Serikyaku S, Saito M, Ishitani R
Group of Neuropharmacology, Josai University, Saitama, Japan.
Jpn J Pharmacol. 1990 Jan;52(1):51-7. doi: 10.1254/jjp.52.51.
To investigate the biochemical nature of temperature-induced high-affinity [3H]tryptamine binding sites, we subjected whole rat brain synaptic membranes to treatment with various protein-modifying reagents and examined the subsequent [3H]tryptamine binding properties of the membranes. Pretreatment of the membrane preparations with NEM, NBS, PCMB, PAPMA and MA, but not with iodoacetamide, DTT, glutathione and cysteine, reduced the [3H]tryptamine binding. In addition, to at least approx. 10(-4) M, the inactivation properties of NEM, PCMB, PAPMA and MA, except for NBS, were temperature-dependent. Furthermore, it was revealed that the Scatchard plot of [3H]tryptamine binding in membranes pretreated with these thiol reagents conformed to a curved line, as well as in the case of the control membranes. Nonlinear regression analysis of these data showed that NEM decreased the Bmax values of both the high and low affinity binding sites with no significant alteration in the KD values, whereas PCMB, PAPMA and MA increased only the KD value of the high affinity sites, accompanying the decrease of the Bmax values of both sites. These results indicate that the temperature-induced high-affinity [3H]tryptamine binding molecule(s) is a thiol protein.
为了研究温度诱导的高亲和力[3H]色胺结合位点的生化性质，我们用各种蛋白质修饰试剂处理大鼠全脑突触膜，并检测随后膜的[3H]色胺结合特性。用NEM、NBS、PCMB、PAPMA和MA预处理膜制剂可降低[3H]色胺结合，但用碘乙酰胺、DTT、谷胱甘肽和半胱氨酸预处理则无此效果。此外，除NBS外，NEM、PCMB、PAPMA和MA在至少约10^(-4) M浓度时的失活特性与温度有关。此外，还发现用这些巯基试剂预处理的膜中[3H]色胺结合的Scatchard图呈曲线，对照膜也是如此。对这些数据进行非线性回归分析表明，NEM降低了高亲和力和低亲和力结合位点的Bmax值，而KD值无显著变化，而PCMB、PAPMA和MA仅增加了高亲和力位点的KD值，同时两个位点的Bmax值均降低。这些结果表明，温度诱导的高亲和力[3H]色胺结合分子是一种巯基蛋白。
