[show abstract][hide abstract] ABSTRACT: Aquaporin 0 (AQP0) is a lens-specific protein comprising more than 30% of lens membrane protein content and is a member of the aquaporin family. Water permeates through AQP0 much more slowly than other aquaporin family members, and other compounds, such as glycerol, also permeate AQP0. In the lens, ascorbic acid (AA) is found at high concentrations, protecting the lens from photochemical events such as photo-oxidation. The aim of the present study was to clarify the function of AQP0. Mouse fibroblast L-cells stably expressing AQP0 were established and incubated in medium containing AA, and intracellular AA levels were measured by high-performance liquid chromatography (HPLC) and 2,6-dichlorophenol-indophenol (DCPIP) analysis. Intracellular AA levels in AQP0-expressing cells quickly rose and reached saturation 10 min after incubation in medium containing 1000 μM AA. In contrast, AA levels in cells slowly decreased when AA was washed out from the medium. Cells overexpressing AQP0 increased the cellular uptake of AA in a time- and concentration-dependent manner. These data suggest that AA as well as water permeates AQP0. AQP0 expression on Xenopus oocyte membranes was achieved by the injection of AQP0 cRNA into oocytes that were incubated in medium containing AA. Intracellular AA levels were then measured by HPLC. AA uptake was demonstrated in the AQP0-expressing oocytes and was shown to quickly reach saturation. Intracellular AA concentration in oocytes increased in a time- and concentration-dependent manner. The data in the present study show that AA permeates AQP0, reveal the role of AQP0 in AA permeability ex vivo, and also indicate that there is a difference between the import and export of AA via AQP0. These findings suggest that AQP0 plays an important role in controlling lens AA content.
Biochemical and Biophysical Research Communications 11/2011; 415(1):125-30. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: To study the interaction between the lens-specific water channel protein, aquaporin 0 (AQP0) and the lens-specific intermediate filament protein, filensin, and the effect of this interaction on the water permeability of AQP0. The effect of other factors on the interaction was also investigated.
Expression plasmids were constructed in which glutathione-S-transferase (GST) was fused to the AQP0 COOH-terminal region (GST-AQP0-C), which contains the major phosphorylation sites of the protein. Plasmids for AQP0 COOH-terminal mutants were also constructed in which one, three or five sites were pseudophosphorylated, and the proteins expressed from these GST-fusion plasmids were assayed for their interaction with lens proteins. Expressed recombinant GST-fusion proteins were purified using glutathione beads and incubated with rat lens extract. Western blotting was used to identify the lens proteins that interacted with the GST-fusion proteins. Filensin tail and rod domains were also expressed as GST-fusion proteins and their interactions with AQPO were analyzed. Additionally, the water permeability of AQP0 was calculated by expressing AQP0 with or without the filensin peptide on the cell membrane of Xenopus oocytes by injecting cRNAs for AQP0 and filensin.
The GST-AQP0-C construct interacted with the tail region of lens filensin and the GST-filensin-tail construct interacted with lens AQP0, but the GST-filensin-rod construct did not interact with AQP0. GST-AQP0-C also interacted with a purified recombinant filensin-tail peptide after cleavage from GST. The AQP0/filensin-tail interaction was not affected by pseudophosphorylation of the AQP0 COOH-terminal tail, nor was it affected by changes in pH. Xenopus oocytes expressing AQP0 on the plasma membrane showed increased water permeability, which was lowered when the filensin COOH-terminal peptide cRNA was coinjected with the cRNA for AQP0.
The filensin COOH-terminal tail region interacted with the AQP0 COOH-terminal region and the results strongly suggested that the interaction was direct. It appears that interactions between AQP0 and filensin helps to regulate the water permeability of AQP0 and to organize the structure of lens fiber cells, and may also help to maintain the transparency of the lens.
