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

Y. Moriyama, N. Nelson

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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.

Original languageEnglish
Pages (from-to)8521-8527
Number of pages7
JournalJournal of Biological Chemistry
Issue number17
StatePublished - 1988
Externally publishedYes


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