Partial purification of active delta and epsilon subunits of the membrane ATPase from escherichia coli.
We have partially purified active delta and epsilon subunits of the E. coli membrane-bound Mg2+-ATPase (ECF1). Treating purified ECF1 with 50% pyridine precipitates the major subunits (alpha, beta, and gamma) of the enzyme, but the two minor subunits (delta and epsilon), which are present in relatively small amounts, remain in solution. The delta and epsilon subunits were then resolved from one another by anion exchange chromatography. The partially purified epsilon strongly inhibits the hydrolytic activity of ECF1. The epsilon fraction inhibits both the highly purified five-subunit ATPase and the enzyme deficient in the delta subunit. The latter result indicates that the delta subunit is not required for inhibition by epsilon. By contrast, two-subunit enzyme, consisting chiefly of the alpha and beta subunits, was insensitive to the ATPase inhibitor, suggesting that the gamma subunit may be required for inhibition by epsilon. The partially purified delta subunit restored the capacity of ATPase deficient in delta to recombine with ATPase-depleted membranes and to reconstitute ATP-dependent transhydrogenase. Previously we reported (Biochem, Biophys. Res. Commun. 62:764 [1975]) that a fraction containing both the delta and epsilon subunits of ECF1 restored the capacity of ATPase missing delta to recombine with depleted membranes and to function as a coupling factor in oxidative phosphorylation and for the energized transhydrogenase. These reconstitution experiments using isolated subunits provide rather substantial evidence that the delta subunit is essential for attaching the ATPase to the membrane and that the epsilon subunit has a regulatory function as an inhibitor of the ATPase activity of ECF1.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010086:Oxidative Phosphorylation Coupling Factors / Q000032:analysis* / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D010086:Oxidative Phosphorylation Coupling Factors; D000255:Adenosine Triphosphate; D009247:NADH, NADPH Oxidoreductases; D014357:Trypsin; D000251:Adenosine Triphosphatases