Localization of ionophore activity in a 20,000-dalton fragment of the adenosine triphosphatase of Sarcoplasmic reticulum.
The (Ca2+ + Mg2+)-dependent ATPase of sarcoplasmic reticulum has been shown to ast as a Ca2+-dependent and selective ionophore in artificial lipid bilayers. Four fragments of 55,000, 45,000, 30,000, and 20,000 daltons have been purified from tryptic digests of the enzyme and it has been shown that the 55,000- and 45,000-dalton fragments are obtained from a single cleavage of the 100,000-dalton ATPase, while the 30,000- and 20,000-dalton fragments are obtained subsequently by a cleavage of the 55,000-dalton fragment. The 55,000- and 20,000-dalton fragments have ionophore activity inhibited by ruthenium red and by mercuric chloride but not by methylmercuric chloride, an inhibitor of the hydrolytic site of the enzyme. Under standard conditions the 45,000-dalton fragment was not active as an ionophore, while the 30,000-dalton fragment acted as a nonselective ionophore. The 55,000- and 30,000-dalton fragments have been shown to contain the site of phosphorylation and of N-ethyl [2-3H]-maleimide binding indicative of the hydrolytic site in the enzyme, and this site is absent from the 20,000-dalton fragment. Therefore, the ionophoric and hydrolytic sites are localized in separate regions of the ATPase molecule and they have now been physically separated. The 20,000-dalton fragment was degraded with cyanogen bromide and fragments were separated by molecular sieving. Ionophore activity was found in fragments of molecular mass less than 2,000 daltons.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004553:Electric Conductivity; D004789:Enzyme Activation / Q000187:drug effects; D007476:Ionophores / Q000378:metabolism*; D007700:Kinetics; D008055:Lipids; D008274:Magnesium / Q000494:pharmacology; D008567:Membranes, Artificial; D008954:Models, Biological; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012430:Ruthenium Red / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D000596:Amino Acids; D007476:Ionophores; D008055:Lipids; D008567:Membranes, Artificial; D012430:Ruthenium Red; D012967:Sodium Dodecyl Sulfate; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium