Bozner P
Department of Pathology, University of South Alabama Medical Center, Mobile 36617, USA.
J Parasitol. 1996 Feb;82(1):103-11.
The responses to heat shock in Tritrichomonas mobilensis, a squirrel monkey parasite and Tritrichomonas augusta, an amphibian trichomonad, were evaluated by means of metabolic labeling with [35S]methionine. Electrophoretically separated trichomonad proteins synthesized at different temperatures were visualized by autoradiography and the label incorporation quantitated by a trichloroacetic acid precipitation procedure. A considerable difference in thermotolerance between the two species was found as the protein synthesis reached a maximum at 41 C in T. mobilensis and 37 C in T. augusta. The latter tolerated temperature increases 13 C above normal cultivation temperatures as compared to only 4 C thermotolerance range above normal in T. mobilensis. Major heat shock proteins (Hsps) were expressed in both T. mobilensis (with apparent Mr 94, 72, and 58 kDa) and T. augusta (Mr 94, 70, and 56 kDa) as revealed by autoradiography. Western blot analysis with polyclonal antibody against DnaK of Escherichia coli showed the presence of antigenic Hsp70 homologs in both trichomonads. Similarly, a polyclonal antibody against Hsp60 with broad interspecies cross-reactivity detected Hsp60 homologs in both T. mobilensis and T. augusta. The anti-DnaK antibody cross-reacted with a T. mobilensis protein localized in Golgi apparatus as demonstrated by immunoelectron microscopy. Immunocytochemistry on trichomonad frozen sections revealed the presence of the Hsp60 homolog in light-microscopic granules corresponding to hydrogenosomes.
通过用[35S]甲硫氨酸进行代谢标记，评估了松鼠猴寄生虫活跃三毛滴虫（Tritrichomonas mobilensis）和两栖类毛滴虫奥古斯塔三毛滴虫（Tritrichomonas augusta）对热休克的反应。通过放射自显影观察在不同温度下合成的经电泳分离的毛滴虫蛋白质，并通过三氯乙酸沉淀法对标记掺入量进行定量。发现这两个物种在耐热性上有很大差异，活跃三毛滴虫的蛋白质合成在41℃达到最大值，而奥古斯塔三毛滴虫在37℃达到最大值。与活跃三毛滴虫仅比正常培养温度高4℃的耐热范围相比，后者能耐受比正常培养温度高13℃的温度升高。放射自显影显示，主要的热休克蛋白（Hsps）在活跃三毛滴虫（表观分子量为94、72和58 kDa）和奥古斯塔三毛滴虫（分子量为94、70和56 kDa）中均有表达。用针对大肠杆菌DnaK的多克隆抗体进行的蛋白质印迹分析表明，两种毛滴虫中均存在抗原性Hsp70同源物。同样，一种具有广泛种间交叉反应性的针对Hsp60的多克隆抗体在活跃三毛滴虫和奥古斯塔三毛滴虫中均检测到了Hsp60同源物。免疫电子显微镜显示，抗DnaK抗体与活跃三毛滴虫中定位于高尔基体的一种蛋白质发生交叉反应。对毛滴虫冰冻切片的免疫细胞化学分析显示，在对应于氢化酶体的光镜颗粒中存在Hsp60同源物。
