McDermott M J, Gawinowicz-Kolks M A, Chiesa R, Spector A
Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Arch Biochem Biophys. 1988 May 1;262(2):609-19. doi: 10.1016/0003-9861(88)90413-4.
The disulfide content of calf gamma-crystallin polypeptides has been investigated. The gamma-crystallin fraction of the soluble lens proteins was separated into five distinct polypeptides and characterized by isoelectric focusing, amino acid composition, and N-terminal sequence analysis to 25 residues. It has been demonstrated that 7 cysteines are present in gamma II, 4 to 5 cysteines in gamma IIIa, gamma IIIb, and gamma IV, and 6 cysteines in gamma I (beta s). Reduction of the total gamma-crystallin fraction with DTT resulted in an increase of approximately 1 to 1.5 mol of free SH per mole of protein. This increase in sulfhydryls was demonstrated to be contributed primarily by gamma II, the major polypeptide representing 50% of the total gamma-crystallin, which showed an increase of approximately 2.5 mol of sulfhydryl per mole of protein upon reduction. Insignificant disulfide content was present in gamma III and gamma IV and only a slight amount of disulfide was found in gamma I (beta s). The observed increase in sulfhydryl content upon reduction was not due to the presence of mixed disulfides of 2-mercaptoethanol, glutathione, or cysteine. The data are consistent with approximately 1 mol of intramolecular disulfide per mole of protein being present in gamma II. X-ray crystallography of gamma II has shown that the spatial location of Cys18 and Cys22 in the tertiary structure permits disulfide bond formation. Sequence analysis of the four major polypeptides of gamma-crystallin, gamma II, gamma IIIa, gamma IIIb, and gamma IV indicates that only gamma II has both Cys18 and Cys22. Cys18 is present in gamma IIIa, gamma IIIb, and gamma IV but Cys22 is replaced by His22. It is probable that the lack of disulfide in gamma IIIa, gamma IIIb, and gamma IV is due to the absence of Cys22.
对小牛γ-晶状体蛋白多肽的二硫键含量进行了研究。可溶性晶状体蛋白的γ-晶状体蛋白组分被分离成五种不同的多肽，并通过等电聚焦、氨基酸组成和N端序列分析（至25个残基）进行表征。已证明γII中有7个半胱氨酸，γIIIa、γIIIb和γIV中有4至5个半胱氨酸，γI（βs）中有6个半胱氨酸。用二硫苏糖醇（DTT）还原总γ-晶状体蛋白组分导致每摩尔蛋白质中游离巯基增加约1至1.5摩尔。巯基的这种增加主要由γII贡献，γII是主要多肽，占总γ-晶状体蛋白的50%，还原后每摩尔蛋白质中巯基增加约2.5摩尔。γIII和γIV中的二硫键含量微不足道，在γI（βs）中仅发现少量二硫键。还原后观察到的巯基含量增加并非由于2-巯基乙醇、谷胱甘肽或半胱氨酸的混合二硫键的存在。数据表明每摩尔γII蛋白质中存在约1摩尔分子内二硫键。γII的X射线晶体学表明，三级结构中Cys18和Cys22的空间位置允许形成二硫键。γ-晶状体蛋白的四种主要多肽γII、γIIIa、γIIIb和γIV的序列分析表明，只有γII同时具有Cys18和Cys22。Cys18存在于γIIIa、γIIIb和γIV中，但Cys22被His22取代。γIIIa、γIIIb和γIV中缺乏二硫键可能是由于缺少Cys22。
