Transmembranous organization of (Ca2(+)-Mg2+)-ATPase from sarcoplasmic reticulum. Evidence for lumenal location of residues 877-888.
An antipeptide antibody was produced against a peptide corresponding to residues 877-888 of fast twitch rabbit sarcoplasmic reticulum ATPase. This antipeptide antibody bound strongly to the ATPase in sarcoplasmic reticulum vesicles only after the vesicles had been solubilized with the detergent C12E8 indicating that its epitope was located in the lumen of the sarcoplasmic reticulum. Digestion of sarcoplasmic reticulum or purified (Ca2(+)-MG2+)-ATPase by proteinase K for up to 1 h resulted in a stable ATPase fragment of 30 kDa containing the epitope for the above antibody and the epitope for an antibody directed against the C terminus. Further proteolysis revealed smaller fragments (Mr 19,000 and 13,000) containing both epitopes. By contrast, small fragments of the ATPase (less than 29 kDa) containing the N-terminal epitope were not observed even after short exposures to proteinase K. These data support the view that the (Ca2(+)-MG2+)-ATPase has 10 transmembranous helices.
Matthews
I
I
Department of Biochemistry, University of Southampton, United Kingdom.
Sharma
R P
RP
Lee
A G
AG
East
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007106:Immune Sera; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010455:Peptides / Q000138:chemical synthesis; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D007106:Immune Sera; D010455:Peptides; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases