Solubilization and separation of ethacrynic acid (EA) highly sensitive and EA less sensitive Mg2+-ATPases in the rat brain.
Rat brain microsomal Mg2+-ATPases with two distinct activities: ethacrynic acid (EA) highly sensitive and EA less sensitive Mg2+-ATPase activities were solubilized by the combined treatment with 10 mM 3-(3-chlolamidopropyl)-dimethylammonio-1-propane-sulfate (CHAPS) and 30 mM octyl-beta-D-glucoside. The solubilized enzymes had properties similar to those of the membrane-bound enzyme in microsomes with respect to the sensitivity to EA and Cl-, although the optimal pH and the affinity to ATP were slightly altered after the solubilization. Fast protein liquid chromatography of the solubilized enzymes on an anion-exchanger (Mono Q) column with a linear NaCl gradient (0-1.0 M) yielded separate peaks for EA highly sensitive and EA less sensitive Mg2+-ATPase activities at 0.1 and 0.35 M NaCl, respectively. Polyacrylamide gradient gel electrophoresis of the samples from the peak-fractions of EA highly sensitive and EA less sensitive Mg2+-ATPase activities yielded prominent bands at 600 and 70 kDa, respectively. These results indicate that EA highly sensitive Mg2+-ATPase is solubilized and separated from EA less sensitive Mg2+-ATPase as a large enzyme molecule with anion-sensitive sites.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004976:Ethacrynic Acid / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains
D003902:Detergents; D017301:Ca(2+) Mg(2+)-ATPase; D004976:Ethacrynic Acid