老化・紫外線・放射線によるタンパク質中のD-β-アスパラギン酸生成とその機構

I)人α - a -晶体蛋白中asp-151和asp-58残基的倒置和异构化。尽管蛋白质通常完全由l -氨基酸组成，但我们发现衰老人类晶状体α -a -晶体蛋白中的Asp-151和Asp-58在衰老过程中高度倒置为生物学上罕见的d - β异构体。这种剧烈的变化从出生时就开始了，在30年的时间里，大约45%的正常L- α - asp消失了。Asp残基的这些修饰可能会影响晶状体蛋白的三维排列。Ii) γ辐照α -晶体蛋白的氧化、异构化和外消旋与伴侣样活性丧失之间的关系。α -晶体蛋白具有分子伴侣样活性，可阻止蛋白质聚集；然而，这种活动的机制尚不清楚。本文对α -晶体蛋白进行γ辐照，研究了分子中氨基酸的氧化、异构化和外消旋等修饰作用与伴侣活性降低的关系。我们发现α -晶体蛋白的伴侣蛋白样活性随着γ辐照的增加而降低。辐照后α -晶体蛋白的二级结构没有发生变化，但在1000Gy辐照后α -晶体蛋白的三级结构发生了变化。α - a -晶体蛋白的Met- 1根据辐照剂量的不同被氧化为蛋氨酸亚砜。此外，α - a-晶蛋白的Asp-151在辐照后被异构化为β - asp形式，Asp-151的外消旋作用减弱。因此，α -晶体蛋白铁伴侣样活性的丧失与其异构化、氧化和外消旋化的变化有关。Iii)太阳损伤皮肤弹性纤维中不常见的d - β -天冬氨酸蛋白。制备了抗含d - β - asp蛋白的抗体，并检测了其在皮肤中的免疫反应性。该抗体识别出暴露在阳光下的皮肤中分解的弹性纤维。Western plot分析表明，含有50kDa的多肽与含有d - β - asp的蛋白和弹性蛋白的抗体具有免疫反应性。结果表明，紫外光照射使弹性纤维蛋白中的Asp残基发生了外消旋和异构化，并在老化过程中积累了异常蛋白。我们提出抗体可能是一个有用的指标，太阳损伤的皮肤。少

I) INVERSION AND ISOMERIZATION OF ASP-151 AND ASP-58 RESIDUE IN HUMAN ALPHA A-CRYSTALLIN FROM NORMALAGED LENSES. Although proteins are generally composed entirely of L-amino acids, we showed that Asp-151 and Asp-58 in alpha A-crystallin from aged human lens is inverted to the biologically uncommon D-beta-isomer to a high degree during aging. The drastic changes started at birth, with about 45 % of normal L- alpha -Asp lost by the 30 year range. These modifications of the Asp residue likely affects the three-dimensional packing array of the lens proteins.II) CORRELATION BETWEEN LOSS OF CHAPERONE-LIKE ACTIVITY, AND OXIDATION, ISOMERIZATION AND RACEMIZATION OF GAMMA-IRRADIATED ALPHA-CRYSTALLIN. 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 gamma-irradiated alpha-crystallin and studied the relationship between the decrease in chaperone-like activity and modific … More ations 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. The secondary structure of the irradiated alpha-crystallin did not change, however its tertiary structure of the alpha-crystallin seems to change more than 1000Gy irradiation. 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 was decreased. Thus, loss of the fe chaperone-like activity of alpha-crystallin is related to changes in its isomerization, oxidation and racemization.III) BIOLOGICALLY UNCOMMON D-BETA-ASPARTIC ACID-CONTAINING PROTEIN IN ELASTIC FIBERS OF SUN DAMAGED SKIN. We have prepared the antibody agaist D-beta-Asp containing protein and examined its immunoreactivity in skin. The antibody recognized disintegrated elastic fibers in sun-exposed skin. Western plot analysis of the proteins isolated from sun-damaged skin demonstrated that the polypeptide with 50kDa was immunoreactive with the antibodies for D-beta-Asp containing protein and elastin. This result suggests that UV irradiation significant racemization and isomerization of Asp resideus in proteins of elastic fibers and the unusual proteins were accumulated during aging. We propose that the antibody could be a useful indicator for sun damage of the skin. Less