Yamaji T, Ishibashi M, Katayama S, Itabashi A, Ohsawa N, Kondo Y, Mizumoto Y, Kosaka K
J Clin Invest. 1981 Dec;68(6):1441-9. doi: 10.1172/jci110396.
The incorporation of labeled compounds into neurophysins of a transplantable human oat cell carcinoma of the lung with ectopic vasopressin production was studied in vitro. Neurophysins in cell extracts and in incubation media were isolated by immunoprecipitation and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. When cells were incubated with L-[35S]cysteine for 12 h, SDS-polyacrylamide gel electrophoresis of the immunoprecipitates from cell extract and medium resolved two forms of neurophysins with apparent molecular mass of 10,000 (10K) and 20,000 (20K). Both forms of [35S]-neurophysins were completely displaced from the immunoprecipitates by excess human neurophysin. Incubation of cells with L-[35S]cysteine and D-[3H]-glucosamine hydrochloride revealed that glucosamine was incorporated into the 20K neurophysin region, but not into 10K species. To observe the kinetics of labeling of the two forms of neurophysins, cells were incubated with L[35S]cysteine for varying periods of time. After short labeling periods, most of the radioactivity resided in 20K species, which plateaued after 1 h, whereas 10K neurophysin progressively increased in its height. When cells were chased with unlabeled cysteine after the exposure to a short pulse of labeling, 20K neurophysin peak gradually decreased with an apparent initial half-life of 1 h. In contrast, the label in 10K neurophysin steadily increased, which exceeded the former by 3 h of chase. Analysis of 20K neurophysin in cell extract by isoelectric focusing on polyacrylamide gel demonstrated that it was principally composed of a protein with an apparent isoelectric point (pI) of 5.7. These results suggest that neurophysin is synthesized in ectopic vasopressin-producing tumors by post-translational processing from a glycosylated proneurophysin with an apparent molecular mass of 20,000 daltons and a pI of 5.7.
对一株可移植的、能异位产生血管加压素的人肺燕麦细胞癌中标记化合物掺入神经垂体素的情况进行了体外研究。通过免疫沉淀法分离细胞提取物和孵育培养基中的神经垂体素，并采用十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳进行分析。当细胞与L-[³⁵S]半胱氨酸孵育12小时后，对细胞提取物和培养基免疫沉淀物进行SDS-聚丙烯酰胺凝胶电泳，可分辨出两种形式的神经垂体素，其表观分子量分别为10,000（10K）和20,000（20K）。两种形式的[³⁵S]-神经垂体素均可被过量的人神经垂体素从免疫沉淀物中完全置换出来。细胞与L-[³⁵S]半胱氨酸和D-[³H]-盐酸葡萄糖胺共同孵育显示，葡萄糖胺掺入到20K神经垂体素区域，但未掺入10K形式中。为观察两种形式神经垂体素的标记动力学，细胞与L-[³⁵S]半胱氨酸孵育不同时间。在短时间标记后，大部分放射性存在于20K形式中，1小时后达到平台期，而10K神经垂体素的高度则逐渐增加。当细胞在短时间脉冲标记后用未标记的半胱氨酸进行追踪时，20K神经垂体素峰逐渐下降，表观初始半衰期为1小时。相比之下，10K神经垂体素中的标记物稳步增加，追踪3小时后超过前者。通过聚丙烯酰胺凝胶等电聚焦分析细胞提取物中的20K神经垂体素表明，它主要由一种表观等电点（pI）为5.7的蛋白质组成。这些结果表明，神经垂体素是由异位产生血管加压素的肿瘤通过对一种表观分子量为20,000道尔顿、pI为5.7的糖基化前神经垂体素进行翻译后加工而合成的。
