Goodman J M, Maher J, Silver P A, Pacifico A, Sanders D
J Biol Chem. 1986 Mar 5;261(7):3464-8.
Peroxisomes are massively induced when methylotrophic yeasts are cultured on methanol as the sole carbon and energy source. An analysis of the protein composition of the peroxisomal membrane and the generation of probes against two peroxisomal membrane proteins (PMPs) have been undertaken. Peroxisomes from Candida boidinii were obtained from sucrose gradients as previously described or from a novel one-step purification of the organelle on a Percoll gradient. The protein composition of the membranes from these two preparations was virtually identical. About 10 proteins comprise nearly all of its protein mass. The most prominent proteins have molecular masses of 120, 100, 47, 31-32 (a triplet), and 20 kDa; significant amounts of alcohol oxidase and dihydroxyacetone synthase, the two abundant matrix proteins, also remain associated with the membrane. Glycosylation of the membrane proteins could not be detected. Exposure of the membrane to chaotropes shows that PMPs 100 and 20 are the most easily removable, whereas PMP 47 appears to be the most tightly associated. Mice were injected with peroxisomal membrane, and hybridoma lines were isolated that produced antibody against PMP 20, PMP 47, and dihydroxyacetone synthase. Indirect immunofluorescence with these monoclonal antibodies confirmed that all three proteins are localized to the peroxisomal cluster. Immunoblotting experiments demonstrated that peroxisomal membrane as well as matrix proteins are induced by methanol.
当甲基营养型酵母以甲醇作为唯一碳源和能源进行培养时，过氧化物酶体会大量诱导产生。我们对过氧化物酶体膜的蛋白质组成进行了分析，并制备了针对两种过氧化物酶体膜蛋白（PMPs）的探针。来自博伊丁假丝酵母的过氧化物酶体可通过先前描述的蔗糖梯度法获得，也可通过在Percoll梯度上对该细胞器进行新型一步纯化法获得。这两种制备物的膜蛋白组成几乎相同。大约10种蛋白质几乎构成了其所有的蛋白质质量。最突出的蛋白质分子量分别为120、100、47、31 - 32（三重峰）和20 kDa；两种丰富的基质蛋白——乙醇氧化酶和二羟基丙酮合酶，也有相当数量与膜相关。未检测到膜蛋白的糖基化。将膜暴露于离液剂表明，PMP 100和20最容易被去除，而PMP 47似乎与膜结合最紧密。给小鼠注射过氧化物酶体膜，分离出了产生针对PMP 20、PMP 47和二羟基丙酮合酶抗体的杂交瘤细胞系。用这些单克隆抗体进行间接免疫荧光证实，这三种蛋白质都定位于过氧化物酶体簇。免疫印迹实验表明，过氧化物酶体膜以及基质蛋白都由甲醇诱导产生。
