Arylisothiocyanate modification of sarcoplasmic Ca2+-stimulated ATPase.

Abstract

The related probes phenylisothiocyanate and p-sulfophenylisothiocyanate possess comparable reactivity with nucleophiles but are dissimilar in their solubility characteristics. The reagents are utilized to topologically characterize the sites of covalent interaction with the Ca2+-stimulated ATPase of sarcoplasmic reticulum membranes. The hydrophobic probe phenylisothiocyanate binds covalently to the membrane-integrated protein. The extent of covalent interaction of this probe is reduced to a limited level of label incorporation by either preincubation with p-sulfophenylisothiocyanate or by exposing the labeled protein to alkaline reductive conditions. With respect to the chemical nature a dual interaction of phenylisothiocyanate is postulated. Phenylisothiocyanate modifies the Ca2+-ATPase hydrophobically. In addition, aqueous-exposed nucleophiles (cysteine thiols) interact with both arylisothiocyanates. Inhibition of the Ca2+-stimulated ATPase activity is effected by either probe.