Jiang X C, Inouchi J, Wang D, Halpern M
Department of Biochemistry, State University of New York Health Science Center, Brooklyn 11203.
J Biol Chem. 1990 May 25;265(15):8736-44.
Following shocks with low voltage electric current, earthworms, Lumbricus terrestris, secrete a yellow mucus that has alarm properties for conspecifics and chemoattractive properties for garter snakes, Thamnophis sirtalis. A proteinaceous chemoattractant for garter snakes has been isolated and purified to homogeneity from such secretions by means of permeation chromatography and semipreparative nondenaturing polyacrylamide gel electrophoresis. The purified protein is highly attractive to garter snakes; it loses its activity after proteolytic digestion. It is a glycoprotein consisting of a single polypeptide chain with an NH2-terminal alanine. This chemoattractant has a minimum molecular mass of 15.4 kDa calculated from its amino acid and carbohydrate contents and an apparent molecular mass of about 20 kDa as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has a pI of about 4.0, and it binds wheat germ agglutinin but not concanavalin A. This chemoattractant shows a protein to carbohydrate ratio of 2.0 +/- 0.08 (n = 5) and a ratio of total sugar to amino sugar of 1.9 +/- 0.08 (n = 3). The sequence of its NH2-terminal 15 amino acid residues has been determined. Studies were also conducted on the chemosignal transduction through the vomeronasal sensory system of the garter snake. Dot blot analysis showed that the purified chemoattractant bound to snake vomeronasal sensory epithelial membrane fractions. It did not bind to membrane extracts of the nonsensory epithelium of the vomeronasal mushroom body. The chemoattractant also bound specifically to vomeronasal sensory epithelial membrane in a reversible and saturable fashion with Kd and Bmax values of about 0.3 microM and 0.4 nmol/mg of protein, respectively. In electrophysiological studies, the chemoattractant applied to the vomeronasal epithelium caused an increase in firing rate of individual neurons in the accessory olfactory bulb of garter snakes, the projection site for vomeronasal neurons. The present results are the first clear biochemical and electrophysiological evidence for a vomeronasal epithelium response to a purified nonvolatile odorant, and this makes the garter snake vomeronasal system ideal for studying the mechanisms of chemosignal transduction.
用低电压电流电击后，蚯蚓（Lumbricus terrestris）会分泌一种黄色黏液，这种黏液对同种个体具有警示特性，对束带蛇（Thamnophis sirtalis）具有化学吸引特性。通过渗透色谱法和半制备非变性聚丙烯酰胺凝胶电泳，已从这种分泌物中分离并纯化出一种对束带蛇具有蛋白质性质的化学引诱剂，使其达到同质状态。纯化后的蛋白质对束带蛇具有高度吸引力；经蛋白水解消化后其活性丧失。它是一种糖蛋白，由一条带有NH2端丙氨酸的单多肽链组成。根据其氨基酸和碳水化合物含量计算，这种化学引诱剂的最小分子量为15.4 kDa，从十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳估计其表观分子量约为20 kDa。它的pI约为4.0，能结合麦胚凝集素但不能结合伴刀豆球蛋白A。这种化学引诱剂的蛋白质与碳水化合物的比例为2.0±0.08（n = 5），总糖与氨基糖的比例为1.9±0.08（n = 3）。已确定其NH2端15个氨基酸残基的序列。还对束带蛇犁鼻感觉系统的化学信号转导进行了研究。斑点印迹分析表明，纯化后的化学引诱剂与蛇的犁鼻感觉上皮细胞膜组分结合。它不与犁鼻蘑菇体非感觉上皮的膜提取物结合。这种化学引诱剂还以可逆和饱和的方式特异性结合犁鼻感觉上皮细胞膜，Kd和Bmax值分别约为0.3 microM和0.4 nmol/mg蛋白质。在电生理研究中，将这种化学引诱剂应用于犁鼻上皮会导致束带蛇副嗅球中单个神经元的放电频率增加，副嗅球是犁鼻神经元的投射部位。目前的结果是关于犁鼻上皮对纯化的非挥发性气味剂产生反应的首个明确的生化和电生理证据，这使得束带蛇犁鼻系统成为研究化学信号转导机制的理想对象。
