Ogawa Y, Harafuji H
Department of Pharmacology, Juntendo University School of Medicine, Tokyo.
J Biochem. 1990 Jun;107(6):894-8. doi: 10.1093/oxfordjournals.jbchem.a123144.
While many reports have shown that Ca2+ alone causes ryanodine binding to the heavy fraction of the sarcoplasmic reticulum (HFSR), our results demonstrate that caffeine or beta,gamma-methylene adenosine triphosphate (AMPOPCP) in addition to Ca2+ is necessary for ryanodine binding, although Ca2+ is indispensable for it. While clarifying the reasons for this discrepancy, we found that a high osmolarity of the reaction medium, but not ionic strength, is a crucial factor. In a hypertonic solution containing 1 M NaCl, Ca2+ alone causes a sizable extent of ryanodine binding. Caffeine and AMPOPCP independently stimulate it, unlike the case of 0.17 M KCl (or NaCl) medium, in which they show a potentiating interaction. Ryanodine binding in the hypertonic solution was markedly enhanced not only as to the binding rate but also the extent. The Scatchard plot was linear, indicating a single class of homogeneous binding sites. The maximum number of binding sites as well as the affinity was also increased in 1 M NaCl-medium. The presence of AMPOPCP and/or caffeine did not affect the magnitudes of them so much, especially that of the affinity, in the hypertonic medium, as in the isotonic medium. The Ca2(+)-dependence of ryanodine binding in the stimulatory range was similar to that in 0.17 M KCl- (or NaCl-) medium. However, the very weak inhibition at high Ca2+ concentrations is in striking contrast to ryanodine binding in the isotonic medium. The stimulation due to a high osmolarity is distinct, as to the mechanism, from that due to AMPOPCP, caffeine, or temperature. The dissociation of [3H]ryanodine bound was also examined under various experimental conditions.
虽然许多报告表明，单独的Ca2+会导致ryanodine与肌浆网重组分（HFSR）结合，但我们的结果表明，除Ca2+外，咖啡因或β,γ-亚甲基三磷酸腺苷（AMPOPCP）对于ryanodine结合也是必需的，尽管Ca2+对其结合不可或缺。在阐明这种差异的原因时，我们发现反应介质的高渗透压而非离子强度是一个关键因素。在含有1 M NaCl的高渗溶液中，单独的Ca2+会导致相当程度的ryanodine结合。与在0.17 M KCl（或NaCl）介质中不同，在该介质中咖啡因和AMPOPCP表现出增强相互作用，而在高渗溶液中，咖啡因和AMPOPCP分别对ryanodine结合有刺激作用。高渗溶液中的ryanodine结合不仅在结合速率上，而且在结合程度上都显著增强。Scatchard图呈线性，表明存在单一类别的均匀结合位点。在1 M NaCl介质中，结合位点的最大数量以及亲和力也有所增加。与等渗介质相比，在高渗介质中，AMPOPCP和/或咖啡因的存在对它们的大小影响不大，尤其是对亲和力的影响。ryanodine结合在刺激范围内的Ca2+依赖性与在0.17 M KCl -（或NaCl -）介质中的相似。然而，在高Ca2+浓度下非常微弱的抑制作用与等渗介质中的ryanodine结合形成了鲜明对比。高渗透压引起的刺激在机制上与AMPOPCP、咖啡因或温度引起的刺激不同。还在各种实验条件下研究了结合的[3H]ryanodine的解离情况。
