Affinity purification of the ryanodine receptor/calcium release channel from fast twitch skeletal muscle based on its tight association with FKBP12.
The ryanodine receptor (RyR)/calcium release channel isolated from skeletal muscle terminal cisternae (TC) of sarcoplasmic reticulum (SR) is tightly associated with FK506 binding protein of 12.0 kDa (FKBP12) (Jayaraman et al., (1992) J.Biol.Chem. 267, 9474-9477). In this study, we describe a new method of affinity chromatography for purifying the RyR from skeletal muscle SR based on: 1) its tight association with FKBP12; and 2) the finding that bound FKBP on the RyR can be exchanged with soluble FKBP12 (Timerman et al., (1995) J.Biol.Chem. 270, 2451-2459). Soluble glutathione S-transferase/FKBP12 (GST/FKBP12) fusion protein was first exchanged with bound FKBP12 on the RyR of TC. The TC were then solubilized with CHAPS and the complex of RyR.GST/FKBP12 was specifically adsorbed by glutathione Sepharose 4B and then eluted with glutathione. The RyR, purified by this method, has similar characteristics by SDS-PAGE, radioligand binding and immuno-reactivity as the RyR purified by multiple sequential column chromatography.
Xin
H B
HB
Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235, USA.
Timerman
A P
AP
Onoue
H
H
Wiederrecht
G J
GJ
Fleischer
S
S
D000818:Animals; D015220:Calcium Channels / Q000302:isolation & purification* / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism*; D009124:Muscle Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D022021:Tacrolimus Binding Proteins
D015220:Calcium Channels; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D005982:Glutathione Transferase; D022021:Tacrolimus Binding Proteins