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.
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, Ki = 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 Km 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 Km values were 62 microM and 76 microM, respectively. Hydrolysis of either substrate was chloride-dependent and both enzymes were strongly inhibited by captopril.
D000803:Angiotensin I / Q000378:metabolism; D002216:Captopril / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D004151:Dipeptides / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D007703:Peptidyl-Dipeptidase A / Q000302:isolation & purification*
D002712:Chlorides; D004151:Dipeptides; C049883:N-(1-carboxy-5-aminopentyl)glycylglycine; D000803:Angiotensin I; D002216:Captopril; D007703:Peptidyl-Dipeptidase A