Characterization of homogeneous atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor.
We previously reported the discovery and partial characterization of bovine atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor, which is associated with atrial granule membranes. We now report the physicochemical properties of electrophoretically homogeneous enzyme purified by a series of chromatography steps from a subcellular fraction enriched for atrial granules. The enzyme tends to associate during purification to higher molecular weight species, but SDS-PAGE analysis reveals a single polypeptide chain of molecular weight 70,000. The enzyme is activated 2-3 fold by Ca+2 and 1.5-fold by Mg+2 and is nearly 100% inhibited by Zn+2 or Co+2. Thus, the enzyme can be considered a calcium activated, neutral pH, serine proteinase. Based on the hydrolysis of numerous synthetic peptide substrates, the recognition sequence for the enzyme within the pro-hormone has been mapped to A96PRSLRR102; cleavage occurs at the Arg98-Ser99 bond yielding bioactive atrial natriuretic peptide directly from the pro-hormone. The doublet of basic amino acids is part of the recognition sequence but is not the primary cleavage site. It is our hypothesis that the processing site sequence acts as a recognition element for the endoproteinase and resides at the surface of the pro-hormone and thus contributes to the molecular basis for limited proteolysis.
Wypij
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DM
Glaxo Research Laboratories, Research Triangle Park, North Carolina 27709.
Harris
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D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003594:Cytoplasmic Granules / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D009318:Natriuresis; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D011498:Protein Precursors; D009320:Atrial Natriuretic Factor; D012697:Serine Endopeptidases