The long term objective is to contribute to our understanding of the mechanisms involved in the insertion and final topology of microsomal cytochrome P-450 in the membrane.
The specific aims are to identify domains of the cytochrome P-450 molecule that function as either membrane-insertion signals or membrane-stop signals. The approach to this problem will be to produce hybrids of hydrophobic potentially membrane-active regions of rabbit cytochrome P-450 and bovine preproparathyroid hormone, a protein that is normally secreted. The signal sequence of preProPTH will be replaced by fragments of cytochrome P-450 to assay for membrane-insertion activity. Fragments of cytochrome P-450 will be inserted in the middle of preProPTH to assay for membrane-stop signals. To produce the hybrid proteins, appropriate combinations of the cloned cDNAs for these two proteins will be constructed in plasmids containing promoters for SP6 polymerase. RNA will be transcribed from the cDNA by SP6 RNA polymerase and the RNA will be translated in wheat germ and reticulocyte cell-free systems supplemented with microsomal membranes. Proteolytic processing of the signal sequence will be detected by polyacrylamide gel electrophoresis. The extent of translocation across the membrane will be analyzed by protection from proteolytic enzymes. The cellular location of hybrid proteins containing membrane-active regions as determined by the in vitro assay will also be analyzed by transfection of the cDNA into intact cells using SV40-based vectors. Cytochrome P-450 is the terminal oxidase of the liver microsomal drug metabolizing system. This system is responsible for the oxidative metabolism of thousands of compounds. Usually the metabolism leads to detoxification or inactivation of the substrate, but in some important cases, for example, carcinogens, activation occurs. The inducibility of cytochrome P-450s with broad substrate specificity forms the basis for drug interactions in man. Since most of the substrates for cytochrome P-450 are lipophilic, an understanding of the interactions of the protein with the membrane will probably be crucial to ultimately describing its mechanism of action.
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