Physiological and enzymatic properties of the ram epididymal soluble form of germinal angiotensin I-converting enzyme.
The 94-kDa ram epididymal fluid form of the sperm membrane-derived germinal angiotensin I-converting enzyme (ACE) was purified by chromatography, and some of its enzymatic properties were studied. For the artificial substrate furanacryloyl-L-phenylalanylglycylglycine (FAPGG), the enzyme exhibited a Michaelis constant (K(m)) of 0.18 mM and a V(max) of 34 micromoles/(min x mg) and for hippuryl-L-histidyl-L-leucine a K(m) of 2.65 mM and a V(max) of 163 micromoles/(min x mg) under the defined standard conditions (300 mM NaCl and 50 mM Tris; pH 7.5 and 8.3, respectively). The FAPGG hydrolysis was decreased by 82.5% and 67.5% by EDTA and dithioerythritol, respectively, and was totally inhibited by specific ACE inhibitors such as captopril, P-Glu-Trp-Pro-Arg-Pro-Glu-Ile-Pro-Pro, and lisinopril. Optimum activity for FAPGG was with pH 6.0, 50 mM chloride, and 500 microM zinc. Under the various conditions tested, bradykinin, angiotensin (Ang) I, Ang II, and LHRH were competitors for FAPGG. Bradykinin and angiotensin I were the best competitors. The enzyme cleaved Ang I into Ang II, and the optimal conditions were with pH 7.5 and 300 mM chloride. The relationship between the carboxypeptidase activity in seminal plasma and the prediction of fertility of young rams was also studied. These results indicated a correlation between sperm concentration and ACE activity in semen but showed no statistically significant correlation between such activity and fertility of the animal. Finally, we tested the role of ACE in fertilization; no difference in the in vitro fertilization rate was observed in the presence of 10(-4) M captopril.
Métayer
S
S
UMR 6073 INRA-CNRS, Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, 37380 Monnaie, France.
Dacheux
F
F
Guérin
Y
Y
Dacheux
J L
JL
Gatti
J L
JL
D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D002216:Captopril / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D004226:Dithioerythritol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004822:Epididymis / Q000201:enzymology*; D005307:Fertilization in Vitro / Q000662:veterinary; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D009842:Oligopeptides / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism* / Q000502:physiology; D012661:Semen / Q000201:enzymology; D012756:Sheep; D012995:Solubility; D013076:Sperm Count; D013094:Spermatozoa / Q000648:ultrastructure
D000806:Angiotensin-Converting Enzyme Inhibitors; D009842:Oligopeptides; C038275:2-furanacryloyl-phenylalanyl-glycyl-glycine; D004226:Dithioerythritol; D004492:Edetic Acid; D002216:Captopril; D007703:Peptidyl-Dipeptidase A