Solubilization and Separation of Ethacrynic Acid (EA) Highly Sensitive and EA Less Sensitive Mg2+-ATPases in the Rat Brain

[摘要] References(20)Cited-By(12)Rat brain microsomal Mgt2+-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-β-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 anionexchanger (Mono Q) column with a linear NaCI 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.

[学科分类] 药理学