Characterization of angiotensin-converting enzyme in canine testis.

ABSTRACT

The aim of this study was to characterize angiotensin-converting enzyme (ACE) in canine testis. Detergent-extracted canine testes were sonicated in the presence of protease inhibitors and purified on an affinity column with the ACE inhibitor, lisinopril, as an affinity ligand for ACE. The fractions recovered were assessed for ACE enzyme activity via an enzyme kinetic microplate assay (at 330 nm) based on the hydrolysis of Fa-Phe-Gly-Gly (FAPGG) at pH 7.5 during an 8 min incubation. The specific activity of ACE in the starting testicular extracts was 3.53 +/- 0.99 mU mg(-1) protein with a 1588 times enrichment in ACE activity after lisinopril affinity chromatography (4239 +/- 2600 mU mg(-1) protein). The recovery efficiency of ACE after lisinopril affinity chromatography was 71.2%. The ACE activity in the detergent extracts and the purified fractions was inhibited significantly by 10 micromol captopril l(-1), a specific ACE inhibitor, and was restored to 88% of normal activity by the addition of the thiol-alkylating agent N-ethylmaleimide (0.5 mmol l(-1)) in the detergent extracts and the purified fractions incubated with captopril. The treatment of testicular extracts with 10 mmol EDTA l(-1) reduced the ACE activity significantly (5.40 +/- 1.26 versus 0.58 +/- 0.23 mU mg(-1)). The ACE activity was restored fully in the presence of zinc (5.28 +/- 0.70 mU mg(-1)). The anti-ACE antibody (raised against a 70 kDa protein from the periacrosomal plasma membrane of equine spermatozoa) recognized a 65-70 kDa protein in the detergent-extracted testes as well as in the affinity-purified fractions. This antibody also recognized a protein of similar molecular mass in ejaculated spermatozoa. ACE was localized in the periacrosomal area of the ejaculated spermatozoa and in spermatids in the seminiferous tubules. The results of this study demonstrate that ACE 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 EDTA and is restored in the presence of N-ethylmaleimide and zinc.