Transmembrane orientation of the N-terminal and C-terminal ends of the ryanodine receptor in the sarcoplasmic reticulum of rabbit skeletal muscle

Antibodies were raised against synthetic peptides corresponding to the N-terminal (residues 2-15) and the C-terminal (residues 5027-5037) parts of the rabbit skeletal muscle ryanodine receptor. The specificity of the antibodies generated was tested by e.l.i.s.a., Western blotting and immunofluoresce...

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Bibliographic Details
Published in:Biochemical journal 1994-03, Vol.298 (3), p.743-749
Main Authors: MARTY, I, VILLAZ, M, ARLAUD, G, BALLY, I, RONJAT, M
Format: Article
Language:English
Subjects:
Animals
Antibodies - immunology
Antibody Specificity
Biological and medical sciences
Blotting, Western
Calcium Channels - analysis
Calcium Channels - immunology
Carboxypeptidases - metabolism
Carboxypeptidases A
Cell Membrane - chemistry
Cell receptors
Cell structures and functions
Cytoplasm - chemistry
Enzyme-Linked Immunosorbent Assay
Fluorescent Antibody Technique
Fundamental and applied biological sciences. Psychology
Miscellaneous
Molecular and cellular biology
Muscle Proteins - analysis
Muscle Proteins - immunology
Muscles - chemistry
Peptide Fragments - analysis
Peptide Fragments - immunology
Rabbits
Ryanodine Receptor Calcium Release Channel
Sarcoplasmic Reticulum - chemistry
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Summary:Antibodies were raised against synthetic peptides corresponding to the N-terminal (residues 2-15) and the C-terminal (residues 5027-5037) parts of the rabbit skeletal muscle ryanodine receptor. The specificity of the antibodies generated was tested by e.l.i.s.a., Western blotting and immunofluorescence. All these tests demonstrated the specificity of the antibodies and their ability to react with both the native and the denaturated ryanodine receptor. Both the anti-N-terminus and the anti-C-terminus antibodies bound to sarcoplasmic reticulum vesicles, indicating that each end of the membrane-embedded ryanodine receptor is exposed to the cytoplasmic side of the vesicles. These immunological data were complemented with proteolysis experiments using carboxypeptidase A. Carboxypeptidase A induced degradation of the C-terminal end of the ryanodine receptor in sarcoplasmic reticulum vesicles and a concomitant loss of reactivity of the anti-C-terminus antibodies in Western blots, providing extra evidence for the cytoplasmic localization of the C-terminal end of the ryanodine receptor.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj2980743