Luteinizing hormone-releasing hormone peptidase activities in the female rat: Characterization by an assay based on high-performance liquid chromatography

So far, the presence of two peptidase activities which degrade luteinizing hormone-releasing hormone (LHRH) have been found in hypothalamic tissue: an endopeptidase and a postproline cleaving enzyme (PPCE). Although it is possible that degradation of LHRH plays a physiological role in the control of gonadotropin secretion, evidence in favor of this hypothesis remains controversial, since only indirect measurements of peptidase activity have been reported. The present paper describes an assay that directly estimates LHRH degradation. This includes fractionation of the degradation products of LHRH by high-performance liquid chromatography (HPLC) and the use of digital integration to measure the disappearance of the LHRH molecule and account for the products deriving from its cleavage. The assay allows for measurement of total LHRH peptidase activity in discrete hypothalamic regions, such as median eminence (ME) and ventromedial preoptic area (POA), of individual animals. These peptidase activities are linear with respect to tissue concentration and time of incubation for up to 40 min for POA and anterior pituitary (AP) and for 2-2.5 h for ME. The major degradation product after HPLC separation and amino acid analysis is the LHRH_1-5 fragment whose appearance in the incubation medium is highly correlated with the disappearance of the LHRH molecule. With the use of this assay, various inhibitors of this activity were examined; the sulfhydryl reagents PCMS and ZnCl₂, the peptide antibiotic bacitracin, and the metalloendopeptidase inhibitor, 1,10-phenanthroline were the most effective.