Preisig-Müller R, Muster G, Kindl H
Biochemie, FB Chemie, Marburg, Germany.
Eur J Biochem. 1994 Jan 15;219(1-2):57-63. doi: 10.1111/j.1432-1033.1994.tb19914.x.
Proteins similar to the bacterial Dnaj protein have been implicated as molecular chaperones in different compartments of eukaryots. A plant equivalent is now described in tissues of dark-grown cucumber seedlings. Using a cucumber Dnaj protein produced by expression in bacteria, we raised polyclonal antibodies against the protein and used them for localization studies. In etiolated cucumber seedlings, both cotyledons and hypocotyledons were found to contain Dnaj proteins. Cell fractionation of etiolated cotyledons showed that Dnaj proteins were detectable mainly in the postnuclear cell fraction after sedimentation at 10,000 x g, and in the microsomes. Following subfractionation by sucrose density gradient centrifugation and analysis by immunoblotting, a 53-kDa protein was attributed to the glyoxysomal fraction and an 80-kDa protein to the mitochondrial fraction. The glyoxysomal Dnaj protein behaved as a membrane-bound form. Upon heat shock, a slight increase in the content of the glyoxysomal Dnaj protein was found. When glyoxysomes were treated with protease and subsequently isolated by gradient centrifugation, virtually all immunologically detectable Dnaj protein was removed. Administration of radiolabelled mevalonic acid to cotyledons and isolation of glyoxysomes yielded labelled Dnaj protein which remained membrane bound during the purification of glyoxysomal membranes by floatation in a density gradient.
与细菌Dnaj蛋白相似的蛋白质已被认为是真核生物不同区室中的分子伴侣。现在在黑暗生长的黄瓜幼苗组织中描述了一种植物等同物。利用在细菌中表达产生的黄瓜Dnaj蛋白，我们制备了针对该蛋白的多克隆抗体，并将其用于定位研究。在黄化黄瓜幼苗中，发现子叶和下胚轴均含有Dnaj蛋白。黄化子叶的细胞分级分离表明，Dnaj蛋白主要在10,000×g沉降后的核后细胞分级分离物和微粒体中可检测到。通过蔗糖密度梯度离心进行亚分级分离并通过免疫印迹分析后，一种53 kDa的蛋白被归为乙醛酸循环体分级分离物，一种80 kDa的蛋白归为线粒体分级分离物。乙醛酸循环体Dnaj蛋白表现为膜结合形式。热激后，发现乙醛酸循环体Dnaj蛋白的含量略有增加。当用蛋白酶处理乙醛酸循环体并随后通过梯度离心分离时，几乎所有免疫可检测的Dnaj蛋白都被去除。将放射性标记的甲羟戊酸施用于子叶并分离乙醛酸循环体，得到标记的Dnaj蛋白，在通过密度梯度漂浮纯化乙醛酸循环体膜的过程中，该蛋白仍与膜结合。
