Differences in α-Crystallin isomerization reveal the activity of protein isoaspartyl methyltransferase (PIMT) in the nucleus and cortex of human lenses.

Although it is well-known that protein turnover essentially stops in mature lens fiber cells, mapping out the ensuing protein degradation and its effects on lens function over time remains challenging. In particular, isomerization is a common, spontaneous post-translational modification that occurs over long timescales and generates products invisible to most analytical methods. Nevertheless, isomerization can significantly impact protein structure, function, and solubility, which are all necessary to maintain clarity and proper refractive index within the lens. Herein, we examine the degree of isomerization occurring in crystallin proteins in the human eye lens as a function of both age and location within the lens. A novel mass spectrometric technique leveraging radical chemistry enables detailed characterization of proteins extracted from the cortex and nucleus of the lens. It is observed that the degree of isomerization increases significantly between the cortex and nucleus and between water-soluble and water-insoluble fractions. Interestingly, the abundance of L-isoAsp is low in the water-soluble cortex despite being the dominant product generated by isomerization of Asp in vitro, suggesting that Protein L-isoaspartyl methyltransferase (PIMT) is active in the cortex and suppresses the accumulation of L-isoAsp. The abundance of L-isoAsp increases dramatically in the nucleus, revealing that PIMT activity decreases over time in the center of the lens. In addition, the growth of L-isoAsp in the nuclear fraction suggests protein isomerization continues within the nucleus, despite the fact that most of the protein within the nucleus has become insoluble. Additionally, it is demonstrated that sequential Asp residues lead to isomerization hotspots in human crystallin proteins and that the isomerization profiles for αA and αB crystallin are notably different. Although αA is more prone to isomerization, αB loses solubility more rapidly upon modification. These differences are likely related to the distribution of Asp residues within αA and αB, which are in turn connected to refractive index. The high Asp content of αA is a hazard in terms of isomerization and aging, but it serves to enhance the refractive index of αA relative to αB, and may explain why αA is only found in the eye.

1-Hydroxypyrazole as a bioisostere of the acetic acid moiety in a series of aldose reductase inhibitors.

Therapeutic intervention with aldose reductase inhibitors appears to be promising for major pathological conditions (i.e., long-term diabetic complications and inflammatory pathologies). So far, however, clinical candidates have failed due to adverse side-effects (spiroimides) or poor bioavailability (carboxylic acids). In this work, we succeeded in the bioisosteric replacement of an acetic acid moiety with that of 1-hydroxypyrazole. This new scaffold appears to have a superior physicochemical profile, while attaining inhibitory activity in the submicromolar range.

Medicinal flowers. XXXVIII. structures of acylated sucroses and inhibitory effects of constituents on aldose reducatase from the flower buds of Prunus mume.

The methanolic extract from the flower buds of Prunus mume, cultivated in Zhejiang province, China, showed an inhibitory effect on aldose reductase. From the methanolic extract, five new acylated sucroses, mumeoses F-J, were isolated together with 29 known compounds. The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects of the isolated compounds on aldose reductase were also investigated. Acylated quinic acid analogs, which are one of the major compounds of the flower buds of P. mume, were shown to substantially inhibit aldose reductase. In particular, mumeic acid-A was found to exhibit a potent inhibitory effect [IC50=0.4 µm].

Inhibitory effects of chlorogenic acid on aldose reductase activity in vitro and cataractogenesis in galactose-fed rats.

Chlorogenic acid (5-O-caffeoylquinic acid, CA), a phenolic compound found ubiquitously in plants, has antidiabetic effect in diabetic animal models. In this study, we investigated the inhibitory effect of CA on diabetic cataractogenesis. We evaluated the aldose reductase (AR) activity during cataract development in 50% galactose-fed rats, an animal model of sugar cataract. Galactose-fed rats were treated orally with CA (10 and 50 mg/kg body weight) once a day for 2 weeks. In vehicle-treated galactose-fed rats, lens opacity was increased, and lens fiber swelling and membrane rupture were observed. In addition, AR protein was highly expressed in lens epithelial cells and lens cortical fibers of galactose-fed rats. However, CA inhibited the rat AR activity in vitro, and the administration of CA prevented the development of sugar cataract through the inhibition of AR activity. These observations suggest that CA is useful for the treatment of sugar cataract.

MAPK/ERK1/2 and PI3-kinase signalling pathways are required for vitreous-induced lens fibre cell differentiation.

