A multicatalytic high-molecular-weight neutral endopeptidase from human kidney

Abstract

A multicatalytic endopeptidase (ME) with three distinct activities, chymotrypsin-like, cucumisin-like, and trypsin-like, occurred in all rat tissues examined with highest activities in kidney, testes, liver, and spleen; they were assayed with benzyloxycarbonyl-Gly-Gly-Leu-p-nitroanilide (Z-Gly-Gly-Leu-pNA), benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide (Z-Leu-Leu-Glu-2NA), and benzyloxycarbonyl-Gly-Gly-Arg-2-naphthylamide (Z-Gly-Gly-Arg-2NA), respectively. All three activities were recovered from a single protein band on a polyacrylamide gel after electrophoresis of purified human kidney ME. The native enzyme had a Mr of 650,000, and it consisted of about 5,135 amino acid residues. After denaturation and electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels kidney ME dissociated into several low Mr components ranging from 23,000 to 33,000. Kidney ME had a pH optimum of 7.6-8.1 with Z-Gly-Gly-Leu-pNA, 7.3 with Z-Leu-Leu-Glu-2NA, and 9.8 with Z-Gly-Gly-Arg-2NA. SDS enhanced chymotrypsin- and cucumisin-like activities by two to three times whereas trypsin-like activity was not enhanced. The specificity constant (kappa cat/Km) of human kidney ME for Z-Gly-Gly-Leu-pNA was 6.7 X 10(3) M-1 S-1; Z-Gly-Gly-Leu-2NA was not hydrolyzed. The specificity constant for Z-Leu-Leu-Glu-2NA was similar to, and for Z-Gly-Gly-Arg-2NA was one half of that for Z-Gly-Gly-Leu-pNA. ME cleaves only the Phe5-Ser6 bond of bradykinin (BK); however, all three ME activities were inhibited by BK. Strong inhibition of ME by albumin suggests that ME is involved in cleavage of larger polypeptides. Antipain and leupeptin almost completely inactivated the trypsin-like activity whereas they had no significant effect on the other two activities. ME is not a metal-loenzyme nor is the serine residue essential for its activities; however, thiol groups are involved. Na+ and K+ inhibited all ME activities. Trypsin-like activity was more sensitive to divalent cations than the other two.