Characterization of D-amino acid containing protein from sun-damaged skin of elderly donors.

1)日晒损伤皮肤弹性纤维中含有生物学上罕见的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残基发生了明显的外消旋和异构化，并在老化过程中积累了异常蛋白。随后，我们利用基质辅助激光解吸/电离飞行时间质谱法和Mascot在线数据库搜索算法鉴定了一些含有d - β - asp的蛋白质。我们提出抗体可能是一个有用的指标，太阳损伤的皮肤。2)弹性蛋白片段对应的模型肽中天冬氨酸残基消旋动力学研究我们合成了三个弹性蛋白片段对应的含asp的模型肽：1)GVADAAAA, 2) REGDPSSS, 3) AGADEGVR。我们计算了三个肽中Asp残基外消旋化的活化能，以及在体温（37℃）下Asp D/L比接近1.0所需的时间。Asp在多肽中的外消旋是在一级反应的基础上进行的。肽No2中的Asp残基对外消旋最敏感，而肽No1中的Asp残基对外消旋最不敏感。而三种多肽中Asp的外消旋率差异不大。在37℃条件下，这些肽中Asp的D/L比达到0.999所需的时间为50 ~ 100年。这些结果表明Asp在老年人皮肤中发生外消旋化。

1)Biologically uncommon D-beta-aspartic acid-containing protein in elastic fibers of sun damaged skin.We have prepared the antibody against 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 siginificant racemization and isomerization of Asp resideus in proteins of elastic fibers and the unusual proteins were accumulated during aging. Subsequnetly, we identified some D-beta-Asp containing proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm.We propose that the antibody could be a useful indicator for sun damage of the skin.2) Kinetic Study of racemization of aspartyl residues in model peptides corresponding to sections of elastinWe synthesized three Asp-containing model peptides corresponding to sections of elastin:1) GVADAAAA, 2) REGDPSSS, 3) AGADEGVR. We calculated the activation energy of the racemization of Asp residues in the three peptide and the time required for the Asp D/L ratio to approximate to 1.0 at body temperature(37℃). The racemization of Asp in the peptides progressed on the basis of the first order reaction. The Asp residue in the peptide No2 is the most susceptible to racemization, while the Asp residue in the peptide No1 is the least susceptible. However, there was little difference the racemization rate of Asp among three peptides. At 37℃, the time required for the D/L ratios of Asp in these peptides to 0.999 was calculated to be 50-100 years. These results suggested that the racemization of Asp occurred in skin of elderly humans.