The reactivity of the thiol groups of the adenosine triphosphatase of sarcoplasmic reticulum and their location on tryptic fragments of the molecule

Abstract

The ATPase from sarcoplasmic reticulum contains 20 thiol groups (115,000 daltons, measured by using N-ethyl[14C]maleimide or 5,5''-dithiobis-(2-nitrobenzoate) in sodium dodecyl sulfate. After reduction there were 26 thiol groups in good agreement with 26.5 residues of cysteic acid found by amino acid analysis. The difference between this and the 20 residues measured before reduction implies the presence of 3 disulfide residues. The same number of disulfide residues was found by direct measurement. Three-6 fewer thiol groups were found in preparations made in the absence of dithiothreitol. Missing residues were accounted for as cysteic acid. Distribution of disulfide bonds and of exposed and buried thiol groups among tryptic fragments of the molecule was measured after labeling with N-ethyl[14C]-maleimide. Disulfides were confined to fragment B (MW 55,000), whereas several thiol groups were present on each of the fragments (A, B, A1 and A2). Kinetics of the reaction of the ATPase with 5,5''-dithiobis-(2-nitrobenzoate) showed that 4 or 5 of the thiol groups were unreactive in the absence of detergent, and 13 of the remainder reacted with a single 1st-order rate constant. In the presence of ATP and Ca2+ the reaction rate of all but 2 groups of this class was uniformly decreased. In the presence or absence of ATP and Ca2+ the rate constant for inactivation was close to the rate constant for this class, but was not identical with it. No selective protection of a specific active-site-thiol group was observed. Parallel experiments with sarcoplasmic reticulum gave similar results, except that reaction rates were a little lower and there were 2 more buried groups. Solution of ATPase of sarcoplasmic reticulum in detergent greatly increased the reactivity of all thiol groups. Effects of low concentrations of deoxycholate were reversible. EGTA or low concentrations (0.02 mM) of Ca2+ or Mg2+ had very little effect on reactivity.