Purification and properties of a cytosolic V1-ATPase.
The native V1 complex of the tobacco hornworm vacuolar type ATPase (V-ATPase) was purified from cytosolic extracts of molting larval midgut. It consisted of the established V-ATPase subunits A, B, and E along with the 14-kDa subunit F and the novel 13-kDa subunit G. The final amount of purified V1 complex made up an unexpectedly high 2% of the total cytosolic protein, with a yield of approximately 0.4 mg/g of tissue. An equally high amount of cytosolic V1 complex was obtained from starving intermolt larvae. By contrast, the cytosolic V1 pool was reduced drastically in feeding intermolt larvae or in larvae that had been refed after starvation. The activity of the membrane-bound V-ATPase holoenzyme was inversely related to the size of the cytosolic V1 pool, suggesting that the insect plasma membrane V-ATPase is regulated by reversible disassembly of the V1 complex as a function of the feeding condition of the larvae. Like F1-ATPases, the purified V1 complex exhibited Ca2+-dependent ATPase activity and, in the presence of 25% methanol, exhibited Mg2+-dependent ATPase activity. Therefore, we designate the native V1 complex, V1-ATPase. Both enzyme activities were completely inhibited by micromolar N-ethylmaleimide. In contrast to the Ca2+-dependent V1-ATPase activity, the Mg2+/methanol-dependent V1-ATPase activity did not decrease with the incubation time and thus was not inhibited by ADP. Methanol appears to induce a conformational change of the V1 complex, leading to enzymatic properties of the V1-ATPase that are similar to those of the membrane-bound V-ATPase holoenzyme. This is the first time that a native and enzymatically active V1 complex has been purified from the cytosol.
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Zoologisches Institut der Universität München, Luisenstrasse 14, D-80333 München, Germany.
Harvey
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Wieczorek
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D000818:Animals; D002118:Calcium / Q000378:metabolism; D002451:Cell Compartmentation; D003600:Cytosol / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D007425:Intracellular Membranes / Q000201:enzymology; D007814:Larva / Q000201:enzymology; D018402:Manduca / Q000201:enzymology*; D008675:Metamorphosis, Biological; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D025262:Vacuolar Proton-Translocating ATPases
D013439:Sulfhydryl Reagents; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide; D002118:Calcium