Oberg S G, Kelly R B
Biochim Biophys Acta. 1976 May 21;433(3):662-73. doi: 10.1016/0005-2736(76)90289-3.
Brief exposure to the protein neurotoxin, beta-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using 125I-labelled toxin. At low membrane protein concentration 125I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration 125I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes. In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of 125I-labeled toxin binding sites on brain mitochondrial preparations (3.3 +/- 0.3 pmol/mg) exceeded by a factor of ten the density on liver mitochondrial preparations (0.3 +/- 0.05 pmol/mg). It is also shown that labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude that beta-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.
已知短暂接触蛋白质神经毒素β-银环蛇毒素会通过阻断神经末梢递质的释放不可逆地破坏神经肌肉传递。这种神经毒素还具有磷脂酶A2活性，尽管一般来说磷脂酶毒性不强。为了确定该分子的毒性是否可能源于与神经组织的特异性结合，我们使用125I标记的毒素寻找高亲和力、可饱和的结合。在低膜蛋白浓度下，125I标记的毒素结合与膜的量成正比；在固定膜浓度下，125I标记的毒素表现出可饱和结合。碘化不太可能显著改变毒素的性质，因为通过竞争实验判断，碘化毒素与天然毒素具有相当的结合亲和力。毒素与脑、肝和红细胞膜结合的比较表明，所有膜都有高亲和力结合位点，解离常数在1至2纳摩尔之间。这与先前显示抑制线粒体功能的浓度相当。然而，这些位点的密度显示出显著差异，例如，脑细胞膜制剂的位点密度为13.0皮摩尔/毫克蛋白，肝细胞膜为2.4皮摩尔/毫克，红细胞膜为0.25皮摩尔/毫克。在早期工作中我们已经表明，脑线粒体摄取钙受到抑制时的毒素浓度比肝线粒体摄取时低得多。肝和脑线粒体都能特异性结合毒素，但脑线粒体制剂上125I标记的毒素结合位点密度（3.3±0.3皮摩尔/毫克）比肝线粒体制剂上的密度（0.3±0.05皮摩尔/毫克）高出10倍。还表明标记的毒素不会穿过突触体膜，这表明线粒体可能不是毒素在体内的作用部位。我们得出结论，β-银环蛇毒素是一种能以高亲和力与细胞膜特异性结合的酶。
