Wang J P, Needleman D H, Hamilton S L
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1993 Oct 5;268(28):20974-82.
Both high and low affinity binding sites for [3H]ryanodine exist in sarcoplasmic reticulum membranes derived from rabbit skeletal muscle. Negatively cooperative binding of [3H]ryanodine at one of four initially identical sites cannot account for some of the kinetic features of the binding to high and low affinity sites. The presence of excess unlabeled ryanodine greatly slows the rate at which [3H]ryanodine bound at the high affinity site dissociates. An examination of the rate of dissociation of [3H]ryanodine bound at increasing [3H]ryanodine concentrations reveals the existence of a second site, occupied only at high ligand concentrations. The occupation of this site correlates well with the conversion of the high affinity site from a site with a dissociation rate constant of approximately 0.0025 min-1 to one with a dissociation rate constant of less than 0.00025 min-1. The low affinity site itself has a dissociation rate constant of 0.013 min-1 and dissociation from this site is unaffected by the presence of 100 microM unlabeled ryanodine. These data suggest that the two binding sites are different but are either allosterically or sterically coupled. Association experiments support this interpretation. Low affinity binding sites for [3H]ryanodine exist in transverse tubule (t-tubule) as well as sarcoplasmic reticulum membranes. High concentrations of both ryanodine and ruthenium red inhibit the binding of [3H]PN200-110 to the dihydropyridine-binding protein in t-tubule membranes. Whether the low affinity site in t-tubule membranes is related to that found in sarcoplasmic reticulum membranes is not yet known.
从兔骨骼肌提取的肌浆网膜中存在[3H]ryanodine的高亲和力和低亲和力结合位点。[3H]ryanodine在四个初始相同位点之一的负协同结合无法解释其与高亲和力和低亲和力位点结合的一些动力学特征。过量未标记的ryanodine的存在极大地减慢了[3H]ryanodine在高亲和力位点解离的速率。对[3H]ryanodine在不断增加的[3H]ryanodine浓度下结合后的解离速率进行检测，发现存在第二个位点，该位点仅在高配体浓度时被占据。该位点的占据与高亲和力位点从解离速率常数约为0.0025 min-1的位点转变为解离速率常数小于0.00025 min-1的位点密切相关。低亲和力位点本身的解离速率常数为0.013 min-1，100 microM未标记的ryanodine的存在不影响其从该位点的解离。这些数据表明这两个结合位点不同，但可能通过变构或空间方式耦合。结合实验支持这一解释。[3H]ryanodine的低亲和力结合位点存在于横管（t-小管）以及肌浆网膜中。高浓度的ryanodine和钌红均抑制[3H]PN200-110与t-小管膜中二氢吡啶结合蛋白的结合。t-小管膜中的低亲和力位点是否与肌浆网膜中的低亲和力位点相关尚不清楚。
