Purification of N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes.
An N-ethylmaleimide-sensitive ATPase was purified 100-fold from chromaffin granule membranes. The purification procedure included solubilization with polyoxyethylene 9 lauryl ether, chromatography on hydroxylapatite and DEAE-cellulose columns, and glycerol gradient centrifugations. Inclusion of phosphatidylserine and a mixture of protease inhibitors during the purification procedure was necessary to maintain the activity of the preparation. The purified preparation contained four major polypeptides with molecular masses of about 115, 72, 57, and 39 kDa, which were copurified with the ATPase activity. The 115-kDa subunit binds [14C]dicyclohexylcarbodiimide and the subunits of 115 and 39 kDa bind [14C]N-ethylmaleimide. The ATP-dependent proton uptake activity of chromaffin granule membranes is inhibited 50% with about 20 microM N-ethylmaleimide, while over 5 mM concentrations of the inhibitor were required to block the ATPase activity of the membranes. The ATPase activity of the purified enzyme was inhibited via two different affinities: a high affinity site with a Ki in the microM range and a low affinity site in the mM range, each contributing to about 50% inhibition of the enzyme. It is concluded that the proton-ATPase of chromaffin granule membranes contains at least four subunits with the 115-kDa polypeptide being the main subunit having the active site for the ATPase activity of the enzyme.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000311:Adrenal Glands / Q000648:ultrastructure; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D002845:Chromatography; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000201:enzymology; D011522:Protons
D011522:Protons; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases; D005033:Ethylmaleimide