Peptidase activity in the hypothalamus of the rat: utilization of leucine-p-nitroanilide to monitor the activity degrading luteinizing hormone releasing hormone.
Previous studies by other investigators have shown that luteinizing hormone releasing hormone (LHRH) and amino acid derivatives of p-nitroanilide are probably degraded by a common enzymatic activity; however, most of these studies are inferential in that they are largely based upon kinetic inhibition data derived from relatively crude tissue preparations. The purpose of this work was to determine whether the synthetic substrate leucine-p- nitroanilide (Leu-p-NA) and LHRH were degraded by the same peptidase activity. Supernatants (10,000 X g) from homogenates of rat hypothalami were eluted from Sephadex G-200, and the resultant fractions were assayed for degrading activity toward LHRH and Leu-p-NA. Radioimmunoassay (RIA) indicated that loss of immunologically active LHRH occurred in the same fractions in which maximal Leu-p-NA degrading activity eluted. Kinetically, exogenous LHRH inhibited degradation of Leu-p-NA in a concentration-dependent manner. When fractions evidencing Leu-p-NA degrading activity were incubated with 125I-LHRH, polyacrylamide gel electrophoresis (PAGE) indicated a time-dependent loss of LHRH with the concomitant production of a radioactive peptide fragment. High-performance liquid chromatography (HPLC) analysis of unlabeled LHRH incubations revealed, within the Leu-p-NA degrading fractions, the formation of two peptide fragments. These studies have further substantiated the likelihood that LHRH and Leu-p-NA are degraded by a common enzyme activity as indicated not only by kinetic inhibition data, but also by cofractionation of activity toward both substrates and by two analytical methods capable of detecting LHRH fragmentation (PAGE and HPLC).
D000818:Animals; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D007031:Hypothalamus / Q000201:enzymology*; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D010446:Peptide Fragments; D007987:Gonadotropin-Releasing Hormone; D010447:Peptide Hydrolases