PURIFICATION AND CHARACTERIZATION OF 2 RYANODINE-BINDING PROTEIN ISOFORMS FROM SARCOPLASMIC-RETICULUM OF BULLFROG SKELETAL-MUSCLE

The two ryanodine-binding proteins (RyBPs) have been purified from sarcoplasmic reticulum of bullfrog skeletal muscle by Mono Q column chromatography following solubilization of SR by CHAPS and sucrose density gradient centrifugation. We conclude that the two RyBPs (alpha- and beta-RyBP) are isoforms on the basis (i) that each RyBP is distinguished by a specific polyclonal antibody and (ii) that distinct polypeptides are generated by limited tryptic digestion of the two RyBPs. Monomeric molecular weights for alpha- and beta-RyBP are estimated to be (690+/-10) and (570+/-10) kDa, respectively, as determined from mobilities on disc SDS-PAGE using the Weber-Osborn buffer system without 6 M urea, which gives an estimate of (590+/-10) kDa for RyBP of rabbit skeletal muscle. Similar determination in the presence of 6 M urea gave 630 kDa for alpha-RyBP and unchanged estimates for the other RyBPs. Both RyBPs show [H-3]ryanodine-binding activities which are activated by Ca2+, AMPOPCP, and caffeine, and inhibited by ruthenium red, MgCl2, and procaine. Beta-RyBP, however, has higher affinity for Ca2+. In the presence of Ca2+ and AMPOPCP, both RyBPs show single homogeneous binding sites for [H-3]ryanodine with K(d)=2-5 nM. The values of B(max) for alpha- and beta-RyBP were 320-340 and 320-375 pmol/mg protein, respectively. These results are consistent with the conclusion that a homo-tetramer of each RyBP binds one ryanodine molecule, taking account of the estimated molecular weight. Corresponding to ryanodine binding, alpha and beta-RyBP show Ca2+-dependent channel currents which are activated by ATP and inhibited by ruthenium red on planar lipid bilayers. These results demonstrate that the two RyBP isoforms, which occur in approximately equal amounts in bullfrog skeletal muscle, constitute Ca2+-induced Ca2+ release channels.