EFFECTS OF A NONIONIC DETERGENT ON CALCIUM-UPTAKE BY CARDIAC-MICROSOMES

We investigated the effects of the nonionic detergent octaethylene gly col monododecyl ether (C(12)E(8)) on the sarcoplasmic reticulum calciu m pump in cardiac microsomes in view of its specific effects on differ ent ATP-accelerated steps in the catalytic cycle of the Ca-ATPase in l eaky fast skeletal muscle microsomes. At low concentrations of MgATP(2 -) (<2.5 mu M), a nonsolubilizing concentration of added C(12)E(8) (15 mu M) increased apparent V-max(MgATP) of oxalate-facilitated calcium uptake associated with MgATP(2-) binding to the high affinity catalyti c site. An ATP induced acceleration of calcium uptake, attributable to regulatory nucleotide binding, was seen between 2 and 3 mu M MgATP(2- ) in both C(12)E(8)-treated and control microsomes. These effects of C (12)E(8) are similar to those seen previously with trypsin treatment o f microsomes [Lu, Y.-Z., Xu, Z.-C., and Kirchberger, M. A, (1993) Bioc hemistry 32, 3105-3111]. However, at a saturating Ca2+ between 3 and 1 0 mu M MgATP(2-), C(12)E(8) produced a greater reduction in the magnit ude of the ATP-induced acceleration of calcium uptake seen with trypsi n. At 1 mM MgATP(2-), C(12)E(8) and trypsin as well as protein kinase A-catalyzed microsomal phosphorylation all increased the Ca2+ affinity of the pump, but only the latter two treatments significantly increas ed apparent V-max(Ca). In fact in trypsin-treated and phosphorylated m icrosomes, C(12)E(8) reduced V-max(Ca) to close to the control values; it reduced V-max(Ca) only slightly in control microsomes. Under our e xperimental conditions, comparable effects of 15 mu M C(12)E(8) on cal cium uptake were absent in fast skeletal muscle microsomes, which lack phospholamban. The present results show that the inhibitory effects o f phospholamban with respect to Ca2+ affinity and V-max(Ca) of the cal cium pump in cardiac microsomes may be dissociated by C(12)E(8), which allows rationalizing these effects in terms of ligand-induced changes in the rate-limiting step of the reaction cycle.