Localization and partial characterization of the oligomeric disulfide-linked molecular weight 95,000 protein (triadin) which binds the ryanodine and dihydropyridine receptors in skeletal muscle triadic vesicles.
A monoclonal antibody, GE 4.90, has been produced following immunization of mice with the 95-kDa protein (triadin) of terminal cisternae of rabbit fast skeletal muscle isolated in nondenaturing detergent. The antibody binds to a protein of Mr95K in Western blots of microsomal vesicles electrophoresed in the presence of mercaptoethanol. The greatest intensity of the immunoblot reaction is to enriched terminal cisternae vesicles while little binding is seen to longitudinal reticulum and transverse tubules. The content of antigen in different microsomal subfractions has been estimated by immunoassay: terminal cisternae/triads contain 5.6 micrograms/mg of protein while heavy terminal cisternae contain 32 micrograms/mg. The molar content of triadin in vesicles is approximately the same as that of the ryanodine receptor. When Western blots of gels of terminal cisternae are run in nonreducing conditions, little protein of Mr95K is visible. A number of bands, however, forming a ladder of higher molecular weight are discerned, indicating that the 95-kDa protein forms a disulfide-linked homopolymer. A biotinylated aromatic disulfide reagent (biotin-HPDP) labels the 95-kDa protein, the junctional foot protein, and the Mr 106K protein described by others as a Ca(2+)-release channel (SG 106). This latter protein migrates in gel electrophoresis under nonreducing conditions at a molecular weight different from that of the 95-kDa protein. We did not detect any alteration of binding of the 95-kDa protein to the dihydropyridine receptor or junctional foot protein dependent on the state of oxidation of cysteine residues of either triadin or receptor protein used as the overlay probe.
Caswell
A H
AH
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101.
Brandt
N R
NR
Brunschwig
J P
JP
Purkerson
S
S
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D015220:Calcium Channels; D002352:Carrier Proteins; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides; D046911:Macromolecular Substances; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D009124:Muscle Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D009132:Muscles; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000737:chemistry* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D002352:Carrier Proteins; D004220:Disulfides; D046911:Macromolecular Substances; D009124:Muscle Proteins; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D019837:Ryanodine Receptor Calcium Release Channel; C070048:triadin; D003545:Cysteine