Conformational responses of the tryptic cleavage products of the Ca2+-ATPase of sarcoplasmic reticulum.

Abstract

Trypsin cleaves the Ca2+-ATPase of sarcoplasmic reticulum into two major fragments (A and B), followed by subsequent cleavage into smaller peptides. Although the ATP-dependent Ca2+ transport is still observed after cleavage of the ATPase into the A and B fragments, the Ca2+ transport energized by acetyl phosphate is strongly inhibited. Covalent labeling of the Ca2+-ATPase by fluorescein 5'-isothiocyanate inhibited both the ATP and acetyl phosphate-dependent Ca2+ transport. Vanadate protected the A and B fragments from further hydrolysis and preserved the ability of the cleaved Ca2+-ATPase to form crystals and to show the characteristic conformational changes in response to Ca2+ and EGTA that are observed with the intact enzyme. The protective effect of vanadate may be useful for the isolation of the A and B fragments in functional form.