Purification and characterization of two 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 [3H]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 [3H]ryanodine with Kd = 2-5 nM. The values of Bmax 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Murayama
T
T
Department of Pharmacology, Juntendo University School of Medicine, Tokyo.
Ogawa
Y
Y
D000818:Animals; D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D015220:Calcium Channels / Q000378:metabolism / Q000502:physiology; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008566:Membranes / Q000502:physiology; D009132:Muscles / Q000737:chemistry*; D011485:Protein Binding; D011892:Rana catesbeiana; D011950:Receptors, Cholinergic / Q000302:isolation & purification*; D012433:Ryanodine / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D014316:Tritium
D015220:Calcium Channels; D008051:Lipid Bilayers; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium; D012433:Ryanodine; D002118:Calcium