title: Affinity chromatographic purification of angiotensin converting enzyme.
abstract: The compounds N-[1 (S)-carboxy-5-amino-pentyl]-L-phenylalanylglycine and N-[1 (S)-carboxy-5-aminopentyl]-DL-alanyl-L-proline were synthesized and explored as potential ligands for the affinity chromatography of angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1) (ACE), a membrane-bound zinc metalloprotease. The N-alkylated Ala-Pro derivative has an apparent Ki less than 1 nM (at pH 7.5, 0.50 M NaCl) while the Phe-Gly derivative is a much less potent competitive inhibitor with an apparent Ki = 0.20 microM under the same conditions and thus more suitable for use as an affinity ligand. Immobilization of these compounds via a 28-A spacer to agarose yields resins with binding capacities of greater than 7 mg of enzyme/mL of resin, while spacers of 22 A or less result in binding capacities at least 350 times smaller. Immobilized N-[1 (S)-carboxy-5-amino-pentyl]-L-Phe-Gly is superior to the Ala-Pro derivative because elution can be affected by raising the pH to 8.9 with 98% yields compared with only 20% from the latter. Thus, a three-step process involving detergent extraction, concentration by ammonium sulfate precipitation, and affinity chromatography on the resin-immobilized Phe-Gly derivative provides 30 mg of homogeneous ACE from 640 g of rabbit lung tissue. An ACE-like metalloprotease has also been isolated from testicular tissue by this same technique.
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D005260:Female; D007202:Indicators and Reagents; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D007703:Peptidyl-Dipeptidase A / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D013737:Testis / Q000201:enzymology*
chemical_list: D007202:Indicators and Reagents; D007703:Peptidyl-Dipeptidase A