Masayasu Bando

Acceleration of calcium-induced aggregation of rat lens soluble protein by photosensitization with 8-methoxypsoralen and 3-Hydroxy-l-kynurenine O-β-glucoside

Abstract The present investigation shows a relationship between near ultraviolet light and calcium-induced aggregation of rat lens protein. Ca-induced aggregation of rat lens soluble protein was accelerated by 8-methoxypsoralen plus near ultraviolet light. This photosensitization effect increased with concentration of 8-methoxypsoralen and with light irradiation time. 3-Hydroxy- l -Kynurenine O -β-glucoside also had a similar photosensitizing action as 8-methoxypsoralen on the Ca-induced aggregation of the lens protein. Furthermore, it was found that acceleration of the Ca-induced protein aggregation by photosensitization was inhibited by glutathione, and that the Ca-induced aggregation of the lens protein was not reversed when calcium ion was removed from the protein solution with dialysis.

Activities of ascorbate free radical reductase and H2O2-dependent NADH oxidation in senile cataractous human lenses

Changes in activities of ascorbate free radical (AFR) reductase (NADH:AFR oxidoreductase) and H2O2-dependent NADH oxidation were correlated with levels of insoluble protein in senile cataractous human lenses. The H2O2-dependent NADH oxidation activity was measured to reflect the content of free glutathione. AFR reductase activities in all the cataractous lenses assayed here tended to decrease with increase of insoluble protein contents. A similar tendency in the relationship between lens protein aggregation and H2O2-dependent NADH oxidation activities, i.e. free glutathione contents was recognized in the lenses with pale yellow, yellow or dark yellow nucleus. However, for the highest levels of insoluble protein, some of the brunescent cataractous lenses exhibited very high activities of H2O2-dependent NADH oxidation, and some brunescent lenses had very low activities. From the above results, it is suggested that lens protein aggregates in the brunescent and non-brunescent cataractous lenses may be formed through significantly different oxidation processes, respectively. The possible mechanisms such as free radical reaction and disulfide bond formation are discussed.

Calcium-induced lens protein aggregation accelerated by reactive oxygen species photosensitized in the presence of hydroxykynurenines.

Photo-oxidation with 3-OH L-kynurenine O-beta-glucoside or 3-OH L-kynurenine accelerates the aggregation of water-soluble protein in the rat lens due to the presence of calcium ion. The present report demonstrates that hydrogen peroxide and superoxide anion mediate a large part of the photodynamic acceleration with hydroxykynurenine compounds. In addition, 3-OH L-kynurenine O-beta-glucoside seems to photosensitize more of the two reactive oxygen species than 3-OH L-kynurenine. The findings are discussed together with other photodynamic effects of hydroxykynurenine compounds.

Scavenging of chlorpromazine cation radical by ascorbic acid or glutathione.

The report presented here demonstrates that scavenging of chlorpromazine cation radical (an absorption maximum = 530 nm) by ascorbic acid or glutathione can be kinetically and stoichiometrically analyzed at pH 1.5 but not at pH 3.0 and 6.0 using a conventional absorption spectrophotometer. The cation radical decays spontaneously about 10 and 200 times faster at pH 3.0 and 6.0, respectively, than at pH 1.5. At pH 1.5, ascorbic acid scavenges the cation radical faster than glutathione does, and the following different scavenging mechanisms are postulated from the above kinetic and stoichiometric analysis. The reaction of the cation radical with ascorbic acid is second order. The ascorbic acid free radical, which decays mainly by dismutation, is generated by the bimolecular reaction. In the case of glutathione, on the other hand, about 70% of the scavenged cation radical disappears through free radical chain reactions that glutathione thiol anion and glutathione free radical probably initiate. The remaining (about 30%) disappears by conjugation with glutathione. It may be due to relative nonreactivity of ascorbic acid free radical that free radical chain reactions, found commonly in radical chemistry, do not occur in the scavenging reaction by ascorbic acid. Based on the above results, the physiological scavenging mechanisms of the cation radical by the two reducing substances are discussed briefly.

