Novel activity of human angiotensin I converting enzyme: release of the NH2- and COOH-terminal tripeptides from the luteinizing hormone-releasing hormone.

Abstract

Angiotensin I converting enzyme (ACE; kininase II; peptidyldipeptide hydrolase, EC 3.4.15.1) cleaves COOH-terminal dipeptides from active peptides containing a free COOH terminus. We investigated the hydrolysis of luteinizing hormone-releasing hormone (LH-RH) by homogeneous human ACE. Although this decapeptide is blocked at both the NH2 and COOH termini, it was metabolized to several peptides, which were separated by HPLC and identified by amino acid analysis. A major product was the NH2-terminal tripep-tide, <Glu-His-Trp, and another was LH-RH-(4-10) hep-tapeptide, indicating that the Trp-Ser bond is cleaved to release the NH2-terminal tripeptide. ACE also released the 8 9 10COOH-terminal tripeptide, Arg-Pro-Gly-NH2, and then 6 7 4 5 sequentially the dipeptides Gly-Leu and Ser-Tyr, leaving <Glu-His-Trp intact. Thus, <Glu-His-Trp was formed by both NH2-and COOH-terminal hydrolysis. The cleavage of LH-RH was inhibited by specific ACE inhibitors and by antibody to ACE but not by inhibitors of other enzymes, showing that the hydrolysis was indeed due to ACE. In the absence of chloride, the hydrolysis proceeded at only 16% of the maximal rate (in 500 mM NaCl), but in 10 mM NaCl it increased to 64%. In 500 mM NaCl solution, 86% of the hydrolysis was accounted for by the release of the NH2-terminal tripeptide, whereas in 10 mM NaCl, the COOH-terminal and NH2-terminal cleavage occurred about equally. Enzyme Purification. ACE was solubilized from a membrane fraction of human kidney with trypsin and purified by DEAE-cellulose and hydroxylapatite chromatography, followed by reverse immunoadsorption chromatography (17). The preparation was homogeneous as determined by NaDodSO4/polyacrylamide gel electrophoresis with Coomassie blue staining; however, when the more sensitive silver staining technique (18) was used, minor contaminant bands became evident in the stored preparation. Thus, an additional step of purification was included. Highly purified fractions of ACE were obtained by gel filtration on a 0.75 x 30 cm TSK-G3000SW column (Varian) in an HPLC system previously described (19). The column was run at room temperature with 0.07 M sodium phosphate buffer (pH 7.2) at a flow rate of 0.3 ml/min, and fractions (0.15 ml) were collected.ACE was eluted at 19.7 min, and the first three fractions containing pure enzyme (as determined by NaDodSO4/polyAbbreviations: LH-RH, luteinizing hormone-releasing hormone; ACE, angiotensin I converting enzyme. 1025The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

“…At least in vitro, it can hydrolyze a wide range of substrates (Skidgel et al, 1984;Skidgel and Erdös, 1985;Skidgel and Erdös, 1987). ACE acts as a C-terminal dipeptidase for angiotensin I, bradykinin, and other small peptide hormones, including neurotensin, substance P, enkephalins, N-formyl-Met-Leu-Phe, acetyl Ser-Asp-Lys-Pro (AcSDKP) and angiotensin 1-7.…”

Effects of the N-Terminal Sequence of ACE on the Properties of Its C-Domain

“…The most important substrates, bradykinin (BK) and angiotensin (Ang) I, are hydrolyzed at both sites, 2,5 but some of the others are cleaved preferentially by 1 of the sites, which is frequently on the N-domain. Of peptide hormones, for example, the luteinizing hormone-releasing hormone (LHRH) is inactivated mostly by the release of ϽGlu 1 -His 2 -Trp 3 , 6,7 and the enkephalin precursor Met 5 -Enk-Arg 6 -Phe 7 is converted primarily to enkephalin by the N-domain site. 8,9 Ang 1-7 10 and the tetrapeptide AcSer-Asp-Lys-Pro (AcSDKP) 11,12 are also cleaved by the N-domain.…”

Action of Three Ectopeptidases on Corticotropin-Releasing Factor: Metabolism and Functional Aspects

“…From structure-activity studies (Rivier et al, 1984), it is clear that all the recovered fragment peptides would have reduced CRF activity. Two of the fragments (1-7 and 1-18) (Skidgel and Erdos, 1985). The specificity indices (k cat /K m ) of ACE for CRF and GnRH are 0.011 and 1.3, respectively.…”