Valtorta F, Madeddu L, Meldolesi J, Ceccarelli B
J Cell Biol. 1984 Jul;99(1 Pt 1):124-32. doi: 10.1083/jcb.99.1.124.
The receptor for alpha-latrotoxin, the major protein component of the black widow spider venom, was investigated by the use of the purified toxin and of polyclonal, monospecific anti-alpha-latrotoxin antibodies. Experiments on rat brain synaptosomes (where the existence of alpha-latrotoxin receptors was known from previous studies) demonstrated that the toxin-receptor complex is made stable by glutaraldehyde fixation. At saturation, each such complex was found to bind on the average five antitoxin antibody molecules. In frog cutaneous pectoris muscles, the existence of a finite number of high-affinity receptors was revealed by binding experiments with 125I-alpha-latrotoxin (Kd = 5 X 10(-10) M; bmax = 1.36 +/- 0.16 [SE] X 10(9) sites/mg tissue, dry weight). Nonpermeabilized muscles were first treated with alpha-latrotoxin, and then washed, fixed, dissociated into individual fibers, and treated with anti-alpha-latrotoxin antibodies and finally with rhodamine-conjugated sheep anti-rabbit antibodies. In these preparations, muscle fibers and unmyelinated preterminal nerve branches were consistently negative, whereas bright specific fluorescent images, indicative of concentrated alpha-latrotoxin binding sites, appeared in the junctional region. These images closely correspond in size, shape, and localization to endplates decorated by the acetylcholinesterase reaction. The presynaptic localization of the specific fluorescence found at frog neuromuscular junctions is supported by two sets of findings: (a) fluorescent endplate images were not seen in muscles that had been denervated; and (b) the distribution of fluorescence in many fibers treated with alpha-latrotoxin at room temperature was the one expected from swollen terminal branches. Swelling of terminals is a known morphological change induced by alpha-latrotoxin in this preparation. When muscles were treated with either proteolytic enzymes (trypsin, collagenase) or detergents (Triton X-100) before exposure to alpha-latrotoxin, the specific fluorescent endplate images failed to appear. Taken together these findings indicate that the alpha-latrotoxin receptor is an externally exposed protein highly concentrated in the nerve terminal plasma membrane. Its density (number per unit area) at the frog neuromuscular junction can be calculated to be approximately 2,400/micron2.
利用纯化毒素以及多克隆、单特异性抗α-拉托毒素抗体，对黑寡妇蜘蛛毒液的主要蛋白质成分α-拉托毒素的受体进行了研究。在大鼠脑突触体上开展的实验（此前研究已证实α-拉托毒素受体的存在）表明，戊二醛固定可使毒素-受体复合物稳定。在饱和状态下，发现每个此类复合物平均结合五个抗毒素抗体分子。在青蛙胸皮肌中，通过用125I-α-拉托毒素进行结合实验揭示了存在有限数量的高亲和力受体（解离常数Kd = 5×10⁻¹⁰ M；最大结合量bmax = 1.36±0.16 [标准误]×10⁹个位点/毫克组织，干重）。未通透处理的肌肉先经α-拉托毒素处理，然后洗涤、固定，解离成单根纤维，再用抗α-拉托毒素抗体处理，最后用罗丹明偶联的羊抗兔抗体处理。在这些标本中，肌纤维和无髓鞘的终末前神经分支始终呈阴性，而在连接区域出现了明亮的特异性荧光图像，表明存在浓缩的α-拉托毒素结合位点。这些图像在大小、形状和定位上与经乙酰胆碱酯酶反应修饰的终板紧密对应。在青蛙神经肌肉接头处发现的特异性荧光的突触前定位得到了两组研究结果的支持：（a）在去神经支配的肌肉中未见到荧光终板图像；（b）在室温下用α-拉托毒素处理的许多纤维中，荧光分布符合终末分支肿胀所预期的情况。终末肿胀是该标本中α-拉托毒素诱导的一种已知形态学变化。当肌肉在暴露于α-拉托毒素之前先用蛋白水解酶（胰蛋白酶、胶原酶）或去污剂（曲拉通X-100）处理时，特异性荧光终板图像未能出现。综合这些研究结果表明，α-拉托毒素受体是一种高度浓缩于神经末梢质膜外部的暴露蛋白。其在青蛙神经肌肉接头处的密度（每单位面积的数量）经计算约为2400/微米²。
