Physicochemical characterization of the 68 000-dalton protein of bovine neurofilaments.
Physicochemical characterization of the 68 000-dalton protein of bovine neurofilaments.
复制标题
牛神经丝 68 000 道尔顿蛋白质的物理化学特征。
DOI：
10.1021/bi00308a004
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发表时间：
1984
期刊：
影响因子：
2.9
通讯作者：
WilliamsJr,RC
中科院分区：
文献类型：
--
作者：
Lifsics,MR;WilliamsJr,RC
Miriam R. Lifsics*和Robley C. Williams, Jr.*摘要：采用deae -纤维素和羟基磷灰石相结合的色谱法，在含有8 M尿素的缓冲液中纯化了牛神经纤维中68000道尔顿的蛋白。虽然这种蛋白与神经丝的其他蛋白的分离似乎受到几种成分混合结合的阻碍，但获得了一种几乎均匀的产品用于研究。在含有8 M尿素的缓冲液中进行沉降平衡实验，结果表明该分子为单体，分子量为70 600±2000。在相同条件下的圆二向色光谱没有残留a-螺旋的证据。在控制孔玻璃上对8 M尿素进行分子筛层析，结果表明该分子以意想不到的小体积洗脱。小洗脱体积不依赖于蛋白质浓度，也不太可能是分子间结合的结果。更确切地说，单体的构象可能比经典的随机线圈更刚性或延伸。当分解成0.01 M三（羟甲基）氨基甲烷/l mM乙二醇双（/3-氨基乙基）神经细丝时，哺乳动物神经元的中间细丝由三种类型的亚基多肽组成：一种是68000道尔顿（NF68），一种是160000道尔顿（NF160），一种是210000道尔顿（NF210）[评论见Lazarides (1982) Schlaepfer (1979) and Williams & Runge(1983)]。目前来自抗体标记（Willard & Simon, 1981； Sharp等，1982）和重组研究（Geisler & Weber, 1981; Liem, 1982; Liem & Hutchison, 1982； Zackroff等，1982）的证据表明，纤维丝由NF68的内核组成，NF160和NF210以一种未知的方式排列在周围。与其他类型的中间纤维一样，神经纤维在中等离子强度的中性缓冲液中相当稳定（Schlaepfer, 1978），但它们可以在变性溶剂中溶解，如8 M尿素或6 M胍盐酸盐。亚基在变性溶剂中被色谱分离（Willard et al., 1980; Moon et al., 1981; Geisler & Weber, 1981; Chiu & Norton, 1982; hoguangelletti et al., 1982; Liem, 1982; Liem & Hutchison, 1982），尽管这种分离显然是困难的，并且没有完整的文献记录。纯化的NF68溶液在ap-缓冲液中透析时，会自发地重新组装成直径约10nm的相当均匀的细丝
Miriam R. Lifsics* and Robley C. Williams, Jr.* abstract: The 68 000-dalton protein from bovine neurofi-laments was purified by a combination of chromatography on DEAE-cellulose and on hydroxylapatite in buffers containing 8 M urea. Although the separation of this protein from the other proteins of the neurofilament appeared to be hampered by a mixed association of the several components, a nearly homogeneous product was obtained for study. Sedimentation equilibrium experiments in buffers containing 8 M urea showed the moleculeto be a monomer with a molecular weight of 70 600±2000. Circular dichroic spectra taken under the same conditions gave no evidence of residual a-helix. Mo-lecular sieve chromatography in 8 M urea on controlled-pore glass showed that themolecule eluted at an unexpectedly small volume. The small elution volume did notdepend significantly on protein concentration and is unlikely to be the result of intermolecular association. Rather, the monomer probably has a conformation more rigid or extended than a classical random coil. When dialyzed into 0.01 M tris (hydroxy-methyl) aminomethane/l mM ethylene glycol bis (/3-aminoethyl eurofilaments, the intermediate filaments of mammalian neurons, are composed of three types of subunit polypeptides: one of 68000daltons (NF68), 1 one of 160000 daltons (NF160), and one of 210000 daltons (NF210)[for reviews, see Lazarides (1982) Schlaepfer (1979), and Williams & Runge (1983)]. Current evidence from antibody labeling (Willard & Simon, 1981; Sharp et al., 1982) and from reassembly studies (Geisler & Weber, 1981; Liem, 1982; Liem & Hutchison, 1982; Zackroff et al., 1982) suggests that the filament consists of an inner core of NF68, with NF160 and NF210 arrayed peripherally in a still unknown fashion. In common with other types of intermediatefilaments, neuro-filaments are quite stable in neutral buffers of moderate ionic strength (Schlaepfer, 1978), but they can be solubilized in denaturing solventssuch as 8 M urea or 6 M guanidine hydrochloride. The subunits have been chromatographically resolved from each other (Willard et al., 1980; Moon et al., 1981; Geisler & Weber, 1981; Chiu & Norton, 1982; Hogue-Angelletti et al., 1982; Liem, 1982; Liem & Hutchison, 1982) in denaturing solvents, although such separations have evidently been difficult and have not been thoroughly documented. Solutions of purified NF68 will spontaneously reassemble into fairly uniform filaments of approximately 10-nm diameter when they are dialyzed into buffers of ap-