[show abstract][hide abstract] ABSTRACT: Ascorbic acid (AA) is found in high concentrations in the lens and protects the lens from photochemical events such as photo-oxidation. Diabetes reduces the concentration of AA in the lens, and it has been suggested that AA supplementation may rescue lens AA in diabetic patients. In the present study, the effects of dietary supplementation with AA were studied to assess changes in AA concentration and AA transporter expression in the lenses of streptozotocin (STZ)-induced diabetic rats. AA was lower in the aqueous humors and lenses of diabetic rats than in control rats, and low AA levels could be normalized with dietary AA supplementation. The sodium-dependent ascorbic acid transporter2, SVCT2, is transporter of AA, and glucose transporter 1 and 3 (GLUT1 and GLUT3) are transporters of dehydroascobic acid. Transcription of GLUT1 and GLUT3 was not changed as a result of diabetes or AA treatment, and the SVCT2 was increased in diabetic rats and normalized by AA supplementation in diabetic rats. Aquaporin 0 (AQP0) expression was increased in diabetic rats and by AA treatment. The rate of AA transport of vesicle originally from diabetic lens and that with AA treatment were greater than that from control rats with or without AA treatment. These results suggest that ascorbates are transported by AQP0 as well as by SVCT2, GLUT1 and GLUT3, and that supplementation with AA may help prevent diabetic cataracts.
[show abstract][hide abstract] ABSTRACT: The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.bionut.2010.09.008. The duplicate article has therefore been withdrawn.
[show abstract][hide abstract] ABSTRACT: Side population (SP) cells were isolated and the possibility whether lens epithelial cells contain stem cells was investigated.
Mouse lens epithelial cells were stained by Hoechst 33342 and then sorted by fluorescence-activated cell sorting (FACS). The expression of stem cell markers in sorted SP cells and the main population of epithelial cells were analyzed by quantitative real-time PCR. Localization of SP cells in the mouse lens was studied by fluorescence microscopy.
The sorted cells contained SP cells, which were no longer separable by FACS following treatment with verapamil. The number of SP cells decreased with aging, but the adult mouse lens still contained SP cells. Phase contrast microscopy revealed that SP cells were smaller in size than non-SP cells. SP cells were localized near the equator region in lens epithelial cell layers. SP cells expressed higher levels of the stem cell markers ATP-binding cassette transporter G2 (ABCG2), p75 neurotrophin receptor (p75NTR), nestin (nes), B-cell lymphoma 2 (Bcl2), and cell surface antigen Sca-1 mRNA than the main population cells. These results suggest that SP cells contain a high proportion of stem cells.
The SP cells in the lens contain stem cells, and these stem cells are localized around the germinative zone.
[show abstract][hide abstract] ABSTRACT: Kynurenine metabolites are derived from the oxidation of tryptophan (Trp) and are involved in many diseases. Previous reports have suggested that some kynurenine metabolites produce superoxide radicals and singlet oxygen molecules under UV illumination. We used electron spin resonance (ESR) spectroscopy to analyze the formation of radicals following ultraviolet-B (UV-B) irradiation of Trp and kynurenine metabolites. As the results, strong phenoxyl radical formation was detected following UV-B irradiation of 3-hydroxy-kynuirenine (3HK), a kynurenine metabolite. Alpha-crystallin, the major lens protein, undergoes many post-translational modifications, including oxidation. Oxidation occurs selectively at methionine, histidine, Trp and lysine residues and associated with the development of age-related cataracts. We examined the effect of these amino acids, as well as tyrosine (which forms tyrosyl radicals in vivo) and aspartic acid (which undergoes racemization following exposure to UV irradiation), on radical formation by 3HK. The combination of Trp and 3HK generated a stronger ESR signal than did 3HK alone. The phenoxyl radical formed by 3HK was strongly induced by UV illumination in the presence of Trp. These results indicated that 3HK could easily produce the phenoxyl radicals via electron-transfer interactions with Trp, which act as a photosensitizer induced UV excitation. In addition, this radical promoted photo-oxidation and may enhance the ability of 3HK to cause disease.
[show abstract][hide abstract] ABSTRACT: Beaded filaments are lens cell-specific intermediate filaments composed of two proteins: filensin and phakinin (CP49). Filensin and phakinin are believed to function in the maintenance of lens transparency. To elucidate the function of filensin and phakinin at the molecular level, we examined the degradation of these two proteins in normal and cataractous rat lenses.
