Chemical modification of sarcoplasmic reticulum with methylbenzimidate. Stimulation of Ca²⁺ efflux

Treatment of sarcoplasmic reticulum membranes with 12 mM-methylbenzimidate (MBI) for 5 min, in the presence of 5 mM-ATP at pH 8.5, resulted in a 2-3-fold stimulation of ATP hydrolysis and over 90% inhibition of Ca²⁺ accumulation. This phenomenon was strictly dependent upon the presence of nucleotides with the following order of effectiveness: adenosine 5'-[β,γ-imido]triphosphate ≥ ATP > UTP > ADP > AMP. Divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, when present during the MBI treatment, prevented both the stimulation of ATPase activity and the inhibition of Ca²⁺ accumulation. Modification with MBI had no effect on E-P formation from ATP, ADP-ATP exchange, Ca²⁺ binding or ATP-P(i) exchange catalysed by the membranes. Membranes modified with MBI in the presence of ATP and then passively loaded with Ca²⁺ released about 80% of their Ca²⁺ content within 3 s. Control membranes released only 3% of their Ca²⁺ during the same time period. MBI modification inhibited Ca²⁺ accumulation by proteoliposomes reconstituted with the partially purified ATPase but not with the purified ATPase fraction. These results suggest that the MBI in the presence of ATP stimulates Ca²⁺ release by modifying a protein factor(s) other than the (Ca²⁺ + Mg²⁺)-ATPase.