The proposed project will study hen oviduct signal peptidase (SP), a membrane-bound proteolytic enzyme that removes amino terminal signal peptides from nascent secretory proteins as they are transported into the lumen of the endoplasmic reticulum. Purified oviduct SP is composed of two proteins: a 19 kDa polypeptide and an electrophoretically heterogenous 23 kDa glycopeptide. Amino acid sequence analysis of tryptic peptides derived from the electrophoretically distinct glycoproteins have shown them to be differentially glycosylated forms of the same polypeptide. The proposed study will determine the complete primary structure of oviduct SP using a combination of direct amino acid sequence analysis of purified peptides and by molecular cloning of SP cDNAs. These studies will provide a detailed description of the primary structure of a eukaryotic signal peptidase, one of the components absolutely required for successful biosynthesis of most secreted proteins. The proposed studies will examine the association of the two proteins of oviduct SP to determine the nature of their association and to identify whether both are required for enzymatic activity. Attempts will be made to dissociate the two proteins without loss of enzymatic activity by treating the active, purified enzyme with concentrations of denaturants determined to preserve enzymatic activity. Photocrosslinking experiments using photoactivated synthetic signal peptide analogs will be used to identify the protein(s) of oviduct SP that interact with the signal peptide. Experimental efforts will focus on characterization of the enzymology of oviduct SP. Studies with synthetic signal peptide analogs and mutant signal peptides will identify the structural requirements for signal peptide cleavage. Active site- directed inhibitors of oviduct SP catalysis will be identified. Identification of an irreversible inhibitor will lead to the identification of the active site of the enzyme and yield information about the catalytic mechanism. Successful completion of the proposed studies will bring about a detailed understanding of the primary structure and fundamental biochemistry of this key enzyme in the biosynthetic pathway of cells.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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Wake Forest University Health Sciences
Schools of Medicine
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