Srivastava O P, Srivastava K, Silney C
Missouri Lions Eye Research Foundation, Columbia.
Invest Ophthalmol Vis Sci. 1994 Jan;35(1):207-14.
To purify crystallin fragments (degraded polypeptides molecular weight < 18 kD) and identify their parent crystallins.
The purification of polypeptides with apparent molecular weights of 4 and 5 kD was carried out using three sequential steps: Sephadex G-50 chromatography under denaturing conditions, preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and high-performance liquid chromatography using a C-18 column. The parent crystallins of the two polypeptides were identified by the Western blotting method using polyclonal antibodies raised against individual 4 and 5 kD polypeptides and by comparing N-terminal amino acid sequences of the polypeptides with crystallins.
Two polypeptides of 4 and 5 kD were purified by the three sequential steps as described from water-soluble proteins of lenses from 60-80-year-old donors. Both purified polypeptides showed a single major peak during high-performance liquid chromatography on a C-18 column and also a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Western blot analyses showed maximum immunoreactivity of the anti-4 kD polypeptide antibody to a 22 kD species of beta-crystallin, whereas the anti-5 kD polypeptide antibody showed maximum reactivity to only the alpha B crystallin. These results were further confirmed during comparison of the N-terminal amino acid sequences of the two polypeptides with crystallins. Such comparison showed that the 4 kD polypeptide originated from beta A 3/A1 crystallin after cleavage at His187-His188 bond. Further, the 5 kD polypeptide was a fragment of alpha B crystallin that originated after cleavage at Val145-Asn146 bond.
These results showed that specific bonds of beta A3/A1 and alpha B crystallins are posttranslationally cleaved in vivo to produce 4 kD and 5 kD polypeptides, respectively.
纯化晶状体蛋白片段（分子量<18 kD的降解多肽）并鉴定其亲本晶状体蛋白。
采用三个连续步骤纯化表观分子量为4 kD和5 kD的多肽：变性条件下的葡聚糖凝胶G-50柱层析、制备性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以及使用C-18柱的高效液相色谱。使用针对单个4 kD和5 kD多肽产生的多克隆抗体，通过蛋白质印迹法以及将多肽的N端氨基酸序列与晶状体蛋白进行比较，来鉴定这两种多肽的亲本晶状体蛋白。
按照所述的三个连续步骤，从60至80岁供体的晶状体水溶性蛋白中纯化出了4 kD和5 kD的两种多肽。两种纯化后的多肽在C-18柱上进行高效液相色谱分析时均显示出一个主要峰，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中也均显示出一条带。蛋白质印迹分析表明，抗4 kD多肽抗体对22 kD的β-晶状体蛋白具有最大免疫反应性，而抗5 kD多肽抗体仅对αB晶状体蛋白具有最大反应性。在将这两种多肽的N端氨基酸序列与晶状体蛋白进行比较时，进一步证实了这些结果。这种比较表明，4 kD多肽是βA3/A1晶状体蛋白在His187-His188键处裂解后产生的。此外，5 kD多肽是αB晶状体蛋白在Val145-Asn146键处裂解后产生的一个片段。
这些结果表明，βA3/A1和αB晶状体蛋白的特定键在体内发生翻译后裂解，分别产生4 kD和5 kD的多肽。
