Abstract
Ryanodine, a highly toxic alkaloid, reacts specifically with the Ca2+ release channels which are localized in the terminal cisternae of sarcoplasmic reticulum (SR). In this study, the ryanodine receptor from cardiac SR has been purified, characterized, and compared with that of skeletal muscle SR. The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of phospholipids. Purification was performed by sequential affinity chromatography followed by gel permeation chromatography in the presence of CHAPS and phospholipids. The enrichment of the receptor from cardiac microsomes was about 110-fold. The purified receptor contained a major polypeptide band of Mr 340,000 with a minor band of Mr 300,000 (absorbance ratio 100/8) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron microscopy of the purified receptor from heart showed square structures of 222 +/- 21 A/side, which is the unique characteristic of feet structures of junctional face membrane of terminal cisternae of SR. Recently, we isolated the ryanodine receptor from skeletal muscle (Inui, M., Saito, A., and Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747). The ryanodine receptors from heart and skeletal muscle have similar characteristics in terms of protein composition, morphology, chromatographic behavior, and Ca2+, salt, and phospholipid dependence of ryanodine binding. However, there are distinct differences: 1) the Mr of the receptor is slightly larger for skeletal muscle (Mr approximately 360,000); 2) the purified receptor from heart contains two different affinities for ryanodine binding with Kd values in the nanomolar and micromolar ranges, contrasting with that of skeletal muscle SR which shows only the high affinity binding; 3) the affinity of the purified cardiac receptor for ryanodine was 4-5-fold higher than that of skeletal muscle, measured under identical conditions. The greater sensitivity in ryanodine in intact heart can be directly explained by the tighter binding of the ryanodine receptor from heart. The present study suggests that basically similar machinery (the ryanodine receptor and foot structure) is involved in triggering Ca2+ release from cardiac and skeletal muscle SR, albeit there are distinct differences in the sensitivity to ryanodine and other ligands in heart versus skeletal muscle.
Highlights
The solubilized ryanodine receptor from cardiac microsomes enriched in sarcoplasmic reticulum (SR) showed similar ryanodine binding properties interms of Ca2+,salt, andphospholipid dependence at 100nM r y a n ~ d i n eT. ~herefore, 25 p M CaCl, 1M NaCl, and 5 mg/ml soybean phospholipid were included in the ryanodine assay medium, and the soybean phospholipid concentration was maintained at 5 mg/ml during theentire purification procedure
Thesame purification procedure for skeletal muscle SR could not be directly applied for the isolation of the cardiac ryanodine receptor
Elmax for high and low affinity ryanodinebinding was 474 f 19 pmol/mg and 5.53 f 0.79 nmol/mg, respectively. [3H]Ryanodine binding to the purified ryanodine receptor from junctional terminal cisternaeof skeletal muscle SR (Fig. 5B) was measured at the sameconditions as the cardiac receptor and showed only the high affinity ryanodine binding with K d of 80.7 f 3.1 nM in this concentration range as reported previously (16) with Elmax 438 f 49 pmol/
Summary
Purification of Ryanodine Receptor from Heart SR-We have previously solubilized and purified the ryanodine receptor from junctional terminal cisternae of skeletal muscle SR (16). Further purification was performed on a p-aminobenzamidine-agarose column inCHAPSand soybean phospholipid (Fig. 3).The ryanodine binding activity eluted asa single peakby increasing the NaCl concentration, and most of the remainder of the proteins were separated from the ryanodinereceptor (Fig. 1,lane 4 ) .In the final step, the sample was further purified bygel permeation column chromatography ona Fractogel TSK HW65Fcolumn (Fig. 4) (Fig. 1, lane 5 ). [3H]Ryanodine binding to the purified ryanodine receptor from junctional terminal cisternaeof skeletal muscle SR (Fig. 5B) was measured at the sameconditions as the cardiac receptor and showed only the high affinity ryanodine binding with K d of 80.7 f 3.1 nM (mean f S.E., n = 3) in this concentration range as reported previously (16) with Elmax 438 f 49 pmol/.
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