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 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.
Marty
I
I
Laboratoire de Biophysique Moléculaire et Cellulaire, Département de Biologie Moléculaire et Structurale, Grenoble, France.
Villaz
M
M
Arlaud
G
G
Bally
I
I
Ronjat
M
M
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D015220:Calcium Channels / Q000032:analysis* / Q000276:immunology; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D002462:Cell Membrane / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D009124:Muscle Proteins / Q000032:analysis* / Q000276:immunology; D009132:Muscles / Q000737:chemistry*; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*
D000906:Antibodies; D015220:Calcium Channels; D009124:Muscle Proteins; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; D002268:Carboxypeptidases; D043422:Carboxypeptidases A