Purification and characterization of a peptidyl dipeptidase resembling angiotensin converting enzyme from the electric organ of Torpedo marmorata.
The electric organ of Torpedo marmorata contains a membrane-bound, captopril-sensitive metallopeptidase that resembles mammalian angiotensin converting enzyme (peptidyl dipeptidase A; EC 3.4.15.1). The Torpedo enzyme has now been purified to apparent homogeneity from electric organ by a procedure involving affinity chromatography using the selective inhibitor lisinopril immobilised to Sepharose via a 28-A spacer arm. The purified protein, like the mammalian enzyme, acted as a peptidyl dipeptidase in cleaving dipeptides from the C-terminus of a variety of peptide substrates, including angiotensin I, bradykinin, [Met5]enkephalin, [Leu5]enkephalin, and the model substrate hippuryl (benzoylglycyl; BzGly)-His-Leu. The hydrolysis of BzGly-His-Leu was activated by Cl-. Enzyme activity was inhibited by classical angiotensin converting enzyme inhibitors, including captopril, enalaprilat (MK422), and lisinopril (MK521). Torpedo angiotensin converting enzyme, like its mammalian counterpart, was also able to act as an endopeptidase in hydrolysing the amidated neuropeptide substance P. Hydrolysis of substance P occurred primarily at the Phe8-Gly9 bond with release of the C-terminal tripeptide, Gly-Leu-MetNH2, and this hydrolysis was blocked by selective inhibitors. The Torpedo enzyme was recognised by a polyclonal antibody to pig kidney angiotensin converting enzyme on immunoelectrophoretic (Western) blot analysis. Thus, on the basis of substrate specificity, inhibitor sensitivity, and immunological criteria, the Torpedo enzyme closely resembles mammalian angiotensin converting enzyme. However, the Torpedo enzyme appears somewhat larger (Mr = 190,000) than the pig kidney enzyme (Mr = 180,000) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The endogenous peptide substrate(s) for Torpedo electric organ angiotensin converting enzyme and the physiological role of the enzyme in this tissue remain to be evaluated.
D000806:Angiotensin-Converting Enzyme Inhibitors; D000818:Animals; D002712:Chlorides / Q000494:pharmacology; D002846:Chromatography, Affinity; D004557:Electric Organ / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007122:Immunoelectrophoresis; D007668:Kidney / Q000201:enzymology; D009479:Neuropeptides / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000302:isolation & purification*; D013379:Substrate Specificity; D013552:Swine; D014101:Torpedo / Q000378:metabolism*
D000806:Angiotensin-Converting Enzyme Inhibitors; D002712:Chlorides; D009479:Neuropeptides; D007703:Peptidyl-Dipeptidase A