Characterization of an enzyme that is capable of processing pro-gonadotropin-releasing hormone protein

Abstract

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-napthylamide (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 V max to K m 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 M r is ≈160,000 but is ≈ 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, M r ≈ 70,000.