Determination of the primary structure of intermolecular cross-linking sites on the Ca2(+)-ATPase of sarcoplasmic reticulum using 14C-labeled N,N'-(1,4-phenylene)bismaleimide or N-ethylmaleimide

To determine the intermolecular cross-linking site on the primary structure sarcoplasmic reticulum (SR) Ca-ATPase, the conditions for the specific binding of 14C-labeled 1,4-phenylene bis maleimide (PBM) or 14C-labeled N-ethylmaleimide (NEM) to the ATPase were explored. SR vesicles were preincubated with nonradioactive PBM in the presence of 1 mM vanadate for 1 h, then washed by centrifugation to remove free PBM and vanadate. When the pretreated SR vesicles were allowed to react with 1 mM 14C]PBM in the presence of 1 mM AMPPNP, the amount of 14C]PBM incorporated into the ATPase increased with time in parallel with the formation of dimeric ATPase and reached the maximum labeling density of 1 mol of 14C]PBM per mol of dimeric ATPase at 40 min after the start of the reaction. When the pretreated SR vesicles were allowed to react with 2 mM 14C]NEM in the absence of AMPPNP, a maximum of about 2 mol of NEM was bound per mol of the ATPase monomer. The labeling density of 14C]NEM decreased from 2 to 1 mol per mol of the ATPase when the SR vesicles were allowed to react with 14C]NEM in the presence of AMPPNP. From the analysis of the amino acid composition of the two major 14C]NEM-labeled peptides isolated from the thermolytic digest of the enzyme after the reaction of SR with 14C]NEM in the absence of AMPPNP, we deduced that 14C]NEM was incorporated into Cys377 and Cys614. On the other hand, the labeling of SR in the presence of AMPPNP resulted in inhibition of the 14C]NEM binding to Cys614, leaving Cys377 unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)