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-.
Studies were performed to characterize ethacrynic acid (EA) highly sensitive Mg2+-ATPase isolated from microsomal fractions of the rat brain. The functional molecular sizes of the EA highly sensitive and EA less sensitive Mg2+-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 Mg2+-ATPase activity. The type of inhibition was uncompetitive with respect to ATP, and the inhibition was suppressed by anions such as Cl-, Br- and I-. Chloride ions stimulated enzyme activity with an increase in Vmax, but not in Km, for ATP. Anions tested also increased the enzyme activity in the following order of decreasing potency: Cl- greater than Br- greater than CH3COO- = I- greater than SO4(2-) = HCO3- greater than SO3(2-). These results suggest that EA highly sensitive Mg2+-ATPase is a relatively large molecule with anion-sensitive sites that affect the ATP hydrolyzing activity and the SITS binding capacity through anions, with Cl- being the most potent.
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002712:Chlorides / Q000494:pharmacology*; D004976:Ethacrynic Acid / Q000494:pharmacology*; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D051379:Mice; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D013267:Stilbenes / Q000494:pharmacology*
D002712:Chlorides; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D004976:Ethacrynic Acid