Characterization of an enzyme that is capable of processing pro-gonadotropin-releasing hormone protein.
A new membrane bound protease has been identified in bovine hypothalamic neurosecretory granules using synthetic substrates that we prepared based on the sequence in pro-gonadotropin-releasing hormone protein that overlaps gonadotropin-releasing hormone and gonadotropin-associated peptide (thought to be prolactin-releasing hormone-inhibiting hormone). The enzyme was solubilized from neurosecretory granules using the detergent Triton X-100 and was further purified by high-performance gel permeation liquid chromatography. The enzyme hydrolyzes the Arg-2-naphthylamide (NA) bond of benzoyl(Bz)-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA which contains two likely processing sites, Arg-Pro and Lys-Arg. On the basis of the ratio of Vmax to Km as a measure of substrate specificity, Bz-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA is about 50-fold better than Bz-Gly-Gly-Lys-Arg-2-NA. Bz-Leu-Arg-2-NA and Bz-Gly-Leu-Arg-Pro-Gly-Gly are not hydrolyzed. The pH optimum for hydrolysis is 7.2 (Bz-Gly-Gly-Lys-Arg-2-NA substrate). As determined by gel permeation chromatography, the apparent molecular weight of the enzyme depends on the chromatography conditions; in the absence of NaCl, the Mr is approximately equal to 160,000 but is approximately equal to 80,000 if NaCl is included in the eluting buffer. After high-performance gel permeation liquid chromatography, the peak fraction containing the enzyme was lyophilized and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; silver staining revealed a single protein band, Mr approximately equal to 70,000.
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003594:Cytoplasmic Granules / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006868:Hydrolysis; D007031:Hypothalamus / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D010906:Pituitary Hormone-Releasing Hormones / Q000096:biosynthesis*; D011498:Protein Precursors / Q000378:metabolism*; D013379:Substrate Specificity
D010906:Pituitary Hormone-Releasing Hormones; D011498:Protein Precursors; D010447:Peptide Hydrolases