Identification and characterization of a Mg2+-dependent and an independent Ca+2-ATPase in microsomal membranes of rat testis.
Rat testicular microsomal membrane fraction contains both Mg+2-dependent and Mg+2-independent Ca+2-ATPase activity. The latter activity is about two times higher than the former. Calcium ion required for maximum activation of Mg+2-independent Ca+2-ATPase in 3.0 mM, whereas for the dependent one it is 2.5 mM. Both the enzymes are resistant to cold shock upto seven days. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities, respectively. The pH optima for dependent one is 7.5, whereas for the independent one it is 8.5. Temperature optima for the former is 37 degrees C and for latter one it is 40 degrees C. Among all the nucleotides tested, ATP is found to be the best substrate for both the enzymes. The optimum concentration of ATP for dependent and independent enzyme activities are 3.0 mM and 1.5 mM respectively. Divalent metal ions like Zn+2, Ba+2 and Mn+2 have been found to inhibit Mg+2-dependent Ca+2-ATPase activity whereas Mg+2-independent Ca+2-ATPase activity is inhibited by the divalent ions except zinc which is found to stimulate the enzyme activity. Both the enzymes are inhibited by vanadate, EDTA and EGTA. I50, for vanadate is 0.05 and 0.125 mM for dependent and independent activities, respectively. Sulfhydryl groups modifying agents e.g., NEM, DTNB and chlorpromazine are found to affect the enzyme activities in different ways. Thus NEM and chlorpromazine are found to inhibit and DTNB stimulate the enzyme activities in both the cases.
NagDas
S K
SK
Department of Chemistry, Bose Institute, Calcutta, India.
Mukherjee
S
S
Mazumder
B
B
Sen
P C
PC
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008274:Magnesium / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D051381:Rats; D013379:Substrate Specificity; D013696:Temperature; D013737:Testis / Q000201:enzymology* / Q000378:metabolism
D002413:Cations, Divalent; D007202:Indicators and Reagents; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002122:Calcium Chloride