Ueno E, Rosenberg P
Section of Pharmacology and Toxicology, University of Connecticut, School of Pharmacy, Storrs, CT 06269.
Toxicon. 1990;28(12):1423-37. doi: 10.1016/0041-0101(90)90156-2.
Some snake venom neurotoxins, such as beta-bungarotoxin (beta-BuTX) and notexin, which inhibit the release of neurotransmitter at both peripheral and central presynaptic terminals possess phospholipase A2 activity. In contrast, most snake venom phospholipase A2 enzymes such as those isolated from Naja naja atra and Naja nigricollis are structurally homologous to these neutrotoxins but do not have any specific or potent presynaptic action although they have higher enzymatic activities than the neurotoxins. In order to investigate the mechanisms of presynaptic action of the snake venom neurotoxins, we studied their effects on phosphorylation of rat brain synaptosomal proteins. It is known that phosphorylation of synapsin I, a neuron specific and synaptic vesicle associated phosphoprotein, increases neurotransmitter release. Incubation of cerebral cortical synaptosomes with 32P-orthophosphate at 37 degrees C for 30 min, caused significant phosphorylation of a wide mol. wt range of proteins including most markedly those proteins in the mol. wt range (81,000-86,000) of synapsin I. Both snake venom phospholipase A2 neurotoxins and enzymes (5, 15 and 50 nM) inhibited phosphorylation in a Ca2(+)-dependent manner with the following order of potencies: beta-BuTX greater than N.n. atra phospholipase A2 greater than or equal to notexin greater than N. nigricollis phospholipase A2. Five nanomoles of beta-BuTX, which has the lowest phospholipase A2 activity, inhibited phosphorylation of a wide range of mol. wt proteins (51,000-188,000) by 42-58%. At the same concentration, N.n. atra phospholipase A2 (which possesses the highest enzymatic activity), notexin and N. nigricollis phospholipase A2 caused less inhibition than beta-BuTX, ranging from 0-40% depending on the agent used. These results indicate that there is no correlation between their potencies in inhibiting phosphorylation and the levels of their phospholipase A2 activities. An inhibitory activity on phosphorylation may be at least partially responsible for a presynaptically-induced block of neurotransmitter release.
一些蛇毒神经毒素，如β-银环蛇毒素（β-BuTX）和诺维毒素，可抑制外周和中枢突触前终末神经递质的释放，它们具有磷脂酶A2活性。相比之下，大多数蛇毒磷脂酶A2酶，如从眼镜蛇和黑颈眼镜蛇中分离出的那些酶，在结构上与这些神经毒素同源，但尽管它们的酶活性比神经毒素高，却没有任何特异性或强效的突触前作用。为了研究蛇毒神经毒素的突触前作用机制，我们研究了它们对大鼠脑突触体蛋白磷酸化的影响。已知突触素I是一种神经元特异性且与突触小泡相关的磷蛋白，其磷酸化会增加神经递质的释放。将大脑皮质突触体与32P-正磷酸盐在37℃孵育30分钟，会导致广泛分子量范围的蛋白质发生显著磷酸化，其中最明显的是分子量范围在（8万1千-8万6千）的突触素I蛋白。蛇毒磷脂酶A2神经毒素和酶（5、15和50 nM）均以Ca2+依赖的方式抑制磷酸化，其效力顺序如下：β-BuTX＞眼镜蛇磷脂酶A2≥诺维毒素＞黑颈眼镜蛇磷脂酶A2。5纳摩尔的β-BuTX，其磷脂酶A2活性最低，可抑制广泛分子量范围的蛋白质（5万1千-18万8千）的磷酸化达42%-58%。在相同浓度下，眼镜蛇磷脂酶A2（具有最高的酶活性）、诺维毒素和黑颈眼镜蛇磷脂酶A2的抑制作用比β-BuTX小，根据所用试剂的不同，抑制范围为0%-40%。这些结果表明，它们抑制磷酸化的效力与其磷脂酶A2活性水平之间没有相关性。对磷酸化的抑制活性可能至少部分地导致了突触前诱导的神经递质释放阻滞。
