Arrigo A P, Welch W J
Cold Spring Harbor Laboratory, New York 11724.
J Biol Chem. 1987 Nov 15;262(32):15359-69.
We describe the biochemical characterization and purification of the small 28,000-dalton heat shock protein (28-kDa protein) of mammalian cells. Metabolic pulse labeling of heat shock-treated cells with either [3H]leucine or H3 32PO4 and analysis of the labeled proteins by two-dimensional gel electrophoresis revealed increased levels of three 28-kDa proteins differing only in their relative isoelectric point. Using both peptide mapping and immunological analysis, we demonstrate that all three proteins are related isoforms, with two of the isoforms containing phosphate. Cell fractionation studies revealed that the 28-kDa protein localizes predominantly within the nuclear pellet very shortly after the heat shock treatment. With increasing times of recovery of the heat-treated cells back at 37 degrees C, the majority of the 28-kDa protein was now observed to fractionate within the soluble fraction of the cells. Both gel filtration and velocity sedimentation studies revealed that the 28-kDA protein exists as a higher order structure with an approximate S20,w value of 10-18 S, a Stokes radius of about 60-70 A, and an estimated native molecular mass of at least 500,000 daltons. We describe a relatively simple and rapid purification of the proteins employing both ion-exchange and gel filtration chromatography.
我们描述了哺乳动物细胞中28,000道尔顿小热休克蛋白（28-kDa蛋白）的生化特性及纯化过程。用[3H]亮氨酸或H3 32PO4对热休克处理的细胞进行代谢脉冲标记，并通过二维凝胶电泳分析标记蛋白，结果显示三种28-kDa蛋白的水平升高，它们仅在相对等电点上有所不同。通过肽图谱分析和免疫分析，我们证明这三种蛋白是相关的同工型，其中两种同工型含有磷酸基团。细胞分级分离研究表明，热休克处理后不久，28-kDa蛋白主要定位于核沉淀中。随着热处理细胞在37℃恢复时间的增加，现在观察到大多数28-kDa蛋白在细胞的可溶部分中分级分离。凝胶过滤和速度沉降研究均表明，28-kDa蛋白以更高阶结构存在，其近似的S20,w值为10 - 18 S，斯托克斯半径约为60 - 70 Å，估计天然分子量至少为500,000道尔顿。我们描述了一种使用离子交换和凝胶过滤色谱法相对简单快速地纯化这些蛋白的方法。
