Kinetics and subcellular localization of specific [3H]phorbol 12, 13-dibutyrate binding by mouse brain.
Kinetics and subcellular localization of specific [3H]phorbol 12, 13-dibutyrate binding by mouse brain.
复制标题
小鼠大脑特异性 [3H]佛波醇 12, 13-二丁酸酯结合的动力学和亚细胞定位。
作者：
W. G. Dunphy;R. Kochenburger;M. Castagna;P. Blumberg
[3H]佛波醇 12,13-二丁酸酯 ([3H]-PDBU) 与小鼠大脑颗粒制剂的特异性结合已得到进一步表征。动力学分析使用过滤测定法测量结合，得出 23 度时的二级速率常数为 3.75 X 10(7) M-1 min-1，一级解离速率常数为 0.21 min-1。根据动力学数据计算出的 Kd 为 5.6 nM，与之前在平衡结合研究中确定的值非常吻合。 [3H]PDBU 结合的 Kd 仅随温度略有变化。从其温度依赖性来看，[3H]PDBU 结合似乎与焓的小幅增加（δ H 度 = +0.4 kcal/mol）和熵的大幅增加（δ S 度 = +38 e.u.）相关。这些值是疏水相互作用的特征。与 Kd 相比，结合的解离速率常数随温度变化很大。释放半场时间范围为 30 度时的 1.75 分钟到 4 度时的 62 分钟。结合的 Kd 对 Ca2+ 敏感；乙二醇双(β-氨基乙基醚)N,N'-四乙酸对Ca2+的螯合使Kd增加2.4倍。亚细胞分级分离后，特定的 [3H]PDBU 结合活性完全是颗粒状的；没有检测到与细胞质的结合。结合显然与核或线粒体标记无关。另一方面，蔗糖密度梯度上的结合活性分布比 Na+-K+-腺苷三磷酸酶活性或苯甲酸奎宁环酯（毒蕈碱胆碱能拮抗剂）的结合更广泛。因此，特定[3H]PDBU 与质膜结合的定位仍然不确定。
The specific binding of [3H]phorbol 12,13-dibutyrate ([3H]-PDBU) to particulate preparations from mouse brain has been further characterized. Kinetic analysis, using a filtration assay to measure binding, yielded a second-order rate constant at 23 degrees of 3.75 X 10(7) M-1 min-1 and a first-order dissociation rate constant of 0.21 min-1. The Kd of 5.6 nM calculated from the kinetic data agreed well with the value determined previously in equilibrium binding studies. The Kd for [3H]PDBU binding varied only slightly with temperature. From its temperature dependence, [3H]PDBU binding appeared to be associated with a small increase in enthalpy (delta H degrees = +0.4 kcal/mol) and a large increase in entropy (delta S degrees = +38 e.u.). Such values are characteristic for hydrophobic interactions. The dissociation rate constant for binding, in contrast to the Kd, varied dramatically with temperature. The half-time for release ranged from 1.75 min at 30 degrees to 62 min at 4 degrees. The Kd for binding was Ca2+ sensitive; chelation of Ca2+ by ethyleneglycolbis(beta-aminoethyl ether)N,N'-tetraacetic acid increased the Kd 2.4-fold. Upon subcellular fractionation, the specific [3H]PDBU binding activity was exclusively particulate; no binding to cytosol was detectable. Binding clearly did not correlate with nuclear or mitochondrial markers. On the other hand, a broader distribution of binding activity was seen on sucrose density gradients than for either Na+-K+-adenosine triphosphatase activity or binding of quinuclidinyl benzilate (a muscarinic cholinergic antagonist). The localization of specific [3H]PDBU binding to the plasma membrane therefore remains uncertain.