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Volume 270, Number 28, Issue of July 14, pp. 16926-16931, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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Sheng-Bin Peng

Evidence suggests that ATP hydrolysis catalyzed by the clathrin-coated vesicle proton-translocating ATPase requires at least four polypeptides of molecular masses of 70, 58, 40, and 33 kDa (Xie, X.-S., and Stone, D. K.(1988) J. Biol. Chem. 263, 9859-9867). To further investigate the subunit requirements for ATP hydrolysis, histidine-tagged, 58-kDa polypeptide was expressed in insect Sf9 (Spodoptera frugiperda) cells. After purification by Ni-nitrolotriacetic acid chromatography, the 58-kDa protein was found to lack significant ATPase activity. However, the subunit was photoaffinity labeled with [-P]ATP, [C]ADP, or S-labeled ADP and UV irradiation in a divalent cation-dependent manner. The labeling was saturable with an apparent K of 4 µM for both ATP and ADP. ATP and ADP competition labeling experiments indicate that the two nucleotides share the same binding site. When reconstituted with recombinant 70-kDa subunit and a biochemically prepared catalytic sector (V) depleted of the 70- and 58-kDa subunits, the 58-kDa component restores Ca-activated ATP hydrolysis to a specific activity of 0.19 µmol P mg protein min, thus demonstrating that ATP hydrolysis in vacuolar type proton pumps is dependent upon the 58-kDa subunit as well as multi-subunit interactions.

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