Pfeifle J, Anderer F A
Eur J Biochem. 1984 Mar 1;139(2):417-24. doi: 10.1111/j.1432-1033.1984.tb08021.x.
Phosphoprotein pp 135 is one of the dominant proteins endogenously phosphorylated in cellular sonicates during short-time exposure to [gamma-32P]ATP. Mouse cells growing exponentially show the highest pp 135 level as determined by endogenous phosphorylation and immunobinding assays. Disruption of cells in the absence of calcium at low magnesium concentration renders more than 90% pp 135 into the cytosolic fraction. A five-step purification yields greater than 95% pure pp 135. The cellular location of pp 135 was determined with a rat anti-(mouse pp 135) serum by immunofluorescence in mouse cell lines and cryostat sections of normal mouse tissue. We observed fluorescence predominantly of nucleolar structures, confirmed by studies of isolated nuclei and nucleoli. Cross-reacting nucleolar phosphoproteins were identified in cell lines of other species with molecular masses of 128 kDa (human), 135 kDa (hamster) and 118 kDa (Drosophila). Endogenous phosphorylation of pp 135 investigated with purified mouse nucleoli showed optimal activity at isotonicity, pH 7.3, in the presence of 10 mM magnesium ions.
磷蛋白pp 135是在短时间暴露于[γ-32P]ATP时细胞超声裂解物中内源性磷酸化的主要蛋白质之一。通过内源性磷酸化和免疫结合测定法确定，指数生长的小鼠细胞显示出最高的pp 135水平。在低镁浓度下无钙条件下破坏细胞，可使超过90%的pp 135进入胞质部分。五步纯化可得到纯度大于95%的pp 135。通过在小鼠细胞系和正常小鼠组织的低温切片中进行免疫荧光，用大鼠抗（小鼠pp 135）血清确定了pp 135在细胞内的定位。通过对分离的细胞核和核仁的研究证实，我们观察到荧光主要存在于核仁结构中。在其他物种的细胞系中鉴定出了交叉反应的核仁磷蛋白，其分子量分别为128 kDa（人）、135 kDa（仓鼠）和118 kDa（果蝇）。用纯化的小鼠核仁研究pp 135的内源性磷酸化，结果表明在等渗、pH 7.3以及存在10 mM镁离子的条件下具有最佳活性。
