Miyakawa T, Kaji M, Yasutake T, Jeong Y K, Tsuchiya E, Fukui S
J Bacteriol. 1985 Apr;162(1):294-9. doi: 10.1128/jb.162.1.294-299.1985.
The involvement of protein sulfhydryls for the signaling of rhodotorucine A, a mating pheromone produced by mating type A cells of Rhodosporidium toruloides, was investigated by the use of sulfhydryl compounds. The sulfhydryl-blocking reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; Ellman's reagent) strongly inhibited both the biological effect of the pheromone on the recipient cell and the hydrolysis of the pheromone, which is catalyzed by the mating type-specific surface endopeptidase of the recipient cell. Conversely, the two reactions were markedly enhanced by the presence of the reducing reagent dithiothreitol. The inhibitory effect of DTNB on the pheromone response of the recipient cell was specific to an initial stage of the differentiation; once it had initiated, the reagent had no effect on its progression. The results suggested that dithiothreitol enhances and DTNB impairs the efficiency with which the pheromone triggers sexual d differentiation. The reaction of DTNB with cellular protein sulfhydryls was highly restricted to those at the exterior surface of the membrane due to the impermeability of the reagent through the membrane. Phosphorylation of endogenous proteins, which is modulated by the pheromone added to an in vitro phosphorylation system, was also blocked by DTNB. The results showed that sulfhydryl groups are involved in the pheromone hydrolysis by the surface endopeptidase of the recipient cell and that pheromone metabolism is indispensable for the signaling reaction. We suggest that the modulation of protein phosphorylation of membrane proteins by the pheromone is an initial transmembrane response coupled to pheromone metabolism.
通过使用巯基化合物，研究了蛋白质巯基在罗伦隐球酵母A交配型细胞产生的交配信息素红球氨酸A信号传导中的作用。巯基阻断剂5,5'-二硫代双（2-硝基苯甲酸）（DTNB；埃尔曼试剂）强烈抑制了信息素对受体细胞的生物学效应以及信息素的水解，后者由受体细胞的交配型特异性表面内肽酶催化。相反，还原试剂二硫苏糖醇的存在显著增强了这两个反应。DTNB对受体细胞信息素反应的抑制作用在分化的初始阶段具有特异性；一旦分化开始，该试剂对其进展没有影响。结果表明，二硫苏糖醇增强而DTNB损害了信息素触发有性分化的效率。由于DTNB不能透过细胞膜，它与细胞蛋白质巯基的反应高度局限于膜外表面的巯基。添加到体外磷酸化系统中的信息素所调节的内源性蛋白质的磷酸化也被DTNB阻断。结果表明，巯基参与了受体细胞表面内肽酶对信息素的水解，并且信息素代谢对于信号反应是必不可少的。我们认为，信息素对膜蛋白磷酸化的调节是与信息素代谢相关的初始跨膜反应。
