在中性pH值下对牛脑细胞膜制剂中32P标记蛋白质的分离。钠钾离子激活的三磷酸腺苷酶成分的部分特性分析。

Separation at neutral pH value of 32P-labelled proteins in a membrane preparation from ox brain. Partial characterization of component of the sodium-plus-potassium ion-activited adenosine triphosphatase.

作者信息

Alexander D R, Rodnight R

出版信息

Biochem J. 1974 Feb;137(2):253-62. doi: 10.1042/bj1370253.

DOI:10.1042/bj1370253

PMID:4363110

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1166112/

Abstract

Ox brain microsomal fractions were labelled with [(32)P]ATP in the presence of Na(+) and the reaction was stopped with sodium dodecyl sulphate. The Na(+)-dependent bound phosphate was isolated on Sephadex G-25 and by acetone precipitation. The bound phosphate isolated under these neutral conditions was labile to hydroxylamine and gave the same pH profile of hydrolysis as that isolated by precipitation with strong acids. 2. When membrane protein was labelled with [(32)P]ATP, solubilized with sodium dodecyl sulphate and fractionated on Sepharose 6B, the Na(+)-dependent label emerged in a peak corresponding to protein of molecular weight 570000-580000. On fractionation of this protein peak on polyacrylamide gels containing detergent and urea, the Na(+)-dependent label occurred in a single band corresponding to a protein of molecular weight 102000. 3. Fractionation on Sepharose 6B of protein labelled with [(32)P]ATP in the absence of Na(+) revealed three labelled peaks, one of which corresponded in position to the Na(+)-dependent label. Electrophoresis of this peak material on polyacrylamide gels showed that most of the label occurred in two fast-running bands. Cyclic AMP stimulated the labelling in these two bands, but had no effect on the labelling of the band corresponding in position to the Na(+)-dependent label. 4. Di-isopropyl [(32)P]phosphorofluoridate also labelled the band corresponding to the Na(+)-dependent label on gel electrophoresis. The labelling of this band by the reagent was inhibited by 50-60% by 3mm-ATP, but there was no evidence to suggest that the group labelled is normally phosphorylated by ATP.

摘要

在有Na⁺存在的情况下，用[³²P]ATP标记牛脑微粒体部分，然后用十二烷基硫酸钠终止反应。通过葡聚糖凝胶G - 25和丙酮沉淀分离出依赖Na⁺的结合磷酸盐。在这些中性条件下分离出的结合磷酸盐对羟胺不稳定，并且其水解的pH曲线与用强酸沉淀分离出的相同。2. 当用[³²P]ATP标记膜蛋白，并用十二烷基硫酸钠溶解，然后在琼脂糖6B上进行分级分离时，依赖Na⁺的标记出现在对应分子量为570000 - 580000的蛋白质峰中。在含有去污剂和尿素的聚丙烯酰胺凝胶上对该蛋白质峰进行分级分离时，依赖Na⁺的标记出现在对应分子量为102000的单一蛋白条带中。3. 在没有Na⁺的情况下，用[³²P]ATP标记的蛋白质在琼脂糖6B上进行分级分离，显示出三个标记峰，其中一个峰的位置与依赖Na⁺的标记相对应。该峰物质在聚丙烯酰胺凝胶上进行电泳显示，大部分标记出现在两条快速迁移的条带中。环磷酸腺苷刺激了这两条带中的标记，但对与依赖Na⁺的标记位置相对应的条带的标记没有影响。4. 二异丙基[³²P]磷酰氟化物在凝胶电泳中也标记了与依赖Na⁺的标记相对应的条带。3mM的ATP可使该试剂对这条带的标记抑制50 - 60%，但没有证据表明被标记的基团通常由ATP磷酸化。