Biochemical characterization of a calcium ion stimulated-ATPase from goat spermatozoa

Abstract

The goat spermatozoa membranes isolated after treatment with octa (ethylene glycol) mono n-dodecyl ether (C12E8) followed by discontinuous sucrose density gradient centrifugation have been found to contain an ATPase that is stimulated by externally added Ca2+ only. The membrane fraction has also found to contain Mg(2+)-dependent Ca(2+)-ATPase activity, however the former activity is about 2 fold higher than the latter. The molecular weight of the enzyme is found to be about 97,000 on SDS-polyacrylamide gel. The optimum concentration of Ca2+ required for maximum activity is 3 mM for both Mg(2+)-dependent and Mg(2+)-independent Ca(2+)-ATPase. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities respectively. ATP with an optimum concentration of 4 mM is observed to be the best substrate than any other nucleotides. The inhibitors like trifluoperazine and vanadate and group specific probes e.g. DTNB and TNBS inhibit these two enzymes but at different rates. Ca(2+)-uptake study shows that the uptake in the presence of Ca2+ and ATP is higher than in the presence of Mg2+, Ca2+ and ATP. The findings lead us to believe that the Mg(2+)-independent Ca(2+)-ATPase has some role in Ca2+ transport like Mg(2+)-dependent enzyme.

Expression and localization of PMCA4 in rat testis and epididymis

“…Both, Mg 2+ -dependent Ca 2+ -ATPase-and Mg 2+ -independent Ca 2+ -ATPase activity have also been shown in goat (Sengupta et al 2007;Sikdar et al 1991) and bull spermatozoa (Sanchez-Luengo et al 2004;Triphan et al, 2007) as well as in microsomal membranes of rat testis (NagDas et al 1988). Sen and co workers (NagDas et al 1988;Sikdar et al 1991) showed similar results with a Mg 2+ -independent activity, higher than Mg 2+ -dependent activity in both goat spermatozoa and rat testis microsomal membranes. Whether or not Mg 2+ -independent-and Mg 2+ -dependent Ca 2+ -ATPase are two diVerent enzymes or identical molecules having the same or diVerent catalytic sites has not yet been elucidated.…”

“…Microsomal membranes of rat sperm were prepared according to Sikdar et al (1991). Epididymal sperm of 30 rats were pooled for one preparation of microsomal membranes.…”

“…Mg 2+ -dependent-and Mg 2+ -independent Ca 2+ -ATPase activity assays were performed using microsomal membranes of sperm from the caput or cauda epididymidis according to Sikdar et al (1991). For assay of Mg 2+ -dependent activity, 5 g sperm protein and 3 mM ATP were added to the reaction buVer containing 54.5 mM histidine hydrochloride, 2 mM EGTA, 0.2 mM DTE, 50 mM sucrose, 1 mM MgCl 2 , 4 mM CaCl 2 in a total volume of 250 l. After 30 min at 37°C, the reaction was stopped by adding 70 l of 30% ice-cold trichloric acid (TCA) and the sample was subsequently diluted with 550 l of distilled water.…”

Arrangement of PMCA4 in bovine sperm membrane fractions

“…Microsomal membranes of caput sperm, cauda sperm and capacitated cauda sperm were prepared as described previously (Sikdar et al. , 1991).…”

“…Mg 2+ ‐dependent Ca 2+ ‐ATPase was measured using activity assays as described before using 5 μg sperm membrane fractions as sample (Sikdar et al. , 1991).…”