Reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole with the (Ca 2+ + Mg 2+) -ATPase protein of sarcoplasmic reticulum at low temperature

Abstract

Modification of the (Ca 2+ + Mg 2+)-ATPase protein of rabbit skeletal sarcoplasmic reticulum (SR) with 7-chloro-4-nitrobenzo-2-oxa 1, 3-diazole, NBD-Cl, at 4°C for 5 min caused a 63% loss of the Ca 2+-dependent ATPase activity when 1 mol of the adenine analog was incorporated per 10 5 g of protein. At 25°C, above the lipid phase transition, the extent of labeling was 3-fold higher although the Ca 2+-ATPase activity was inhibited to the saine extent. MgATP protected the ATPase activity at 4°C and 25°C but there was little change in the extent of labeling at 4°C suggesting that changes in the fluidity of the lipid moiety made different sites on the ATPase protein accessible to the reagent. At 4°C, addition of sodium deoxycholate enhanced the inactivation (6% ATPase activity remained) but the labeling of the SR-ATPase protein did not increase significantly. Incubation with MgATP prior to solubilization with deoxycholate resulted in the protection of the Ca 2+-ATPaseactivity and only a small decrease in the labeling occurred. At 25°C, a similar pattern was found with deoxycholate but the loss of ATPase activity was less dramatic and the extent of labeling by NBD-Cl was greater than that at 4°C. MgATP induced changes in the conformation of the ATPase protein protecting essential cysteine residues while shifting the reaction of NBD-Cl with the ATPase protein to non-essential sites in the absence or presence of deoxycholate. An analysis of tryptic digests of the NBD-ATPase protein showed that MgATP shifted the labeling from the A 2 subfragment to the A 1 subfragment in the absence of deoxycholate and from the A 1 subfragment to the A 2 subfragment in the presence of deoxycholate. The reagent, NBD-Cl, can distinguish between different temperature dependent conformational states of the ATPase protein.