纯化骨骼肌α-辅肌动蛋白的一些特性。

Some properties of purified skeletal muscle alpha-actinin.

作者信息

Suzuki A, Goll D E, Singh I, Allen R E, Robson R M, Stromer M H

出版信息

J Biol Chem. 1976 Nov 10;251(21):6860-70.

Abstract

Highly purified alpha-actinin can be made by using the low ionic strength extraction procedure previously described (Arakawa N., Robson, R. M., and Goll, D. E. (1970) Biochim. Biophys. Acta 200, 284-295) and then subjecting the crude alpha-actinin fraction obtained with this extraction procedure to successive chromatography on DEAE-cellulose and hydroxyapatite. Hydrozyapatite chromatography specifically removes a protein having a subunit molecular weight of 42,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Hydroxyapatite-purified alpha-actinin sediments entirely as a 6.21 S boundary in the analytical ultracentrifuge with no trace of the small 9 to 10 S boundary seen in earlier alpha-actinin preparations purified by DEAE-cellulose chromatography. In 100 mM KCl, 20 mM Tris-acetate, pH 7.5, hydroxyapatite-purified alpha-actinin has a diffusion coefficient (D020,w) of 2.71 X 10(-7) cm2-s-1, an intrinsic viscosity of 20.6 ml-g-1, a molecular weight of 201,000 +/- 4,300 (plus or minus least squares standard error) as determined by sedimentation equilibrium, and a molecular weight of 210,000 as determined by sedimentation diffusion. In 6 M guanidine HCl, hydroxyapatite-purified alpha-actinin has a molecular weight of 106,000 +/- 6,300 as determined by sedimentation equilibrium and a molecular weight of 100,000 as determined by a calibrated 4% agarose gel permeation column. SDS-polyacrylamide gel electrophoresis gives a molecular weight of 96,000 to 100,000 for hydroxyapatite-purified alpha-actinin. Rod-shaped particles 44 X 390 to 400 A are seen in electron micrographs of negatively stained alpha-actinin. By assuming 45% hydration and a molecular weight of 206,000, dimensions of approximately 40 X 500 A can be calculated for the alpha-actinin molecule by using either s 020, w, D 020, w, intrinsic viscosity, or a calibrated 6% agarose gel permeation column. Hydroxyapatite-purified alpha-actinin has an alpha-helical content of 74% as measured by circular dichroism at 208 nm.

摘要

高纯度的α-辅肌动蛋白可以通过使用先前描述的低离子强度提取方法制备（荒川N.、罗布森、R.M.和戈尔、D.E.（1970年）《生物化学与生物物理学学报》200，284 - 295），然后将用此提取方法获得的粗α-辅肌动蛋白级分先后在DEAE - 纤维素和羟基磷灰石上进行层析。羟基磷灰石层析能特异性去除在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳上亚基分子量为42,000的一种蛋白质。经羟基磷灰石纯化的α-辅肌动蛋白在分析超速离心机中完全以6.21 S的边界沉降，没有在早期通过DEAE - 纤维素层析纯化的α-辅肌动蛋白制剂中所见的9至10 S小边界的痕迹。在100 mM KCl、20 mM Tris - 乙酸、pH 7.5条件下，经羟基磷灰石纯化的α-辅肌动蛋白的扩散系数（D020,w）为2.71×10(-7) cm2·s-1，特性粘度为20.6 ml·g-1，通过沉降平衡测定的分子量为201,000±4,300（正负最小二乘标准误差），通过沉降扩散测定的分子量为210,000。在6 M盐酸胍中，经羟基磷灰石纯化的α-辅肌动蛋白通过沉降平衡测定的分子量为106,000±6,300，通过校准的4%琼脂糖凝胶渗透柱测定的分子量为100,000。SDS - 聚丙烯酰胺凝胶电泳测得经羟基磷灰石纯化的α-辅肌动蛋白的分子量为96,000至100,000。在负染的α-辅肌动蛋白的电子显微照片中可见44×390至400 Å的杆状颗粒。通过假设45%的水合度和分子量为206,000，使用s 020,w、D 020,w、特性粘度或校准的6%琼脂糖凝胶渗透柱，可以计算出α-辅肌动蛋白分子的尺寸约为40×500 Å。经羟基磷灰石纯化的α-辅肌动蛋白在208 nm处通过圆二色性测量的α-螺旋含量为74%。