Krizaj I, Dolly J O, Gubensek F
Department of Biochemistry and Molecular Biology, J. Stefan Institute, Ljubljana, Slovenia.
Biochemistry. 1994 Nov 22;33(46):13938-45. doi: 10.1021/bi00250a049.
A specific, high-affinity binding site for ammodytoxin C in synaptic membranes from bovine cerebral cortex was detected and partially characterized. Equilibrium binding analysis revealed a single population of [125I]ammodytoxin C acceptors with the following binding parameters: Kd = 6.0 nM and Bmax = 5.7 pmol/mg membrane protein. Such binding was strongly inhibited by three ammodytoxins (A, B, and C) and by crotoxin B. Vipera berus berus phospholipase A2 was a weaker inhibitor; nontoxic phospholipase A2, ammodytin I2, and the myotoxic phospholipase A2 homologue, ammodytin L, both from Vipera ammodytes ammodytes venom, inhibited binding only at very high concentrations, whereas alpha-dendrotoxin, beta-bungarotoxin, and crotoxin A had no influence on the [125I]ammodytoxin C-specific binding. The ammodytoxin C neuronal binding site therefore overlaps, at least partially, with the neuronal acceptors for some of the related presynaptically neurotoxic phospholipases A2 (beta-neurotoxins). [125I]-Ammodytoxin C was covalently attached to its acceptor by chemical cross-linking. Subsequent SDS-PAGE analysis followed by autoradiography revealed saturably labeled membrane components with apparent M(r) values of 51,000 (weaker band) and 53,000-56,000 (stronger band). Pretreatment of synaptic membranes with Staphylococcus aureus V-8 proteinase and proteinase K, heat, or low pH decreased the [125I]ammodytoxin C-specific binding to various extents, but never abolished it completely. Membrane protein and certain phospholipids residing in its vicinity are therefore most likely involved in the binding of ammodytoxin C to bovine synaptic membranes.
在牛大脑皮层突触膜中检测到了沙蝰毒素C的一个特异性、高亲和力结合位点，并对其进行了部分特性鉴定。平衡结合分析显示存在单一群体的[125I]沙蝰毒素C受体，其结合参数如下：解离常数（Kd）= 6.0 nM，最大结合量（Bmax）= 5.7 pmol/mg膜蛋白。三种沙蝰毒素（A、B和C）以及响尾蛇毒素B能强烈抑制这种结合。贝氏蝰蛇磷脂酶A2是较弱的抑制剂；无毒磷脂酶A2、沙蝰肽I2以及同样来自极北蝰蛇毒液的肌毒性磷脂酶A2同系物沙蝰肽L，仅在非常高的浓度下才抑制结合，而α-树眼镜蛇毒素、β-银环蛇毒素和响尾蛇毒素A对[125I]沙蝰毒素C特异性结合没有影响。因此，沙蝰毒素C神经元结合位点至少部分地与一些相关的突触前神经毒性磷脂酶A2（β-神经毒素）的神经元受体重叠。通过化学交联将[125I]-沙蝰毒素C共价连接到其受体上。随后的SDS-PAGE分析及放射自显影显示，膜成分被饱和标记，表观分子量（M(r)）值为51,000（较弱条带）和53,000 - 56,000（较强条带）。用金黄色葡萄球菌V-8蛋白酶、蛋白酶K、加热或低pH预处理突触膜，会不同程度地降低[125I]沙蝰毒素C特异性结合，但从未完全消除。因此，膜蛋白及其附近的某些磷脂很可能参与了沙蝰毒素C与牛突触膜的结合。
