A procedure for purification of the ryanodine receptor from skeletal muscle.
In this paper, we describe a simple and reproducible method for purifying large quantities of ryanodine receptor from skeletal muscle membranes. The procedure involves the use of ion exchange chromatography and sucrose gradient centrifugation to purify the protein which has been identified as the calcium release protein of the sarcoplasmic reticulum (Imagawa, T., Smith, J., Coronado, R. and Campbell, K. (1987) J. Biol. Chem. 262:16,636-16,643). Addition of micromolar quantities of unlabeled ryanodine prior to solubilization and throughout the isolation procedure appears to stabilize the tetrameric structure of the ryanodine receptor. The purified receptor, consisting predominantly of a 400K polypeptide on SDS-PAGE, binds [3H]ryanodine with a binding affinity similar to that in membranes. Overall recovery of ryanodine binding activity was 21% of the initial activity with a 30-fold purification of the receptor.
Hawkes
M J
MJ
Department of Physiology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030.
Díaz-Muñoz
M
M
Hamilton
S L
SL
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008566:Membranes / Q000032:analysis / Q000648:ultrastructure; D009132:Muscles / Q000032:analysis* / Q000648:ultrastructure; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000302:isolation & purification* / Q000378:metabolism; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012967:Sodium Dodecyl Sulfate; D014316:Tritium
D002352:Carrier Proteins; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium; D012433:Ryanodine; D012967:Sodium Dodecyl Sulfate