Purification and properties of a vanadate- and N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes

A vanadate- and N-ethylmaleimide-sensitive ATPase was purified about 500-fold from chromaffin granule membranes. The purified preparation contained a single major polypeptide with an apparent molecular mass of about 115 kDa, which was copurified with the ATPase activity. Immunological studies revealed that this polypeptide has no relation to subunit 1 (115 kDa) of the H⁺-ATPase from chromaffin granules. The ATPase activity of the enzyme is inhibited about 50% by 100 μM N-ethylmaleimide or 5 μM vanadate. The enzyme is not sensitive to dicyclohexylcarbodiimide, ouabain, SCH28080, and omeprazole, which distinguishes it from Na⁺/K⁺-ATPase and the gastric K⁺/H⁺-ATPase. ATP and 2-deoxy ATP are equally effective substrates for the enzyme. However, the enzyme exhibited only 10% activity with GTP as a substrate. UV illumination of the purified enzyme in the presence of [α-³²P]ATP exclusively labeled the 115 kDa protein. This labeling was increased by Mg²⁺ and strongly inhibited by Ca²⁺ ions. Similarly, the ATPase activity was dependent on Mg²⁺ and inhibited by the presence of Ca²⁺ions. The ATPase activity of the enzyme was largely insensitive to monovalent anions and cations, except for F^-, which inhibited the vanadate sensitive ATPase. Incubation of the enzyme in the presence of [¹⁴C]N-ethylmaleimide labeled the 115-kDa polypeptide, and this labeling could be prevented by the addition of ATP during the incubation. A reciprocal experiment showed that preincubation with N-ethylmaleimide inhibited the labeling of the 115-kDa polypeptide by [α-³²]ATP by UV illumination. This suggests a close proximity between the ATP-binding site and an essential sulfhydryl group. A possible connection between the isolated ATPase and organelle movement is discussed.