INTERACTIONS OF DIHYDROXYBENZENES WITH THE CA2-ATPASE - SEPARATE BINDING-SITES FOR DIHYDROXYBENZENES AND SESQUITERPENE()

The Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by 2,5-di-tert-butyl-1,4-dihydroxybenzene (BHQ) and other hydrophobic 1,4-dihydroxybenzenes. Inhibitory potency increases on increasing sub stituent chain length from 2,5-dipropyl-1,4-dihydroxybenzene to 2,5-di -tert-amyl-1,4-dihydroxybenzene, the most potent inhibitor, but then d ecreases for 2,5-bis(7-methylheptyl)-1,4-dihydroxybenzene. Kinetic mea surements are consistent with isomerization following the initial bind ing of BHQ to the ATPase to give a modified E2 conformation, E2(A)I, a s for the binding of sesquiterpene lactones, such as thapsigargin. Bin ding of BHQ to the ATPase shifts the E1-E2 equilibrium toward E2 becau se of the formation of E2(A)I. Measurements of Ca2+ binding as a funct ion of BHQ concentration suggest that BHQ can bind to the El conformat ion of the ATPase (but without the subsequent conformational change ob served on binding to E2) and that the binding constants of El for Ca2 are unaffected by binding of BHQ. Binding of BHQ to the ATPase in the presence of substoichiometric amounts of thapsivillosin A and effects of mixtures of BHQ and thapsivillosin A show that these two inhibitor s have separate binding sites on the ATPase.