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.

Isoforms of angiotensin I-converting enzyme in the development and differentiation of human testis and epididymis

“…The folding of the outer plasma membrane prior to sperm release, furthermore, can explain the occurrence of tACE on residual bodies and the neck and midpiece region of released and ejaculated spermatozoa (Vanha-Perttula et al, 1985;Gatti et al, 1999;Pauls et al, 1999). Again, in the latter, we did not find tACE immunoreactivity in the head or acrosomal region as recently proposed in dogs (Sabeur et al, 2001), except for malformed cells with nuclei entrapped in larger cytoplasmic remnants (FE Franke and FM Köhn, unpublished observations). It is still not clear, however, if tACE maintains this cellular position on spermatozoa (Köhn et al, 1998;Gatti et al, 1999).…”

Purification of angiotensin‐converting enzyme from human plasma and investigation of the effect of some active ingredients isolated from Nigella sativa L. extract on the enzyme activity

“…Two forms of ACE were designated the somatic form with a higher molecular weight (130-180 kDa) present in somatic tissues, inclusive of the epididymal epithelium and prostate; the testicular form was of a smaller size (70-110 kDa), which is described only in germ cells (Bull, Thornberry, & Cordes, 1985;El-Dorry, Bull, Iwata, & Soffer, 1982;Sabeur et al, 2001). The molecular weight of the ACE purified by Lanzillo, Stevens, Dasarathy, Yotsumoto, and Fandburg (1985) from human lung, kidney, blood plasma and seminal plasma was 140 kDa and that from testis consisted of both a 90 and a 140 kDa form in a 4:l ratio.…”

“…The activity-containing tubes were pooled. All of the procedures were performed at 4°C (Pantoliano, Holmquist, & Riordan, 1984;Sabeur, Vo, & Ball, 2001).…”

Characterization of NADPH oxidase 5 in equine testis and spermatozoa

“…Protease inhibitors were added immediately to the Tris buffer: phenylmethylsulphonyl fluoride (500 mM), pepstatin (1 mM), leupeptin (5 mM) and antipain (25 mM). The homogenized sample was passed through cheesecloth and centrifuged twice at 20 000 g for 30 min (Sabeur et al 2001). The resulting pellet was suspended in 6-10 ml Tris buffer with protease inhibitors and triton X-100 was added to a final concentration of 1%.…”