Partial purification and characterization of an anion-activated ATPase from radish microsomes.
Previous investigation showed two distinct ATP-dependent proton-transporting systems in microsomal vesicle from radish seedlings, one inhibited by vanadate and one inhibited by NO-3. On the bases of the effects of these inhibitors we could discriminate two distinct ATPase activities in the same material. The NO-3 sensitive activity was separated from the vanadate-sensitive activity and partially purified by a single-step chromatographic method, which lead to approx 35-fold purification from the microsomes and to a specific activity of 2.3 mumol Pi X min-1 X mg protein-1, at 30 degrees C. The partially purified activity was specific for ATP, some activity being observed toward GTP, and even less toward CTP, UTP and ITP. No significant Pi hydrolysis was found with ADP, AMP, p-nitrophenylphosphate and glucose 6-phosphate. ADP but not AMP was inhibiting in the presence of ATP. The activity was dependent on divalent cations in the order of preference: Mg2+ greater than Mn2+ greater than Co2+ greater than Ca2+ greater than Zn2+. The activity was unaffected by monovalent cations, strongly activated by Cl-, inhibited by 90% by 50 mM NO-3, virtually unaffected by oligomycin and NaN3. At least 90% of the activity was abolished in the presence of each: 10 microM N,N'-dicyclohexylcarbodiimide, 10 microM erythrosin B, 10 mu mersalyl, 100 microM trimethyltin, 100 microM diethylstilbestrol, 100 microM N-ethylmaleimide. No inhibition has been found in the presence of Ca2+, at a concentration blocking the vanadate-sensitive activity. Nigericin, gramicidin and carbonylcyanide p-trifluoromethoxyphenylhydrazone stimulated the activity of this preparation after it was incubated in the presence of sonicated phospholipids, suggesting the capacity of the ATPase to function as a H+-transporting system. All characteristics mentioned were closely similar to those described in the vacuolar ATPases.
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D000838:Anions / Q000494:pharmacology; D002412:Cations / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology*; D009566:Nitrates / Q000494:pharmacology; D010944:Plants / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D012995:Solubility; D013379:Substrate Specificity; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D000838:Anions; D002412:Cations; D009566:Nitrates; D014639:Vanadium; D014638:Vanadates; D006180:Proton-Translocating ATPases; D008274:Magnesium