A hereditary cataract model, the Shumiya cataract rat (SCR), was used for these studies. Anti-filensin antibodies were raised against three different regions of the protein, the rod domain, the inner region of the tail domain, and the outer region of the tail domain. Anti-filensin and anti-phakinin antibodies were used to examine the conformation of degradation of filensin and phakinin by western blot analysis and fluorescent immunocytochemistry of cryosectioned lenses.
In the normal lens, filensin was processed from a 94 kDa protein to proteins of 50 kDa and 38 kDa. Similarly, phakinin was processed from a 49 kDa protein to one of 40 kDa. The concentrations of filensin and phakinin in the rat lens cortex fluctuated with age and decreased during cataractogenesis. The 50 kDa form of filensin decreased significantly before opacification. In the normal lens, phakinin, the filensin rod domain, and the filensin inner tail domain localized to membrane lining regions in the shallow cortex and to the central region of the cytoplasm in the deep cortex. The COOH-terminal domain of filensin localized to the membrane lining region in the deep cortex. In pre-cataractous lenses, phakinin and the filensin rod domain localized primarily to the membranes lining the shallow cortex region and were distributed throughout the cytoplasm of lens fiber cells in the deep cortex.
The 50 kDa form of filensin is important for the localization of beaded filaments in lens fiber cells and for lens transparency.
[show abstract][hide abstract] ABSTRACT: In the rabbit lens, high levels of reduced nicotinamide adenine dinucleotide (NADH) can function as a near-ultraviolet light (near-UV) filter, an effect apparently achieved by specific nucleotide binding to lambda-crystallin. The present investigation asks whether lambda-crystallin enhances NADH photo-oxidation by superoxide radicals produced via a photosensitization reaction of near-UV with NADH.
Lambda-crystallin was partially purified from rabbit lens soluble fraction by a two-step gel filtration and affinity column chromatography procedure. NADH solutions with or without partially purified lambda-crystallin were subjected to near-UV irradiation or exposed to superoxide generated enzymatically by the xanthine/xanthine oxidase system. NADH oxidation was determined by assaying the decrease of absorbance at 340 nm.
When irradiated with near-UV, free NADH was oxidized very little in the absence of lambda-crystallin. In contrast, NADH photo-oxidation was rapidly initiated in the presence of partially purified lambda-crystallin. This lambda-crystallin-enhanced NADH photo-oxidation was totally inhibited by adding superoxide dismutase. We also found that lambda-crystallin largely increased NADH oxidation by a superoxide that is generated enzymatically. These results indicate that NADH bound to lambda-crystallin rapidly reacts with superoxides. The reactivity of bound NADH with superoxide was almost equivalent to that of ascorbic acid. However, lambda-crystallin-enhanced NADH oxidation exceeded the superoxide levels generated by NADH photosensitization and xanthine/xanthine oxidase.
We conclude that NADH binding to lambda-crystallin enhances NADH photo-oxidation through a radical chain reaction mechanism that is initiated by superoxides generated by NADH photosensitization and propagated by another superoxide from the molecule oxygen. High concentrations of NADH bound to lambda-crystallin may be beneficial to the rabbit lens in scavenging the low amounts of superoxide produced by near-UV absorption, since oxygen tension in the lens is very low. We also discuss the function of near-UV-filtering and the anti-photo-oxidation systems in other vertebrate lenses.
[show abstract][hide abstract] ABSTRACT: The present investigation aims to evaluate the NADH binding ability of lambda-crystallin, a taxon-specific enzyme-crystallin, in the rabbit lens.
A lambda/betaL1-crystallin fraction was separated from the rabbit lens soluble fraction by gel filtration and the enzyme-crystallin was partially purified by subsequent affinity column chromatography. Analysis of NADH bound to the lambda-crystallin preparation was performed using spectrophotometric and enzymological methods. Binding of added NADH to the enzyme-crystallin preparation was also analyzed using a simple ultrafiltration method, which was theoretically equivalent to equilibrium dialysis, to study additional NADH binding to the protein.
