Rosenfeld M G, Barrieux A
Biochemistry. 1977 Feb 8;16(3):514-8. doi: 10.1021/bi00622a027.
One of the proteins in the 0.5 M KCl eluate of rabbit reticulocyte polyribosomes which bind poly(A)-rich mRNA has been purified to apparent homogeneity using ammonium sulfate fractionation and phosphocellulose, hydroxylapatite, and diethylaminoethylcellulose column chromatography. The protein appears to contain two subunits of 66 700 and 56 400 apparent molecular weights with a 1:1 stoichiometry, since an apparent molecular weight of 110 000 was determined using Sephadex G-200 chromatography and an s020,w of 5.6 was obtained with rate-zonal sedimentation. The mRNA binding activity banded at pH 5.2-5.5 on isoelectric-focusing polyacrylamide gel electrophoresis. Protein-dependent binding appeared to be specific, since other natural or synthetic RNAs, including tRNA, ribosomal RNA, and poly(riboadenylic acid), were 90- to 250-fold less effective than mRNA at competing for binding of [3H]poly(adenylic acid)-rich mRNA. Poly(riboguanylic acid), however, was even more efficiently bound by this protein than mRNA.
利用硫酸铵分级分离以及磷酸纤维素、羟基磷灰石和二乙氨基乙基纤维素柱层析，已将兔网织红细胞多核糖体0.5M KCl洗脱液中一种能结合富含多聚腺苷酸mRNA的蛋白质纯化至表观均一。该蛋白质似乎包含两个表观分子量分别为66700和56400的亚基，化学计量比为1:1，因为使用葡聚糖G - 200层析测定的表观分子量为110000，速率区带沉降得到的s020,w为5.6。在等电聚焦聚丙烯酰胺凝胶电泳上，mRNA结合活性在pH 5.2 - 5.5处出现条带。蛋白质依赖性结合似乎具有特异性，因为包括tRNA、核糖体RNA和聚（核糖腺苷酸）在内的其他天然或合成RNA在竞争结合[³H]富含聚（腺苷酸）的mRNA时，其效力比mRNA低90至250倍。然而，聚（核糖鸟苷酸）被该蛋白质结合的效率甚至比mRNA更高。
