Rabbit pulmonary angiotensin-converting enzyme: The NH 2-terminal fragment with enzymatic activity and its formation from the native enzyme by NH 4OH treatment

Abstract

The NH 2-terminal sequence of 22 residues of rabbit lung angiotensin-converting enzyme has been determined as (NH 2)ThrLeuAspProGlyLeuLeuProGlyAsp PheAlaAlaAspAsnAlaGlyAlaArgLeuPheAla. In the course of purification of the enzyme for structural analysis a protein of M r = 82,000 with angiotensin-converting activity was separated from the major fraction containing the native enzyme ( M r = 140,000). This low-molecular-weight enzyme catalyzed the hydrolysis of the synthetic substrate HipHisLeu at a rate 23% of that with the native enzyme, and exhibited a similar K m value as well as behaviors towards various effectors of angiotensin-converting enzyme. Edman degradation of both the native and the 82K enzymes revealed that they contain identical amino acid sequences from the NH 2-termini. This result and those of peptide mapping and carbohydrate and amino acid analyses indicate that the 82K enzyme is a fragment derived from the NH 2-terminal portion of the native enzyme, and hence contains its catalytic site. Evidence has been obtained indicating that the active fragment was formed from the native enzyme during its elution from the antibodyaffinity column with NH 4OH: on treatment of the native enzyme (140K M r ) with l n NH 4OH at room temperature, a cleavage occurred and two proteins with M r = 82 K and M r = 62 K were obtained. The 82K M r fragment was found to be enzymatically active and to contain the same NH 2-terminal sequence as the native enzyme. The other fragment (62K M r ) was devoid of the activity and was shown to derive from the COOH-terminal portion of the native enzyme by the peptide mapping and terminal analyses. Cleavage of a peptide bond with NH 4OH is unusual and appears to be specific for the native angiotensin-converting enzyme from rabbit lung.