Effect of Cl − on the function of the Cl −-activated arginine aminopeptidase purified from human erythrocytes

Abstract

The Cl −-activated arginine aminopeptidase was purified from human erythrocytes using electrofocusing in granulated gel, gel permeation chromatography, and affinity chromatography. The purified enzyme showed a molecular weight of 105,000 ± 3000 and was homogenous according to several criteria. A subunit structure was revealed during sodium lauryl sulfate electrophoresis, the main form being of M r 24,500 ± 1300. The enzyme was considered to be a tetramer consisting of four monomers of equal molecular weight. Cl − affected the hydrolysis of peptides and synthetic substrates differently, the Cl − activation being less marked with peptide substrates. The catalysis obeyed regular Michaelis-Menten kinetics and Cl − affected both the K m and V values. Arg-Phe and bradykinin showed no cooperativity in the hydrolysis of Arg-2-naphthylamide catalyzed by the Cl −-activated arginine aminopeptidase. Cl − affected the enzyme structure reflected by changes in the uv-absorption spectra in the presence and without added Cl −.