A simple, fast, one-step method for the purification of the skeletal-muscle ryanodine receptor.
In this paper we describe a simple, fast, one-step method for the purification of the skeletal-muscle ryanodine receptor. The ryanodine receptor from CHAPS-solubilized junctional sarcoplasmic-reticulum membranes was adsorbed to a spermine-agarose column and eluted by 2 mM-spermine. The purified receptor, consisting predominantly of a 450 kDa polypeptide on SDS/PAGE, binds [3H]ryanodine with a specific activity of approximately 300 pmol/mg of protein and with a high affinity (KD = 4.7 +/- 2 nM). The purified receptor appears to retain the pharmacological properties of the receptor in the original membranes. The purification resulted in over 80% recovery of the initial ryanodine-binding sites and about 30-96-fold purification. This simple and fast method is highly reproducible and suitable for purification of small as well as large quantities of ryanodine receptor.
Shoshan-Barmatz
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Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel.
Zarka
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A
D000818:Animals; D002793:Cholic Acids; D002846:Chromatography, Affinity / Q000379:methods*; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D009124:Muscle Proteins / Q000302:isolation & purification*; D009132:Muscles / Q000737:chemistry*; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000302:isolation & purification*; D015203:Reproducibility of Results; D019837:Ryanodine Receptor Calcium Release Channel; D013096:Spermine
D002793:Cholic Acids; D003902:Detergents; D009124:Muscle Proteins; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D013096:Spermine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate