Purification and physical characterization of nucleic acid helix-unwinding proteins from calf thymus.
Purification and physical characterization of nucleic acid helix-unwinding proteins from calf thymus.
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DOI：
10.1016/s0021-9258(17)33665-7
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发表时间：
1976-04
期刊：
影响因子：
--
通讯作者：
G. Herrick;B. Alberts
中科院分区：
文献类型：
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作者：
G. Herrick;B. Alberts
我们设计了一个通用的蛋白质分级分离程序，选择真核生物的DNA结合蛋白，其中一些类似于原核生物的DNA解旋蛋白。选择这样的蛋白质：（a）通过天然DNA-纤维素柱，（B）与变性DNA-纤维素柱结合，和（c）在用聚阴离子硫酸葡聚糖钠盐的稀溶液冲洗期间保持与变性DNA-纤维素柱结合。当这种分级应用于小牛胸腺的可溶性蛋白质时，回收了三种主要的蛋白质种类。占主导地位的一个具有约24，000的表观分子量在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，是近中性等电，并洗脱作为一个单体从变性DNA-纤维素在中等NaCl浓度。这种蛋白质，命名为小牛解旋蛋白1（UP 1），已被纯化至同质。然而，等电聚焦揭示了四个或五个亚种（显然由单电荷差异分开），它们对DNA的亲和力明显不同。获得了另外两种主要蛋白质，它们在十二烷基硫酸钠中的表观分子量为33，000：第一种蛋白质用低盐从DNA-纤维素中洗脱，作为均匀的制剂，似乎是碱性蛋白质（虽然它显然不是组蛋白）;另一种是从DNA-纤维素中洗脱出来的“高盐洗脱级分”的主要成分，“是一种酸性蛋白质，可与较低的高分子量物质共同纯化。我们和其他人在小鼠、仓鼠、猴和人类的组织培养细胞中观察到了与这三种主要小牛胸腺蛋白相似的蛋白质，这表明它们在真核生物中广泛存在。
We have devised a general protein fractionation procedure which selects for eukaryotic DNA-binding proteins, some of which resemble DNA-unwinding proteins from prokaryotes. Proteins were selected which (a) pass through a native DNA-cellulose column, (b) bind to a denatured DNA-cellulose column, and (c) remain bound to the latter column during a rinse with a dilute solution of the sodium salt of the polyanion dextran sulfate. When this fractionation was applied to the soluble proteins fo calf thymus, three major protein species were recovered. The predominant one has an apparent molecular weight of about 24,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is isoelectric near neutrality, and elutes as a monomer from denatured DNA-cellulose at moderate NaCl concentrations. This protein, designated calf-unwinding protein 1 (UP1), has been purified to homogeneity. However, isoelectric focusing reveals four or five subspecies (apparently separated by single-charge differences) which differ appreciably in their affinities for DNA. Two other major proteins are obtained which have apparent molecular weights in sodium dodecyl sulfate of 33,000: the first, which elutes with low salt from DNA-cellulose as a homogeneous preparation, appears to be a basic protein (although it is clearly not a histone); the other, which elutes from DNA-cellulose as the major component of a "high salt eluting fraction," is an acidic protein which co-purifies with less prominent species of higher molecular weights. Proteins similar to each of these three major calf thymus proteins have been observed by us and others in tissue culture cells of mouse, hamster, monkey, and humans, suggesting their wide occurrence among eukaryotes.