Sarcoplasmic reticulum ATPase. Spin labeling detection of ligand-induced changes in the relative reactivities of certain sulfhydryl groups

In sarcoplasmic reticulum fragments, chemical reactivity of calcium ATPase -SH groups toward N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-iodoacetamide (ISL) was estimated by measuring the steady reduction in free label spectrum intensity during the labeling reaction. A few -SH groups reacted easily with ISL and activity was not inhibited. The reaction rate was highly sensitive to pH and temperature. Calcium chelation in the presence of magnesium accelerated the reaction slightly, and nucleotides accelerated if severalfold in the presence of calcium. The resulting spectra were also studied for the bound labels, after extensive washing of the nonreacted label. Compared to the spectrum obtained after labeling in the control calcium medium, the "weakly immobilized signal" of the spectrum of vesicles labeled in a chelated calcium medium was enhanced. On the other hand, the "strongly immobilized signal" was enhanced when vesicles were labeled in a medium containing calcium and nucleotides. This was taken as evidence that different -SH groups are selectively alkylated, according to the labeling medium. The present study confirms the calcium-induced modifications in the -SH environment reported previously and suggests new ways of searching for possible conformational events during the transport cycle in the membrane.