Rapid affinity chromatographic purification of human lung and kidney angiotensin-converting enzyme with the novel N-carboxyalkyl dipeptide inhibitor N-[1( S)-carboxy-5-aminopentyl]glycylglycine

Abstract

Human angiotensin-converting enzyme has been purified, in a single chromatographic step, using a novel N-carboxyalkyl dipeptide CA-GlyGly ( N-[1( S)-carboxy-5-aminopentyl]glycylglycine) synthesised in our laboratory. CA-GlyGly is a weak competitive inhibitor, K i=0.18 mM, and its inhibitory profile is markedly pH-dependent. Human lung and kidney angiotensin-converting enzyme were solubilised with Triton X-100 and after ammonium sulphate fractionation the crude extract was applied to a column containing CA-GlyGly coupled to agarose via a 2.8 nm spacer group. Electrophoretically pure human angiotensin-converting enzyme could be eluted by raising the pH of the chromatography buffer from 7.50 to 9.50. The specific activity of human angiotensin-converting enzyme purified from lung was 104 units/mg, while that from kidney was 88 units/mg. Molecular weight for both enzymes was estimated to be 160 000. The K m with respect to hippuryl- l-histidyl- l-leucine was 1.9 mM in the case of lung angiotensin-converting enzyme and 1.7 mM in that of kidney angiotensin-converting enzyme, while for the substrate angiotensin I K m values were 62 μM and 76 μM, respectively. Hydrolysis of either substrate was chloride-dependent and both enzymes were strongly inhibited by captopril.