Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle.
Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle.
复制标题
小热休克蛋白与来自人骨骼肌的 α B 晶状体蛋白的共纯化。
DOI：
10.1016/s0021-9258(18)42574-4
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Tomiko Asano
中科院分区：
文献类型：
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作者：
Kanefusa Kato;H. Shinohara;Satoshi Goto;Y. Inaguma;R. Morishita;Tomiko Asano
用饱和度为40%的（NH_4）_2SO_4沉淀人胸肌提取物中的免疫反应性α B晶体蛋白和28-kDa蛋白，并在DEAE-Sepharose和Bio-Gel A-5 m柱层析过程中共洗脱。在7 M尿素存在下，在S-Sepharose HP柱上分离两种蛋白质。在Superdex 75 μ g柱和TSK-SP 5 PW柱上，在尿素存在下，对两种所得级分中的每一种进一步层析，得到α B晶体蛋白和28-kDa蛋白的制备物，它们在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上各产生一条带。最终制备的28-kDa蛋白含有至少两种亚型，其在TSK-SP柱上是可分离的。然而，两个主要的28 kDa的蛋白质与内切蛋白酶Asp-N消化后的碎片模式是相同的。通过切割纯化的28-kDa蛋白质和α B晶状体蛋白形成的肽的氨基酸序列分别与人小热休克蛋白（HSP 28）和透镜α B晶状体蛋白的推导的氨基酸序列的特定区域的氨基酸序列相同。使用免疫测定方法，用兔子中产生的抗体，我们发现HSP 28存在于所有测试的人体组织中，并且在心脏和其他由横纹肌和平滑肌组成的组织中处于高水平（大于1微克/毫克蛋白质）。在几种人和牛组织的提取物中发现的与α B晶状体蛋白一起存在的HSP 28被捕获在α B晶状体蛋白上，并与α B晶状体蛋白从用抗α B晶状体蛋白的抗体制备的亲和柱中共洗脱。这一结果表明，这两种蛋白质在细胞中是相关的。
Immunoreactive alpha B crystallin and a 28-kDa protein in an extract of human pectoral muscle were precipitated by (NH4)2SO4 at 40% saturation, and coeluted during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. The two proteins were separated on a column of S-Sepharose HP in the presence of 7 M urea. Further chromatography of each of the two resultant fractions on a column of Superdex 75 pg and on a TSK-SP 5PW column in the presence of urea yielded preparations of alpha B crystallin and the 28-kDa protein each of which gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final preparation of 28-kDa protein contained at least two subtypes, which were separable on the TSK-SP column. However, fragmentation patterns of the two major 28-kDa proteins after digestion with endoproteinase Asp-N were identical. Amino acid sequences of peptides formed by cleavage of the purified 28-kDa protein and alpha B crystallin were identical to those of particular regions of the deduced amino acid sequences of human small heat shock protein (HSP28) and lens alpha B crystallin, respectively. Using an immunoassay method, with antibodies raised in rabbits, we found that HSP28 was present in all human tissues tested and at high levels (greater than 1 micrograms/mg protein) in the heart and other tissues composed of striated and smooth muscles. HSP28, found with alpha B crystallin, in extracts of several human and bovine tissues was trapped on and coeluted with alpha B crystallin from an affinity column prepared with antibodies against alpha B crystallin. This result suggests that the two proteins are associated in cells.