Different amino acid residues in cardiac sarcolemmal vesicles were modified by incubation with various chemical reagents. The effects of these modifications on sarcolemmal Na+-Ca2+ exchange were examined. Dithiothreitol, an agent that maintains sulfur-containing residues in a reduced state, caused a time- and concentration-dependent decrease in Na+-Ca2+ exchange. The treatment with dithiothreitol resulted in a decrease in Vmax values but did not alter the Km for Ca2+ for the Na2+-Ca2+ exchange reaction. If Na+ replaced K+ as the ion present during the modification of sarcolemmal membranes with dithiothreitol, there was substantially less of an inhibitor effect on Na+-Ca2+ exchange. Similar results were obtained with reduced glutathione, a reagent that also maintains sulfur-containing residues in a reduced state. Two sulfhydryl modifying reagents, methylmethanethiosulfonate and N'-ethylmaleimide, were capable of altering Na+-Ca2+ exchange, and the type of ion present during modification significantly affected the extent of this alteration. Almost all of the chemical reagents investigated that modified other amino acid resides (carboxyl, lysyl, histidyl, tyrosyl, tryptophanyl, arginyl and hydroxyl) had the capacity to alter Na+-Ca2+ exchange after preincubation with the sarcolemmal membrane vesicles. However, the sulfur residue-modifying reagents were the only compounds to exhibit significant differences in their action on Na+-Ca2+ exchange, depending on whether Na+ or K+ was present in the preincubation modification medium. The tryptophan modifier, N-bromosuccinimide, was the sole reagent that elicited a substantial increase in membrane permeability. The evidence is consistent with the hypothesis that sulfur-containing residues interact with a Na+-binding site for Na+-Ca2+ exchange in cardiac sarcolemmal vesicles.
Animals Carrier Proteins