Dinitrophenylation of rabbit skeletal sarcoplasmic reticulum ATPase protein.
The ATPase (ATP phosphohydrolase (EC 3.6.1.3)) protein of rabbit skeletal sarcoplasmic reticulum rapidly incorporated three mol of 1-fluoro-2,4-dinitrobenzene per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-dependent ATPase activity was inhibited. The dinitrophenyl group was located mainly in the ATPase protein and a small amount of the label was found in the proteolipid component of the ATPase preparation as judged by dissociation experiments in sodium dodecyl sulfate. Cysteine and tyrosine residues were dinitrophenylated in the modified ATPase protein. Thiolysis of the dinitrophenylated ATPase protein with 2-mercaptoethanol under various conditions did not restore the Ca2+-dependent ATPase activity. Solubilization of the ATPase protein with sodium deoxycholate increased the reactivity of the reagent and the Ca2+-dependent ATPase activity was inhibited to a greater extent. Dinitrophenylation of the ATPase protein was Ca2+-dependent; in the presence of high Ca2+ the incorporation increased by 50% and a large decrease in the Ca2+-ATPase activity was noted. The half-maximal changes for the incorporation of the reagent and the inhibition of the Ca2+-ATPase activity occurred at 3--4 microgram Ca2+-concentration, consistent with the binding of Ca2+ to high affinity sites on the ATPase protein. These results indicate that the ATPase protein as a Ca2+-free and a Ca2+-bound conformation. The reagent, 1-fluoro-2,4-dinitrobenzene reacts differentially and thus characterizes these two conformations.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D004139:Dinitrofluorobenzene; D004140:Dinitrophenols; D008623:Mercaptoethanol; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D004140:Dinitrophenols; D008623:Mercaptoethanol; D004139:Dinitrofluorobenzene; D000251:Adenosine Triphosphatases; D002118:Calcium