Alderson B H, Feher J J
Biochim Biophys Acta. 1987 Jun 30;900(2):221-9. doi: 10.1016/0005-2736(87)90336-1.
The binding of [3H]ryanodine with cardiac sarcoplasmic reticulum vesicles depends on the calcium concentration. Binding in the absence of calcium appears to be non-specific because it shows no saturation up to 20 microM ryanodine. The apparent Km value for calcium varied between 2 and 0.8 microM when the ryanodine concentration varied between 10 and 265 nM. The Hill coefficient for the calcium dependence of [3H]ryanodine binding was near two. Scatchard analysis of ryanodine binding indicated a high-affinity site with a Bmax of 5.2 +/- 0.4 pmol/mg with a Kd of 6.8 +/- 0.1 nM. Preincubation under conditions in which the high-affinity sites were saturated did not result in stimulation of the calcium uptake rate indicative of closure of the calcium channel. Stimulation of calcium uptake rate occurred only at higher concentrations of ryanodine (apparent Km = 17 microM). This stimulation of the calcium uptake rate also required calcium in the submicromolar range. The data obtained support the hypothesis that ryanodine binding to the low-affinity site (Km about 17 microM) is responsible for closure of the calcium release channel and the subsequent increase in the calcium uptake rate of the sarcoplasmic reticulum. Because the number of ryanodine-binding sites is much less than the number of calcium transport pumps the channel is probably distinct from the pump.
[3H]ryanodine与心肌肌浆网囊泡的结合取决于钙浓度。在无钙情况下的结合似乎是非特异性的，因为在高达20微摩尔的ryanodine浓度下都未显示出饱和。当ryanodine浓度在10至265纳摩尔之间变化时，钙的表观Km值在2至0.8微摩尔之间变化。[3H]ryanodine结合的钙依赖性的希尔系数接近2。对ryanodine结合的Scatchard分析表明存在一个高亲和力位点，其Bmax为5.2±0.4皮摩尔/毫克，Kd为6.8±0.1纳摩尔。在高亲和力位点饱和的条件下预孵育并未导致钙摄取速率的刺激，这表明钙通道关闭。仅在较高浓度的ryanodine（表观Km = 17微摩尔）时才发生钙摄取速率的刺激。这种钙摄取速率的刺激也需要亚微摩尔范围内的钙。所获得的数据支持这样的假设，即ryanodine与低亲和力位点（Km约为17微摩尔）的结合导致钙释放通道关闭以及随后肌浆网钙摄取速率增加。由于ryanodine结合位点的数量远少于钙转运泵的数量，该通道可能与泵不同。
