Paradis G, Gaudreau J, Frenette G, Thabet M, Tremblay R R, Dubé J Y
Laboratory of Hormonal Bioregulation, Laval University Hospital Research Centre, Ste-Foy, Que., Canada.
Biochem Cell Biol. 1991 Aug;69(8):561-5. doi: 10.1139/o91-083.
As a first step in understanding the changes in protein synthesis that occur in renal cell carcinoma, we have prepared poly(A)+ RNA from surgically removed tumors and from their normal tissue counterpart. These RNAs were then translated in vitro in the rabbit reticulocyte lysate system and the synthesized labeled polypeptides were separated by one- and two-dimensional gel electrophoresis. A major 25-kDa primary translation product was observed with all renal cell carcinomas. The synthesis of this protein was barely detectable with the RNA from normal tissue adjacent to the tumor. To determine if this protein could be further processed (removal of signal peptide and (or) core glycosylation), canine pancreatic microsomal membranes were added to the system. This addition resulted in the formation of a vertical row of three additional spots, with the same isoelectric point as the primary translation product and with molecular masses ranging from 27 to 31 kDa. The 31-kDa protein was retained on Concanavalin A. After digestion with endoglycosidase H, it was no longer visible on sodium dodecyl sulfate gels and a new 27-kDa band was generated suggesting that the mature protein was indeed a glycoprotein. Future experiments will be aimed at identifying this protein and examining its potential value as a marker of renal cell carcinoma.
作为了解肾细胞癌中蛋白质合成变化的第一步，我们从手术切除的肿瘤及其正常组织对应物中制备了聚腺苷酸加尾（poly(A)+）RNA。然后，这些RNA在兔网织红细胞裂解物系统中进行体外翻译，合成的标记多肽通过一维和二维凝胶电泳进行分离。在所有肾细胞癌中均观察到一种主要的25 kDa初级翻译产物。用肿瘤旁正常组织的RNA几乎检测不到这种蛋白质的合成。为了确定这种蛋白质是否可以进一步加工（去除信号肽和/或核心糖基化），将犬胰腺微粒体膜添加到系统中。这种添加导致形成了垂直排列的另外三个斑点，其等电点与初级翻译产物相同，分子量范围为27至31 kDa。31 kDa的蛋白质保留在伴刀豆球蛋白A上。用内切糖苷酶H消化后，它在十二烷基硫酸钠凝胶上不再可见，并且产生了一条新的27 kDa条带，表明成熟蛋白质确实是一种糖蛋白。未来的实验将旨在鉴定这种蛋白质，并研究其作为肾细胞癌标志物的潜在价值。
