Characteristics of Ethacrynic Acid Highly Sensitive Mg2+-ATPase in Microsomal Fractions of the Rat Brain: Functional Molecular Size, Inhibition by SITS and Stimulation by Cl−

Abstract

Abstract-Studieswere performed to characterize ethacrynic acid (EA) highly sensitive Mgt+-ATPase isolated from microsomal fractions of the rat brain. The functional molecular sizes of the EA highly sensitive and EA less sensitive Mgt+ ATPases, estimated by a radiation inactivation method, were 480 and 80 kDa, respectively.An anion transport inhibitor, 4-acetamido-4'-isothiocyanostilbene 2,2'-disulfonic acid (SITS) inhibited the EA highly sensitive Mgt+-ATPase activity. The type of inhibition was uncompetitive with respect to ATP, and the inhibition was suppressed by anions such as CI-, Br and I-. Chloride ions stimulated enzyme activity with an increase in Vma ,X, but not in Km, for ATP. Anions tested also increased the enzyme activity in the following order of decreasing potency: CI-> Br->CH3000-=I->S042-=HC03->S032-.These results suggest that EA highly sensitive Mgt+-ATPase is a relatively large molecule with anion-sensitive sites that affect the ATP hydrolyzing activity and the SITS binding capacity through anions, with CI being the most potent.

Solubilization and separation of ethacrynic acid(EA) highly sensitive and EA less sensitive Mg2+-ATPases in the rat brain.

“…These results suggest that EA highly sensitive Mgt+-ATPase is solubilized with the intact EA sensitive sites. Stimulation by CI was observed only with EA highly sensitive Mgt+-ATPase in the solubilized enzymes, as well as in the microsomes (12,13). The extent of stimulation by CI of the solubilized enzyme was comparable to that of the enzyme in the microsomes.…”

“…Polyacrylamide gel electrophoresis of the peak fraction of EA highly sensitive or EA less sensitive Mgt+-ATPase yielded a con centrated protein band at approximately 600 or 70 kDa, respectively. In our previous study using the radiation inactivation method (13), functional molecular sizes of EA highly sensitive and EA less sensitive Mgt+-ATPases in the microsomes were estimated to be 480 and 80 kDa, respectively.…”

“…We previously showed that brain micro somes contain two types of Mgt+-ATPases activities: ethacrynic acid (EA) highly sensitive and EA less sensitive Mgt+-ATPase activities (10)(11)(12)(13).…”

“…The former activity was mainly located in plasma membrane fractions and was sensitive to anions, especially to Cl (a stimulatory anion) and NO3 (an inhibitory anion), and the latter was insensitive to the anions and rather widely distributed (12,13 In the combined detergent-treatment, CHAPS was added to the suspension of microsomes (10 mg protein/ml) to achieve a final CHAPS concentration of 10 mM, and the mixture was centrifuged at 20,000 x g for 15 min. To the resulting supernatant, OcGlu up to 50 mM was added.…”

Cl Anion-Dependent Mg-ATPase

“…ATPase activity was represented as micromoles of P i per hour, with milligram protein units. Reagent medium always contained 30 mM Tris–Malate (pH 7.65), 0.4 mM EGTA, 0.2 mM ouabain and 0.3 mM ethacrynic acid (the specific inhibitor of Cl-ATPase (Gassnez and Komnick 1981; Tanaka et al 1986). Concentrations of other ligands in incubation solution are given in the text.…”