Lens epithelial cells withdraw from the cell cycle to differentiate into secondary fibre cells in response to vitreal factors. Fibroblast growth factor (FGF) in the vitreous has been shown to induce lens fibre differentiation in vivo and in vitro through the activation of defined intracellular signalling, namely via MAPK/ERK1/2 and PI3-K/Akt pathways. To better understand the role of these growth factor-activated signalling pathways in lens fibre differentiation, FGF- and vitreous-induced lens fibre differentiation was examined in primary rat lens epithelial cell explants. The induction of cell elongation and fibre specific beta- and gamma-crystallin expression in lens explants was accompanied by distinct phosphorylation profiles for ERK1/2 and Akt. Using selective inhibitors (U0126 and LY294002) in blocking studies, these pathways were shown to be required for different aspects of lens fibre differentiation. Furthermore, a short 'pulse' treatment of explants with FGF showed that the activation of ERK1/2 over 24 h was not sufficient for the progression of lens fibre differentiation and that cyclic ERK1/2 phosphorylation was required throughout the extended differentiation process. In conclusion, these results support a key role for both ERK1/2 and PI3-kinase/Akt signalling pathways in FGF- and vitreous-induced lens fibre differentiation.

Matrix metalloproteinase-9 activity in human lens epithelial cells of cortical, posterior subcapsular, and nuclear cataracts.

PURPOSE: To evaluate the level of matrix metalloproteinase-9 (MMP-9) activity in lens epithelial cells (LECs) derived from different types of cataract in patients having phacoemulsification. SETTING: Iladevi Cataract & IOL Research Centre, Memnagar, Ahmedabad, India. METHODS: This observational study of 275 patients having phacoemulsification was performed to evaluate the level of MMP-9 activity in LECs. All anterior lens capsules harboring the LECs and derived from the surgical curvilinear capsulorhexis were obtained during phacoemulsification. The anterior lens capsule samples were processed to analyze MMP-9 activity using a succinylated gelatin assay. The samples were grouped based on age and on pure cataract type. RESULTS: The level of MMP-9 activity in LECs was highest in eyes with cortical cataract. A significant difference in the level of MMP-9 activity was found in different types of cataract (P<.001). The highest level of MMP-9 activity was in patients older than 60 years. The MMP-9 activity increased gradually with age irrespective of cataract type (P<.001). CONCLUSIONS: A significant difference was observed in the level of MMP-9 activity between different types of cataract. The activity of MMP-9 was highest in eyes with cortical cataract. The level of MMP-9 activity increased with age in the LECs of patients with age-related cataract.

Thioredoxin, thioredoxin reductase, and alpha-crystallin revive inactivated glyceraldehyde 3-phosphate dehydrogenase in human aged and cataract lens extracts.

PURPOSE: To investigate whether mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), with or without alpha-crystallin can revive inactivated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in both the cortex and nucleus of human aged clear and cataract lenses. METHODS: The lens cortex (including capsule-epithelium) and the nucleus were separated from human aged clear and cataract lenses (grade II and grade IV) with similar average age. The activity of GAPDH in the water-soluble fraction after incubation with or without Trx or/and TrxR for 60 min at 30 degrees C was measured spectrophotometrically. In addition, the effect of a combination of Trx/TrxR and bovine lens alpha-crystallin was investigated. RESULTS: GAPDH activity was lower in the nucleus of clear lenses than in the cortex, and considerably diminished in the cataractous lenses, particularly in the nucleus of cataract lenses grade IV. Trx and TrxR were able to revive the activity of GAPDH markedly in both the cortex and nucleus of the clear and cataract lenses. The percentage increase of activity in the cortex of the clear lenses was less than that of the nucleus in the presence of Trx and TrxR, whereas it was opposite in the cataract lenses. The revival of activity in both the cortex and nucleus from the cataract lenses grade II was higher than that of the grade IV. Moreover, Trx alone, but not TrxR, efficiently enhanced GAPDH activity. The combination of Trx and TrxR had greater effect than that of either alone. In addition, alpha(L)-crystallin enhanced the activity in the cortex of cataract grade II with Trx and TrxR present. However, it failed to provide a statistically significant increase of activity in the nucleus. CONCLUSIONS: This is the first evidence to show that mammalian Trx and TrxR are able to revive inactivated GAPDH in human aged clear and cataract lenses, and alpha-crystallin helped this effect. The inactivation of GAPDH during aging and cataract development must be caused in part by disulphide formation and in part by unfolding, and can be recovered by reducing agents and a molecular chaperone.

Inhibition of fiber cell globulization and hyperglycemia-induced lens opacification by aminopeptidase inhibitor bestatin.