Ascorbate free radical reductase activity in vertebrate lenses of certain species

PURPOSE To clarify the function of ascorbate free radical (AFR) reductase in the antioxidation system of different vertebrate lenses. METHODS The soluble and insoluble fractions were prepared from bullfrog, guinea pig, rat, rabbit, swine, and bovine lenses, and membrane-bound enzymes in the insoluble fraction were extracted by 0.3% Triton X-100. Ascorbate free radical reductase and diaphorase activities in each fraction were determined. RESULTS Ascorbate free radical reductase activity in the lens soluble fraction was the highest in the bullfrog. That in the guinea pig and rabbit was at the next level. There was only a little activity in rat and swine lenses, and none was detected in the bovine lenses. However, a large species difference in AFR reductase activity was not observed in the 0.3% Triton X-100 extracts. Diaphorase activity was three to nine higher than AFR reductase activity in the soluble fractions of bullfrog, guinea pig, and rabbit. In the 0.3% Triton X-100 extracts of all animal species used, it was very high, 108 to 311 times the AFR reductase activity. CONCLUSION These results indicate that the lens soluble and membrane-bound AFR reductase in the different animals may be individual enzyme molecules and have different antioxidative functions. Because the lenses of bullfrog, guinea pig, and rabbit are known to contain a near-ultraviolet (UV) light-absorbing compound, reduced pyridine nucleotide, at a high concentration, the soluble AFR reductase activity is expected to be high in the vertebrate lenses with a near-UV light filter, to enhance the antiphoto-oxidation capacity of ascorbate.

λ-crystallin related to dehydroascorbate reductase in the rabbit lens

PURPOSE To evaluate the relationship of lambda-crystallin to reduced nicotinamide adenine dinucleotide (NADH)-dependent dehydroascorbate (DHA) reductase found specifically in the rabbit lens. METHODS DHA reductase Fractions I-IV were separated from the lambda/betaL1-crystallin fraction of rabbit lens soluble protein by diethylaminoethyl (DEAE)-cellulose ion-exchange column chromatography, and then the enzyme was partially purified from Fraction II by rechromatography on the same ion-exchange column. The isolated DHA reductase fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, native isoelectric focusing and two-dimensional gel electrophoresis. RESULTS Using Western blot and a probe of antiserum to recombinant lambda-crystallin, the main 33-kDa protein band was strongly stained in all the rabbit lens DHA reductase fractions, and most of the additional protein bands of approximately 25-30 kDa were also detectable. In the partially purified enzyme, the 33-kDa subunit alone was identified as a distinct protein band by SDS-PAGE, and a main basic protein was found at pI 7.6 by native isoelectric focusing. In addition, many bands of more acidic proteins were separated from other enzyme fractions, and protein spots corresponding to the 33 and/or approximately 25-30-kDa subunits were detected in each of the more acidic proteins by two-dimensional gel electrophoresis. CONCLUSION These results suggest that lambda-crystallin is closely related to the DHA reductase in the rabbit lens. The above heterogeneity of the enzyme-crystallin may arise from posttranslational modifications.

Heterogeneity of ascorbate free radical reductase in the human lens.

The soluble ascorbate free radical (AFR) reductases in the human lens were separated into many isoforms in the range of pI 5-7 by native isoelectric focusing. In the two-dimensional gel electrophoresis, however, two main proteins with molecular weights of 20-25 kD were commonly identified to each isoform. The observed heterogeneity of the human lens AFR reductase is very similar to those reported for beta- and gamma-crystallins in aged and cataractous human lenses. From these results, it is suggested that some of the isoforms of the lens AFR reductase, especially the more acidic isoforms, may be formed by posttranslational modifications.

Ferricyanide Reductase Activity in Cataractous Human Lens

We assayed ferricyanide reductase activity (one of NADH-dependent diaphorase activities) in the soluble and insoluble fractions of cataractous human lenses. Activity of this reductase in both the soluble and insoluble fractions tended to decrease in order of cortex > nucleus periphery > nucleus center, and it was suggested that a decrease of the reductase activity is closely correlated with lens protein aggregation, and to some extent associated with the development of nuclear sclerosis (coloration) and cortical cataract. Furthermore, insoluble fraction had very high specific activity per mg insoluble protein in cortex, and the activity decreased sharply with an increase in the level of insoluble protein. The reductase activity in the insoluble fraction may be also related to the metabolic activity of plasma membranes.