The prepared lambda-crystallin samples clearly exhibited an absorption maximum at 340 nm, even though they were thoroughly dialyzed. This was due to the presence of nondialyzable NADH bound tightly to the protein. The bound NADH was removed by charcoal treatment, and extracted by 0.1% SDS or 70 degrees C heat treatment. A dissociation constant (Kd) of less than 5 nM indicated tight binding of NADH. The quantity of bound NADH in the 88% purified 33 kDa enzyme-crystallin was estimated to be 20.5 nmol/mg protein, suggesting a stoichiometry of 0.7 mol of the nucleotide/mol of the 33 kDa protein. Additional looser binding of added NADH to lambda-crystallin was observed in both the lambda/betaL1-crystallin fraction (including the full-length 33 kDa protein: 34%; 25-30 kDa proteins, most of which might be generated by cleavage of the 33 kDa protein: 64%) and the partially purified enzyme-crystallin. It was assumed from the analysis of binding titration that some (about 30%) of the 33 kDa protein and most of the lower molecular weight proteins still possessed the ability to loosely bind NADH. Kd values of their lower affinity binding were determined to be 2 or 6 microM.
From the present study, we conclude that lambda-crystallin plays a sufficiently important role as a NADH binding protein to maintain high levels of this nucleotide in the rabbit lens.
[show abstract][hide abstract] ABSTRACT: The Shumiya cataract rat (SCR) is a hereditary cataract model in which lens opacity appears sponta-neously in both the nuclear and perinuclear regions of 10–11 week old animals. The expression of drug me-tabolizing enzymes, including cytochrome P450 (CYP) and conjugation enzymes, was investigated in the ocu-lar tissues of SCRs during cataractogenesis. Signifi-cantly high levels of gene expression were detected for CYP1A1, CYP2B2, CYP2C11 and CYP2E1 in normal lenses, whereas CYP1A1 and CYP2C11 were at lower levels in the cataractous lenses. The gene expression profiles of conjugation enzymes were also compared between normal and cataractous ocular tissues in which a moderate reduction in ST1A1 and ST1B1 ex-pression, and an extensive loss of UGT1A1 expression, were observed in the SCR animals that had developed cataracts. In contrast, the elevated expression of the GSTA family of genes was evident in cataractous lenses. The reduced expression of several drug metabolizing enzymes in the cataractous ocular tissues might there-fore be related to cataractogenesis via the accumula-tion of endogenous or exogenous toxic substances.
[show abstract][hide abstract] ABSTRACT: Ascorbate free radical (AFR) reductase with diaphorase activity was isolated from the rabbit lens soluble fraction to characterise some molecular properties of the enzyme. The isolation was accomplished using gel filtration (Sephadex G-75 superfine or Sephacryl S-200 HR), affinity chromatography (Affi-Gel Blue), native isoelectric focusing and two-dimensional gel electrophoresis. A major soluble AFR reductase was found at an isoelectric point of 8.4 and a molecular weight of 31 kDa, and a few minor enzymes were also detected in the range of pI 7.0-8.6. An unknown N-terminal partial amino acid sequence was determined in one peptide fragment prepared from the major enzyme fraction. From the sequence analysis, it is discussed that the lens soluble AFR reductase may differ from NADH-cytochrome b5 reductase reported to be involved in the membrane-bound AFR reductase activity of mitochondria, microsomes and plasma membrane.
Experimental Eye Research 01/2005; 79(6):869-73. · 3.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: We studied the intraocular pharmacokinetics of dorzolamide hydrochloride eye drops and the effect of dorzolamide on carbonic anhydrase activity and localization in ocular tissues. Carbonic anhydrase activity was detected in normal ocular tissues. The activity was inhibited in corneal endothelial cells, the ciliary body, lens epithelial cells, or the retina 1 to 8 hours after instillation of dorzolamide eye drops. In lens epithelial cells and the retina, the enzyme activity had not recovered even 10 hours after instillation of the drug. Immunostaining did not reveal any differences between the group administered dorzolamide eye drops and the control group administered a physiologically balanced solution. Time-related changes in dorzolamide concentrations in ocular tissues were measured by high-performance liquid chromatography (HPLC). In the cornea, anterior aqueous, iris, ciliary body and retina, drug concentrations increased 15 minutes after the instillation and peaked within 1 hour. These results suggest that dorzolamide immediately suppresses carbonic anhydrase activity in ocular tissues, and is rapidly distributed among the tissues of the eye when administered as eye drops.
