The basic goal of this research program is to contribute to the understanding of the molecular basis of the biological activities of the liver microsomal membrane proteins. The following strategy will be used to approach these goals. Proteins present in the microsomal membranes are isolated and sequenced. Amino acid sequenced data, together with the methodology for isolating peptide fragments is used to define their topology in the membrane and to identify the residues involved in the function of these enzymes. Microsomal membranes, under controlled conditions, are treated with proteolytic enzymes and group specific reagents. The peptides and the proteins of the digests re characterized by means of electrophoresis and high performance liquid chromatography. Micro- sequence analysis of the isolated fragments and the transblottead proteins identifies the cleavage site or the position of the label in the particular protein. The orientation studies will be focused on the microsomal preparations enriched with cytochromes, deacylases, and epoxide hydrolase. Proteins present in microsomal membranes will be separated by high resolution two-dimensional electrophoresis. N-terminal sequences of these proteins will be obtained by direct blotting of the separated proteins from electrophoresis gels to chemically inert supports followed by excision of the protein band and sequencing. Attempts will be made to isolate and characterize some of the enzymes, involved in the posttranslational processing reactions that occur in the microsomal membranes. The health-relatedness of this project is that a preponderance of xenobiotics including chemical carcinogens and drugs as well as physiological compounds such as steriods and prostaglandins are metabolized by the microsomal cytochromes and the luminal esterases. Many intrinsic, as well as, viral proteins pass through the microsomal membrane to reach their destination. Many undergo proteolysis, glycosylation, acylation and deacylation reactions. Thus, the basic studies addressed in this proposal provide fundamental knowledge relevant to the clinical problems of atherosclerosis (cytochrome b5-desaturase system), and cancer (cytochrome P-450 system and esterases, chemical carcinogenesis). Elucidation of the mechanism of acylation, glycosylation and proteolysis steps. Many of these are provided by the enzymes of endoplasmic reticulum and all are essential for the assembly of active viral particles.
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