骨骼肌型ryanodine受体放射配基分析法的建立

Establishment of a radioligand assay of skeletal ryanodine receptor

目的 探讨骨骼肌型ryanodine受体 (RyR1)放射配基分析法的建立及适宜条件。方法蔗糖梯度离心提取骨骼肌肌浆网膜蛋白质组分 ,用3H ryanodine进行放射配基结合实验 ,观察RyR1与3H ryanodine结合的适宜条件。结果 ① 37℃恒温水浴下 ,0～ 6 0min内 ,3H ryanodine与骨骼肌肌浆网膜蛋白质的结合量迅速增加 ,90min时基本达平衡 ,90～ 12 0min内无明显变化 ;②3H ryanodine在1～ 30nmol·L- 1 ,匀浆蛋白质质量浓度为 0 .4～ 1.0g·L- 1 范围内 ,3H ryanodine与RyR1结合的效果最佳。结论 用3H ryanodine在适宜的条件下可建立RyR1放射配基分析法。

Objective To establish an appropriate condition for measuring the skeletal ryanodine receptor 1 (RyR1) in rabbit skeletal sarcoplasmic reticulum (SR). Methods A fraction of skeletal SR membrane was extracted via sucrose gradient centrifugation. 3H ryanodine was used as a radioligand to assay its binding to the skeletal SR membrane fraction of adult rabbits in various protein concentrations and incubation time. Results ①The ryanodine binding kept increasing at 37 ℃ incubation from 0 to 60 min, and reached the equilibrium at 90 min, then remained with it at 90～120 min. ②The optimum binding was obtained when the 3H ryanodine concentration was 1～30 nmol·L -1 and the membrane fraction concentration ranging from 0.4 to 1.0 g·L -1 . Conclusion 3H ryanodine can be used as a radioligand for analysing the RyR1 under appropriate condition.