Journal of Ocular Pharmacology and Therapeutics 03/2004; 20(1):1-13. · 1.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Purpose: To evaluate the relationship of λ-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 λ/βL1-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 λ-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 λ-crystallin is closely related to the DHA reductase in the rabbit lens. The above heterogeneity of the enzyme-crystallin may arise from posttranslational modifications.
Japanese Journal of Ophthalmology - JPN J OPHTHALMOL. 01/2003; 47(5):437-443.
[show abstract][hide abstract] ABSTRACT: It is well known that m-calpain, a ubiquitous calpain, is involved in cataract formation in rodent lens. Involvement of Lp82, a lens-specific calpain, in the cataract formation is also suggested. However, the exact relationship between Lp82-mediated proteolysis and lens opacification has not yet been established. We therefore compared Lp82- and m-calpain-mediated proteolyses of alphaA-crystallin during cataractogenesis to clarify whether Lp82 is involved in cataract formation.
In order to analyze the Lp82- and m-calpain-mediated proteolyses, we developed antibodies exclusively specific to the proteolytic products of alphaA-crystallin produced by Lp82 and m-calpain actions, respectively. The proteolytic profiles of alphaA-crystallin by Lp82 and m-calpain during cataractogenesis in SCR lenses were analyzed by Western blotting and immunohistochemical staining.
While m-calpain-mediated proteolysis was detected predominantly in cataractous lenses, Lp82-mediated proteolysis was detected not only in cataractous but in normal lenses. The m-calpain-mediated proteolysis was observed in restricted areas developing and destined to develop opacification, i.e., the nuclear and perinuclear regions of lens. On the other hand, Lp82-mediated proteolysis was observed not only in the same regions but also in the cortical region where opacity does not develop. Unlike m-calpain-mediated proteolysis, Lp82-mediated proteolysis was not inhibited by the oral administration of aminoguanidine (AG), which acts to prevent lens opacification.
From these results, it is shown that there is no direct contribution of Lp82-mediated proteolysis to cataract formation in SCR. Rather, Lp82 may function in fiber cell development and/or fiber cell remodeling during lens maturation under physiological conditions, since Lp82-mediated proteolysis occurs in the cortical region of normal lens.
Current Eye Research 11/2002; 25(4):207-13. · 1.71 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have previously reported that connective tissue cells in the superficial dermis preferentially express alpha1(XVI) collagen rather than those in the lower dermis. Double immunofluorescence labeling using the antibodies for alpha1(XVI) collagen and factor XIIIa (plasma transglutaminase), which is a marker of dermal dendrocytes, demonstrated that both antibodies reacted with the same cells in the superficial dermis of normal skin as well as the lesional skins of dermal dendrocyte-related disorders, dermatofibroma, and psoriasis. Dermal dendrocytes are considered to be established by a culture of peripheral blood monocytes in the presence of granulocyte macrophage-colony stimulating factor and interleukin-4. Reverse transcription--polymerase chain reaction, metabolic labeling, and immunofluorescence studies demonstrated that treatment of CD14+ peripheral blood monocytes with granulocyte macrophage-colony stimulating factor/interleukin-4 over a period of 8 d resulted in the induction of alpha1(XVI) collagen as well as factor XIIIa. The physiologic significance of colocalization of alpha1(XVI) collagen and factor XIIIa in the tissue and their coordinate induction in CD14+ monocyte-derived dendritic cells in vitro was studied. Considerable incorporation of [3H]putrescine by factor XIIIa into recombinant noncollagenous domain (NC) 11 but not into collagenous domain (COL) 1.NC1 domain of the alpha1(XVI) polypeptide was found. Incubation of recombinant NC11 of alpha1(XVI) polypeptide with factor XIIIa in vitro produced a covalent cross-linking complex on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The results indicate that alpha1(XVI) collagen is constitutively expressed by most dermal dendrocytes in the skin and dendritic cells differentiated from peripheral blood monocytes in vitro. Type XVI collagen is expressed in factor XIIIa+ dermal dendrocytes and may form an intermolecular cross-linking through NC11 domain by the reaction catalyzed by factor XIIIa contributing to the structural integrity of factor XIIIa+ dendritic cell-rich tissues.
