Definition of surface-exposed and trans-membranous regions of the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum using anti-peptide antibodies.
Peptides have been synthesized representing parts of the transduction, phosphorylation, nucleotide-binding and hinge domains of the (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR), and corresponding to segments of all of the postulated short inter-membranous loops of the (Ca(2+)-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-alpha-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.
Mata
A M
AM
Department of Biochemistry, University of Southampton, U.K.
Matthews
I
I
Tunwell
R E
RE
Sharma
R P
RP
Lee
A G
AG
East
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000737:chemistry* / Q000276:immunology; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry*; D009711:Nucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013379:Substrate Specificity
D000906:Antibodies; D008565:Membrane Proteins; D009711:Nucleotides; D010446:Peptide Fragments; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases