Charlene Michaud

Endopeptidase-24.15 Is the Primary Enzyme that Degrades Luteinizing Hormone Releasing Hormone Both In Vitro and In Vivo

Abstract: The concentration of luteinizing hormone releasing hormone (LHRH) (pGlu‐His‐Trp‐Ser‐Tyr‐Gly‐Leu‐Arg‐Pro‐Gly‐NH2), which reaches the anterior pituitary via the hypothalamo‐hypophyseal portal system, appears to be controlled in part by the rate of LHRH degradation within the hypothalamus and/or pituitary. Specific, active site‐directed endopeptidase inhibitors synthesized in our laboratory were used to identify the enzyme(s) involved in LHRH degradation by hypothalamic and pituitary membrane preparations, and by an intact anterior pituitary tumor cell line (AtT20). Incubation of LHRH with pituitary and hypothalamic membrane preparations led to the formation of pGlu‐His‐Trp (LHRH1–3) as the main reaction product. Under the same conditions, addition to the incubation mixtures of captopril, an inhibitor of the angiotensin converting enzyme, led to accumulation of pGlu‐His‐Trp‐Ser‐Tyr (LHRH1–5) and, to a lesser extent, pGlu‐His‐Trp‐Ser‐Tyr‐Gly (LHRH1–6). The degradation of LHRH and the formation of the N‐terminal tri‐ and pentapeptides was blocked by N‐[1‐(R,S)‐carboxy‐3‐phenylpropyl]‐Ala‐AlaPhe‐p‐aminobenzoate (cFP‐AAF‐pAB), a specific, active site directed inhibitor of endopeptidase‐24.15. Some inhibition of LHRH degradation and formation of the N‐terminal hexapeptide was also obtained in the presence of N‐[1‐carboxy‐2‐phenylethyl]‐Phe‐p‐aminobenzoate (cFE‐F‐pAB), an inhibitor of endopeptidase‐24.11. Similar results were obtained with AtT20 cell membranes and with intact AtT20 cells in monolayer culture. Following cleavage by endopeptidases the C‐terminal part of LHRH was rapidly degraded by aminopeptidases. Superactive analogs of LHRH in which Gly6 was replaced by a D‐amino acid are resistant to degradation by both endopeptidase‐24.11 and ‐24.15. In vivo, when LHRH was injected directly into the third ventricle of rats, the presence of cFP‐AAF‐pAB inhibited LHRH degradation. It is concluded that LHRH degradation is primarily initiated by the membrane‐bound form of endopeptidase‐24.15 to yield pGlu‐His‐Trp‐Ser‐Tyr and to a lesser extent by endopeptidase‐24.11 to yield pGlu‐His‐Trp‐Ser‐Tyr‐Gly.

Generation of methionine and leucine-enkephalin from precursor molecules by cation-sensitive neutral endopeptidase of bovine pituitary

Highly purified preparations of cation-sensitive neutral endopeptidase, from bovine pituitary, and also rabbit brain, generate methionine-enkephalin, from α-endorphin, a peptide containing the amino acid sequence 61–76 of β-lipotropin (β-LPH),★ The enzyme also catalyzes the hydrolysis of the Leu-Thr bond in the synthetic peptide Tyr-Gly-Gly-Phe-Leu-Thr-2-naphthylamide with the release of leucine-enkephalin and Thr-2-naphthylamide. Neither Met- nor Leu-enkephalin are degraded. The data indicate that the presence of a free N-terminal group of tyrosine inhibits the further degradation of Leu- and Met-enkephalin by the endopeptidase. It is suggested that cation-sensitive neutral endopeptidase is one of the enzymes capable of generating Met- and Leu-enkephalin in, vivo.