifications have been reported for rat liver SE and rat liver (SPF). Several OSC enzymes have been purified from plants, but the vertebrate enzymes have as yet eluded purification to homogeneity. Our goal is to purify, clone, and sequence these proteins from rat liver and then from human hepatoma cells. Furthermore, photoaffinity labels and irreversible enzyme-activated inhibitors recently discovered in our labs will enable us to selectively and covalently modify the active sites of both enzymes. First, we will synthesize selective inhibitors and radiolabeled substrates and photoaffinity labels for SPF, SE and OSC. Next, using these substrates, selective inhibitors and photoaffinity labels, we propose to purify these three proteins from rat liver. Rats will be induced to increased levels of cholesterol biosynthetic enzymes by ingestion of cholestyramine and an HMG-CoA reductase inhibitor or a SE epoxidase inhibitor. Enzymes will be purified by classical and affinity methods, using photoaffinity labeling and enzyme activity assays to monitor purification. Controlled trypsinization will be used to excise catalytically-active protein substructures from membrane proteins. Purified OSC and SE activity will be employed to obtain N-terminal and internal amino acid sequence data to design corresponding antisense oligonucleotide probes. PCR cloning will be employed to screen a rat liver cDNA library in lambda/gt11 to obtain full-length clones for these enzymes. cDNA probes from this system will then be used to identify SE and OSC cDNAs in a human hepatoma cell (Hep G2) cDNA library. A heterologous expression system will be developed to provide milligram quantities of the human proteins for studies of active site chemistry. Covalently-modified SE and OSC will be subjected to selected proteolysis to define the residues present in the active site.