Submission Number: KHA-4-12-8

Abstract Number: 327

CHARACTERIZATION OF ANGIOTENSIN-CONVERTING ENZYME (ACE) IN CANINE TESTIS.

Khalida Sabeur and Barry A Ball*

Dept. Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA ¹

Abstract:
Objectives: The purpose of this study was to characterize the presence of ACE in canine testis. Materials and Methods: Canine testes were extracted in the presence of protease inhibitors with 1% TX-100 and sonication. The sonicated membrane was centrifuged (45,000xg for 60 min), and the detergent-extracted testicular samples were purified with Lisinopril as an affinity ligand for ACE. Recovered fractions were assessed for ACE enzyme activity via an enzyme kinetic microplate assay (at 330 nm) based upon the hydrolysis of 166-M Fa-Phe-Gly-Gly (FAPGG) at pH 7.5 during an 8-min incubation. The detergent extracts and the purified concentrated fractions were subjected to SDS-PAGE followed by silver staining or western blotting. Blots were incubated with an anti-ACE polyclonal antibody (1:5,000, raised against a 70-kD protein from the periacrosomal plasma membrane of equine sperm). After washing and incubation with an HRP-secondary antibody, the proteins were detected with ECL. Results: Specific activity of ACE in the starting testicular extracts was 1.61 0.4 mU/mg protein with a 3400X enrichment in ACE activity after Lisinopril-affinity chromatography (5.5 2.8 U/mg protein). Recovery efficiency of ACE after Lisinopril-affinity chromatography was 68.8%. The ACE activity in the detergent extracts and the purified fractions was significantly inhibited by 10 M captopril, a specific ACE inhibitor (5.5 2.8 U/mg for the purified fractions vs 0.1 mU/mg in the presence of captopril). The enzyme activity was restored to 85% normal activity by the addition of 0.5 mM N-ethylmaleimide in the detergent extracts and the purified fractions incubated with captopril. The anti-ACE antibody recognized a 70-kD protein in the detergent-extracted testicular tissue as well as in the purified fractions. Conclusions: This study demonstrates that angiotensin-converting enzyme is present in canine testis and retains its enzyme activity after purification with lisinopril-affinity chromatography. Activity of canine ACE is inhibited by captopril and restored in the presence of N-ethylmaleimide. .

Keywords: ACE, testis, lisinopril, canine

This abstract is being presented at: 8:00 AM in session:
Gonadal Function