Sulfhydryl group modification of sarcoplasmic reticulum membranes

Abstract

Modification of Ca-translocating sarcoplasmic reticulum membranes (SR) from rabbit skeletal muscle with 5,5''-dithiobis(2-nitrobenzoate) (Nbs2) reveals 4 classes (kinetic sets) of sulfhydryl groups [SH]. Of the 25 mol/1.5 .times. 105 g of SR protein (i.e., containing 1 mol of ATPase protein) estimated in the presence of sodium dodecyl sulfate, 8 mol are unreactive, while 7, 8 and 2 mol display pseudo-first-order rate constants (k1) of 0.16, 0.68 and 8.3 min-1, respectively (25.degree. C, pH 7.8, 4 mM Nbs2). Under these conditions, the Ca-ATPase activity is lost with k1 = 0.73 min-1, whereas the Ca-independent ATPase activity is essentially unchanged. These results are changed little by the presence of Mg2+ or Ba2+ in the modification mixture, while Ca2+ or Sr2+ causes all 16-17 reactable SH groups to be modified with k1 = 0.50 and 0.53 min-1, respectively. The corresponding values for the loss of Ca-ATPase activity are 0.53 and 0.67 min-1; this suggests that blocking of only 1 of the 16-17 SH groups inactivates the enzyme, i.e., that there is a single essential SH group. The midpoint of the transition between the Ca2+-free and Ca2+-modification patterns occurs at a free Ca2+ concentration of about 0.9 .mu.M, implying that it is Ca2+ binding at the active sites (Kd = 0.1-1.0 .mu.M), rather than at the low-affinity non-specific sites, that effects a conformation change in the ATPase protein (which contains > 90% of the cysteines). A Ca-induced conformation change is also suggested by increased UV absorbance spectrum of the purified ATPase protein upon Ca binding. If protein-lipid interaction is disrupted with deoxycholate or Triton X-100 (which does not destroy the Ca-ATPase activity and hence presumably leaves the tertiary structure of the ATPase protein largely intact), 95% of the SH groups react with Nbs2 considerably faster; thus, at 2 mg/ml of deoxycholate, 14 groups react with k1 > 20, 5 with k1 = 2.3, and 5 with k1 = 0.4 min-1. The inaccessibility of SH groups in the absence of detergents is probably due to extensive interaction of the bilayer phospholipids with the ATPase protein.