Biochemical characterization of a calcium ion stimulated-ATPase from goat spermatozoa.
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.
Sikdar
R
R
Department of Chemistry, Bose Institute, Calcutta, India.
Ganguly
U
U
Pal
P
P
Mazumder
B
B
Sen
P C
PC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005591:Chemical Fractionation; D003902:Detergents / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006041:Goats; D007124:Immunoenzyme Techniques; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D013094:Spermatozoa / Q000201:enzymology*; D013379:Substrate Specificity; D014268:Trifluoperazine / Q000494:pharmacology; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D003902:Detergents; D014268:Trifluoperazine; D014638:Vanadates; D000255:Adenosine Triphosphate; D014302:Trinitrobenzenesulfonic Acid; D004228:Dithionitrobenzoic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases