Huisamen B, Ellis E, van Dyk M, Lochner A
Department of Medical Physiology and Biochemistry, Faculty of Medicine, University of Stellenbosch, Tygerberg, Republic of South Africa.
Mol Cell Biochem. 1996 Sep 6;162(1):1-9. doi: 10.1007/BF00250989.
Although it is well-accepted that the phosphatidylinositol signalling transduction pathway, producing inositol-1,4,5-P3 (InsP3) and inositol-1,3,4,5-P4 (InsP4) as second messengers, functions in heart muscle, virtually nothing is known about the roles of the higher inositol polyphosphates such as inositolhexakisphosphate (InsP6). This study demonstrates that InsP6 has the ability to bind intracellularly, with different binding characteristics, to different myocardial membranes. Binding to purified sarcoplasmic reticulum (SR) membranes, purified sarcolemmal (SL) membranes as well as to viable mitochondria were characterized. Binding to all these membranes display high as well as low affinity binding sites, with differing affinities. Kd values of binding to SR were 32 and 383 nM, to SL 61 and 1312 nM, while those of mitochondrial binding were 230 and 2200 nM respectively. InsP4 binding was also investigated and displayed the following characteristics: to SR, one low affinity binding site (Kd = 203 nM) and to SL, a high as well as a low affinity binding site with Kd values of 41 and 2075 nM respectively. Presence of InsP3, the second messenger for SR calcium release, at concentrations of 1 nM, elevated the binding of InsP4 to SR and SL by a mean of 30% and 20% respectively. Fractionation of SR and SL membranes on sucrose density gradients, after solubilization with CHAPS, indicated that InsP6 bound to two separate protein peaks in both these membranes, while InsP4 bound to only one. In SR membranes, InsP4 bound preferentially to a protein separating at high sucrose density while it bound to a protein separating at low sucrose density in SL membranes.
尽管磷脂酰肌醇信号转导途径可产生作为第二信使的肌醇 -1,4,5 - 三磷酸（InsP3）和肌醇 -1,3,4,5 - 四磷酸（InsP4），且其在心肌中发挥作用已被广泛接受，但对于诸如肌醇六磷酸（InsP6）等更高阶肌醇多磷酸的作用却几乎一无所知。本研究表明，InsP6能够以不同的结合特性在细胞内与不同的心肌膜结合。对其与纯化的肌浆网（SR）膜、纯化的肌膜（SL）膜以及活线粒体的结合特性进行了表征。与所有这些膜的结合均显示出高亲和力和低亲和力结合位点，且亲和力各不相同。与SR结合的解离常数（Kd）值分别为32和383 nM，与SL结合的Kd值为61和1312 nM，而线粒体结合的Kd值分别为230和2200 nM。还对InsP4的结合进行了研究，其表现出以下特性：与SR结合有一个低亲和力结合位点（Kd = 203 nM），与SL结合有一个高亲和力和一个低亲和力结合位点，Kd值分别为41和2075 nM。作为SR钙释放第二信使的InsP3，在浓度为1 nM时，分别使InsP4与SR和SL的结合平均增加了30%和20%。在用CHAPS溶解后，在蔗糖密度梯度上对SR和SL膜进行分级分离，结果表明InsP6在这两种膜中均与两个单独的蛋白峰结合，而InsP4仅与一个蛋白峰结合。在SR膜中，InsP4优先与在高蔗糖密度下分离的一种蛋白结合，而在SL膜中它与在低蔗糖密度下分离的一种蛋白结合。
