Analysis of the ATP-induced conformational changes in sarcoplasmic reticulum

A series of group-specific spin-labeled compounds was used to investigate the mechanism of the ATP-induced conformational changes in rabbit skeletal sarcoplasmic reticulum. The spin labels used can be divided into three classes according to their specificities: (I) N(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide for SH groups; (II) N(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)isothiocyanate for amine or hydroxyl groups; and (III) N-oxyl-4′,4′-dimethyl-oxazolidine derivatives of stearic acid for fatty acids.Of the three classes of compounds tested, only the mobility of probe (I) changed upon addition of ATP to the spin-labeled sarcoplasmic reticulum. This ATP-induced conformational change could be depressed by 5 mm propranolol, a concentration which by itself had no effect on the mobility of the spin label. Since similar concentrations of propranolol inhibited the breakdown but did not influence the formation of a phosphorylated intermediate during the hydrolysis of ATP, these observations suggest that the conformational change takes place at a step in ATP hydrolysis beyond the formation of the phosphorylated intermediate. The same basic series of experiments was also performed with the purified sarcoplasmic reticulum enzyme. Even though similar results were obtained, the sensitivity of the enzyme toward propranolol and also the mobility of probe (I) in the enzyme were different from that of the sarcoplasmic reticulum. Large doses (10–20 mm) of propranolol, however, were found to directly alter the mobilities of all the classes of probes used. The effect of 20 mm propranolol on probe (III) in the sarcoplasmic reticulum was equivalent to a 10 °C rise in temperature of the membrane.