Labeling the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum with maleimidylsalicylic acid

Maleimidylsalicylic acid reacts with the Ca2+-ATPase of skeletal muscle sar coplasmic reticulum with high affinity and inhibits the ATPase activity fol lowing a pseudo-first-order kinetic with a rate constant of 8.3 M-1 s(-1) C alcium binding remains unaffected in the maleimide-inhibited ATPase. Howeve r, the presence of ATP, ADP, and, to a lesser extent, AMP protects the enzy me against inhibition. Furthermore, ATPase inhibition is accompanied by a c oncomitant decrease in ATP binding. The stoichiometry of the nucleotide-dep endent maleimidylsalicylic acid binding is 6-10 nmol/mg ATPase, which corre sponds to the binding of up to one molecule of maleimide/molecule of ATPase . The stoichiometry of maleimide binding is decreased in the presence of nu cleotides and in the ATPase previously labeled with fluorescein-5'-isothioc yanate or N-ethylmaleimide A fluorescent peptide was isolated by high perfo rmance liquid chromatography after trypsin digestion of the maleimide-label ed ATPase. Analysis of the sequence and mass spectrometry of the peptide le ads us to propose Cys(344) as the target for maleimidylsalicylic acid in th e inhibition reaction. The effect of Cys(344) modification on the nucleotid e site is discussed.