Fujii Y, Watanabe K, Hayashi H, Urade Y, Kuramitsu S, Kagamiyama H, Hayaishi O
Hayaishi Bioinformation Transfer Project, Research Development Corporation of Japan, Kyoto.
J Biol Chem. 1990 Jun 15;265(17):9914-23.
In a previous paper, we reported that the partial amino acid sequence (225 residues) from the COOH terminus of rho-crystallin from European common frog lens shows 77% similarity to that of prostaglandin (PG) F synthetase, an aldo-keto reductase, from bovine lung (Watanabe, K., Fujii, Y., Nakayama, K., Ohkubo, H., Kuramitsu, S., Kagamiyama, H., Nakanishi, S., and Hayaishi, O. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 11-15). Here rho-crystallin was purified to apparent homogeneity from the eye lens of the Japanese common bullfrog (Rana catesbeiana) by four sequential chromatographies using Sephadex G-100, Red Sepharose, and dual Mono S. Two types of rho-crystallin, RHO-I and RHO-II, named according to their elution order from a Mono S column, are essentially identical in terms of immunochemical properties, amino acid composition, and partial amino acid sequence. But the NH2-terminal Thr of RHO-I is blocked with an acyl group, while that of RHO-II is free. Both crystallins as well as PGF synthetase are monomeric proteins with a molecular weight of about 35,000 and they have the ability to bind NADPH with a stoichiometry of 0.75 mol of cofactor/mol of protein. Although rho-crystallin does not cross-react with antibody against PGF synthetase, the NH2-terminal amino acid sequence (107 residues) of rho-crystallin shows 77% similarity to that of the enzyme. However, PGD2, PGE2, 9,10-phenanthrenequinone, p-nitrobenzaldehyde, DL-glyceraldehyde, D-glucuronic acid, D-glucose, D-xylose, menadione, p-nitroacetophenone, dihydroxyacetone, succinic semialdehyde, phenylglyoxal, and testosterone were not substrates for these crystallins. PGH2 9,11-endoperoxide reductase activities of RHO-I and RHO-II were 1.3 and 1.0 milliunits/mg of protein, respectively, which are only about 2% of that of bovine lung PGF synthetase. These results indicate that the rho-crystallins RHO-I and RHO-II belong to a group of aldo-keto reductases based on primary structure, molecular properties, and NADPH-binding ability, but show only low PGH2 9,11-endoperoxide reductase activity.
在之前的一篇论文中，我们报道了欧洲普通青蛙晶状体中rho-晶状体蛋白COOH末端的部分氨基酸序列（225个残基）与牛肺中前列腺素（PG）F合成酶（一种醛糖还原酶）的该序列有77%的相似性（渡边健、藤井洋、中山健、大久保博、仓光史、加美山博、中岸史、林石，（1988年）《美国国家科学院院刊》85卷，第11 - 15页）。在此，通过使用Sephadex G - 100、Red Sepharose和双Mono S进行的四次连续色谱法，从日本牛蛙（Rana catesbeiana）的眼晶状体中纯化出了表观上均一的rho-晶状体蛋白。根据它们从Mono S柱上的洗脱顺序命名的两种类型的rho-晶状体蛋白，RHO - I和RHO - II，在免疫化学性质、氨基酸组成和部分氨基酸序列方面基本相同。但是RHO - I的NH2末端苏氨酸被一个酰基封闭，而RHO - II的是游离的。这两种晶状体蛋白以及PGF合成酶都是分子量约为35000的单体蛋白，并且它们都有以0.75摩尔辅因子/摩尔蛋白质的化学计量比结合NADPH的能力。尽管rho-晶状体蛋白与抗PGF合成酶的抗体不发生交叉反应，但其NH2末端氨基酸序列（107个残基）与该酶的序列有77%的相似性。然而，PGD2、PGE2、9,10 - 菲醌、对硝基苯甲醛、DL - 甘油醛、D - 葡萄糖醛酸、D - 葡萄糖、D - 木糖、甲萘醌、对硝基苯乙酮、二羟基丙酮、琥珀酸半醛、苯乙二醛和睾酮都不是这些晶状体蛋白的底物。RHO - I和RHO - II的PGH2 9,11 - 内过氧化物还原酶活性分别为1.3和1.0毫单位/毫克蛋白，仅约为牛肺PGF合成酶活性的2%。这些结果表明，基于一级结构、分子性质和NADPH结合能力，rho-晶状体蛋白RHO - I和RHO - II属于醛糖还原酶家族，但仅表现出低水平的PGH2 9,11 - 内过氧化物还原酶活性。
