Definition of surface-exposed and trans-membranous regions of the (Ca2+-Mg2+)-ATPase of sarcoplasmic reticulum using anti-peptide antibodies

Abstract

Peptides have been synthesized representing parts of the transduction, phosphorylation, nucleotide-binding and hinge domains of the (Ca2+-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR), and corresponding to segments of all of the postulated short inter-membranous loops of the (Ca2+- Mg2+)-ATPase (residues 77-88, 277-287, 780-791, 808-818, 915-=924 and 949-958). A number of antibodies raised to these peptides have been shown to bind to the ATPase, defining surface-exposed regions. Many of these are concentrated in the phosphorylation and nucleotide-binding domains, suggesting that these domains could be exposed on the top surface of the ATPase. The cytoplasmic location of the loop containing residues 808-818 was confirmed by the finding that proteinase K treatment of intact SR vesicles enhanced the binding of antibodies against this segment. These findings support the 10-α-helix model of the ATPase. These results also suggest that only inter-membranous loops larger than about 20 residues are likely to be detected by immunological methods in transmembranous proteins. Binding of anti-peptide antibodies to proteolytic fragments of the ATPase has been used to define the domain structure of the enzyme. Some of the anti-peptide antibodies have been characterized by studying their binding to sets of hexameric peptides synthesized on plastic pegs. A wide pattern of responses is observed, with a restricted range of epitopes being recognized by each anti-peptide antibody.