Fujii N, Hiroki K, Matsumoto S, Masuda K, Inoue M, Tanaka Y, Awakura M, Akaboshi M
Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka 590-0494, Japan.
Photochem Photobiol. 2001 Sep;74(3):477-82. doi: 10.1562/0031-8655(2001)074<0477:cbtlot>2.0.co;2.
Alpha-crystallin possesses a molecular chaperone-like activity that prevents proteins from aggregating; however, the mechanism of this activity is not well known. Here we have taken gamma-irradiated alpha-crystallin and studied the relationship between the decrease in chaperone-like activity and the modifications such as oxidation, isomerization and racemization of amino acids in this molecule. We found that the chaperone-like activity of alpha-crystallin decreased with increasing gamma irradiation. After 4000 Gy gamma irradiation the activity of alpha-crystallin was reduced to 40% of the level of nonirradiated, native alpha-crystallin. The circular dichroism spectrum showed that the secondary structure of the irradiated alpha-crystallin had not changed. However, its tertiary structure appeared to change following more than 1000 Gy irradiation. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis also indicated that cross-linking of alpha-crystallin increased with increasing radiation doses. Irradiated and nonirradiated alpha-crystallin was subjected to trypsin digestion and peptide analysis by reverse-phase high-performance liquid chromatography and mass and sequence analysis. Depending on the radiation dose, Met-1 of alpha A-crystallin was oxidized to methionine sulfoxide. In addition, Asp-151 of alpha A-crystallin was isomerized to the beta-Asp form after irradiation, and racemization of Asp-151 decreased. Thus, the loss of the chaperone-like activity of alpha-crystallin is related to changes in its isomerization, oxidation and racemization.
α-晶状体蛋白具有类似分子伴侣的活性，可防止蛋白质聚集；然而，这种活性的机制尚不清楚。在这里，我们采用了γ射线辐照的α-晶状体蛋白，并研究了其类似分子伴侣活性的降低与该分子中氨基酸的氧化、异构化和消旋化等修饰之间的关系。我们发现，α-晶状体蛋白的类似分子伴侣活性随着γ射线辐照剂量的增加而降低。在4000戈瑞的γ射线辐照后，α-晶状体蛋白的活性降低至未辐照的天然α-晶状体蛋白水平的40%。圆二色光谱表明，辐照后的α-晶状体蛋白的二级结构没有改变。然而，在超过1000戈瑞的辐照后，其三级结构似乎发生了变化。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳也表明，α-晶状体蛋白的交联随着辐射剂量的增加而增加。对辐照和未辐照的α-晶状体蛋白进行胰蛋白酶消化，并通过反相高效液相色谱以及质谱和序列分析进行肽分析。根据辐射剂量的不同，αA-晶状体蛋白的Met-1被氧化为甲硫氨酸亚砜。此外，αA-晶状体蛋白的Asp-151在辐照后异构化为β-Asp形式，且Asp-151的消旋化减少。因此，α-晶状体蛋白类似分子伴侣活性的丧失与其异构化、氧化和消旋化的变化有关。
