Purification and characterization of rho-crystallin from Japanese common bullfrog lens.
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.
Fujii
Y
Y
Hayaishi Bioinformation Transfer Project, Research Development Corporation of Japan, Kyoto.
Watanabe
K
K
Hayashi
H
H
Urade
Y
Y
Kuramitsu
S
S
Kagamiyama
H
H
Hayaishi
O
O
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003459:Crystallins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000378:metabolism; D007564:Japan; D007908:Lens, Crystalline / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011892:Rana catesbeiana; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D003459:Crystallins; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D006911:Hydroxyprostaglandin Dehydrogenases; D000426:Alcohol Dehydrogenase; C031051:prostaglandin-F synthase; D000449:Aldehyde Reductase