title: A comparison of the zinc contents and substrate specificities of the endothelial and testicular forms of porcine angiotensin converting enzyme and the preparation of isoenzyme-specific antisera.
abstract: Angiotensin converting enzyme (ACE; EC 3.4.15.1) was purified from porcine kidney and lung (endothelial isoenzyme) and testis (testicular isoenzyme) by affinity chromatography on lisinopril-2.8 nm-Sepharose. Atomic-absorption spectroscopy revealed that ACE purified from kidney and lung contained 2.58 and 2.35 atoms of zinc per molecule of enzyme (M(r) 147,000) respectively. In contrast, ACE purified from testis contained only 1.58 atoms of zinc per molecule of enzyme (M(r) 80,000). Thus it would appear that both putative zinc-binding sites in endothelial ACE contain zinc and may therefore be catalytically active. No differences were observed in the pattern of products generated on hydrolysis of benzoyl (Bz)-Gly-His-Leu, substance P, luteinizing-hormone-releasing hormone (LH-RH) and its analogue, des-Gly10-LH-RH-ethylamide, by kidney and testicular ACE. There was also no difference in the initial rates of hydrolysis of Bz-Gly-His-Leu or substance P by the two isoenzymes, although LH-RH and its analogue were hydrolysed twice as rapidly by kidney ACE. It is therefore unlikely that the N-terminal catalytic site in porcine endothelial ACE is predominantly responsible for the atypical cleavage of LH-RH generating the N-terminal tripeptide. Two polyclonal antisera were raised to the affinity-purified forms of pig kidney and testicular ACE. Isoenzyme-specific antisera were then isolated from these by absorbing out those antibodies recognizing determinants on the other isoenzyme. Immunoelectrophoretic blot analyses and immunofluorescent staining of sections of pig kidney were used to demonstrate the specificity of the antisera. Immunofluorescent staining of sections of pig testis with the antiserum specific to testicular ACE localized testicular ACE solely to the lumen of the seminiferous tubules, whereas the antiserum specific to endothelial ACE revealed the presence of this isoenzyme only in blood vessels. The antiserum to endothelial ACE, which recognizes determinants in the unique N-terminal domain, was investigated as a possible specific inhibitor of the N-terminal catalytic site. Although this antiserum failed to inhibit testicular ACE, the effect on the activity of endothelial ACE appeared to be due to inhibition of both the N- and C-terminal catalytic sites.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
authors.forename: T A
authors.initials: TA
authors.lastname: Williams
authors.forename: K
authors.initials: K
authors.lastname: Barnes
authors.forename: A J
authors.initials: AJ
authors.lastname: Kenny
authors.forename: A J
authors.initials: AJ
authors.lastname: Turner
authors.forename: N M
authors.initials: NM
authors.lastname: Hooper
mesh_terms: D000595:Amino Acid Sequence; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D000906:Antibodies; D000917:Antibody Formation; D000918:Antibody Specificity; D001665:Binding Sites; D003429:Cross Reactions; D004727:Endothelium / Q000201:enzymology; D004800:Enzymes, Immobilized / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006868:Hydrolysis; D007106:Immune Sera / Q000276:immunology* / Q000494:pharmacology; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000032:analysis* / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity; D013552:Swine; D013737:Testis / Q000201:enzymology*; D015032:Zinc / Q000032:analysis*
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D000906:Antibodies; D004800:Enzymes, Immobilized; D007106:Immune Sera; D007527:Isoenzymes; D010455:Peptides; D007703:Peptidyl-Dipeptidase A; D015032:Zinc