Journal of Investigative Dermatology 03/2002; 118(2):267-74. · 6.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Composition and inducibility of cytochrome P450 (CYP) in ocular tissues were investigated using reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot techniques. Composition of ocular CYPs is more restricted than that of hepatic forms. High levels of CYP2B1/2 and reduced levels of CYP2C11 expression were detected in rat ocular tis- sues. Phenobarbital induced CYP2B1/2 expression in the lens but not in the rest of the eye, whereas CYP2C11 induction was observed in both regions. In addition, CYP2B1/2 proteins were found to be prominent in the retina. Our data indicate eye-specific regulation of CYP expression.
Journal of Health Science - J HEALTH SCI. 01/2002; 48(4):346-349.
[show abstract][hide abstract] ABSTRACT: The present investigation demonstrates the existence of NADH-dependent dehydroascorbate (DHA) reductase activity in the soluble fraction of the rabbit lens. This DHA reductase was specific for NADH, and its apparent Km values for DHA and NADH were 5.7 mM and 4.0 microM, respectively. In a gel filtration of the lens soluble fraction on a Sephadex G-75 superfine column, the NADH-dependent DHA reductase activity was eluted at the oligomeric betaL1-crystallin fraction, which may also contain lambda-crystallin (a rabbit-specific crystallin). Furthermore, about 80% of protein fractions separated from the betaL1-crystallin fraction by DEAE-cellulose ion-exchange column chromatography exhibited DHA reductase activity. In the SDS-PAGE analysis of the protein fractions with DHA reductase activity, 32-33, 27 and 25 kDa protein subunits were commonly identified. These results suggest that oligomers of beta-crystallin and/or lambda-crystallin subunits may be associated with the DHA reductase activity. The present paper also discusses that the function of the reductase may be to enhance the antiphotoxidation capacity of the lens.
The Tokai journal of experimental and clinical medicine 05/2001; 26(1):25-32.
[show abstract][hide abstract] ABSTRACT: 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.
Japanese Journal of Ophthalmology 01/2001; · 1.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: Purpose: To clarify the function of ascorbate free radical (AFR) reductase in the lens antioxidation mechanism, we investigated the difference among species in AFR reductase activity in different vertebrate lenses.Materials and Methods: Soluble and insoluble fractions were prepared from the lenses of frogs, guinea pigs, rats, rabbits, pigs, and calves. AFR reductase and diaphorase activity of each fraction was determined.Results: AFR reductase activity in the lens soluble fraction was the highest in frogs. That of guinea pigs and rabbits was at the next level; there was only a little activity in rats and pigs, and none was detected in calves. Membrane-bound AFR reductase in the lens insoluble fraction was extracted by 0.3% Triton X-100. The membrane-bound enzyme activity was almost at the same level in frogs, rats, rabbits, and calves, and a little higher in guinea pigs and pigs. However, such species-specificity of AFR reductase activity as in the soluble fraction was not observed in 0.3% Triton X-100 extracts. Diaphorase activity was 3 to 9 times as much as AFR reductase activity in the soluble fractions of frogs, guinea pigs, and rabbits, but in 0.3% Triton X-100 extracts of all vertebrate species used, it was very high, 108 to 311 times the AFR reductase activity.Conclusion: These results suggest that the lens soluble and membrane-bound AFR reductases are individual enzyme molecules and have different anti-oxidative functions. The lenses of frogs, guinea pigs, and rabbits contain a near-ultraviolet (UV) light absorbing compound, reduced pyridine nucleotide at a high concentration. Therefore, the soluble AFR reductase activity may be high in the vertebrate lenses with a near-UV light filter, and enhance the antiphotoxidation of ascorbic acid.
Japanese Journal of Ophthalmology 12/2000; 44(6):694. · 1.27 Impact Factor