PURPOSE: To examine the role of calcium-dependent and -independent proteolytic activity in the globulization of isolated fiber cells and glucose-induced lens opacification. METHODS: Fiber cells from rat lens cortex were isolated, and the [Ca(2+)](i) and protease activity in the isolated fibers were determined by using a calcium binding dye and the protease substrate t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin (BOC-Leu-Met-CMAC). The activity of calpain in the lens cortex homogenate was determined with fluorescein-casein in the presence of Ca(2+) and that of fiber cell globulizing aminopeptidase (FCGAP) with BOC-Leu-Met-CMAC and reduced glutathione (GSH) in the absence of Ca(2+). The lens proteases-calpain and the novel aminopeptidase FCGAP were partially purified by diethylaminoethyl (DEAE) gel column chromatography. Single fiber cells were isolated from rat lens, plated on coverslips, and placed in a temperature-controlled chamber. Their globulization time was determined by the appearance of light-scattering globules in the absence and the presence of protease inhibitors including the aminopeptidase inhibitor bestatin. To investigate the effect of the protease inhibitors E-64 and bestatin on the prevention of hyperglycemic cataract, the rat lenses were cultured in medium 199 in the presence of 5.5 and 50 mM glucose and in the absence and the presence of protease inhibitors. Changes in light transmission by the lenses were determined by digital image analysis. RESULTS: Normal levels of lens fiber cell [Ca(2+)](i), determined by using a cell-permeable dye were approximately 100 nM, and the protease activity determined with BOC-Leu-Met-CMAC was maximum at [Ca(2+)](i) of approximately 500 nM. A large fraction of the FCGAP that cleaves BOC-Leu-Met-CMAC was separated from calpain, which cleaves fluorescein-casein, by diethylaminoethyl (DEAE) gel column chromatography. The FCGAP did not bind to the column, whereas calpain bound to the column and was eluted by approximately 180 mM NaCl. Unlike calpain, the FCGAP did not require calcium for activation and did not cleave fluorescein-casein. However, the Ca(2+)-dependent calpain activated FCGAP, indicating that the latter may exist in pro-protease form. The FCGAP was selectively inhibited by the specific aminopeptidase inhibitor bestatin, indicating that FCGAP could be an aminopeptidase. However, the FCGAP was found to be immunologically distinct from leucine aminopeptidase and calpain. Perfusion of the isolated rat lens fiber cells with Ringer's solution led to their globulization in 30 +/- 3 minutes. Addition of 0.5 mM of the protease inhibitors E-64 and leupeptin increased the globulization time to 60 and 100 minutes, respectively, whereas no globulization of the fiber cells was observed for 4 hours in the presence of 0.05 mM bestatin. In rat lens cultured in medium containing 50 mM glucose, both E-64 and bestatin (0.05 mM each) significantly reduced the extent of opacification, indicating that an aminopeptidase, downstream to a Ca(2+)-dependent protease, may be involved in mediating cataractogenic changes. CONCLUSIONS: In addition to calpain, a Ca(2+)-independent novel protease, FCGAP, a novel aminopeptidase, represents a significant fraction of the total proteolytic activity in the lens. Inhibition of FCGAP by bestatin attenuates Ca(2+)-induced globulization of the isolated fiber cells in vitro and hyperglycemia-induced opacification of cultured rat lens.

The role of Src family kinases in cortical cataract formation.

PURPOSE: The goal of this study was to determine the role of Src family kinases (SFKs) in the development of lens cataract. This question was particularly significant, because these tyrosine kinases mediate the stress pathways known to lead to cataract formation. The experiments were focused on whether the inhibition of SFK activity suppresses the formation of lens opacities. METHODS: A whole-lens culture system was developed, in which cortical opacities formed within 5 days, in embryonic day (E)10 lenses grown in medium containing 10% fetal bovine serum. SFK activity was blocked in the cultured lenses by growth in the presence of the SFK-specific inhibitor PP1. Control cultures were grown in medium without inhibitor or in the presence of PP3, the inactive analogue of PP1. Lenses were cultured for 10 days, observed, and photographed daily. Opacification was quantified with image-analysis software. Tissue architecture was determined after hematoxylin and eosin staining and cellular organization by fluorescent localization of filamentous actin with fluorescein-conjugated phalloidin. RESULTS: Almost all lenses in the control cultures developed cortical opacities covering approximately 50% of the lens area by day 10. Similar to control cultures, PP1-treated lenses showed mild posterior opacities during the first 5 days in culture, but then became strikingly transparent. Only 7% of the PP1-treated lenses showed development of cortical cataract, and the average area of opacity was just 0.5% by culture day 10. In all cultured lenses, even in the presence of the PP1 inhibitor, the bow region of the lens extended to the posterior pole, and distribution of nuclei from the posterior pole toward the anterior aspects of the lens suggested that newly added fiber cells were misdirected. However, neither this feature, nor the presence of vacuoles appeared to correlate with the development of opacity in the cultured lenses. Instead, the lens opacities appeared to result from gross abnormalities in the shape and organization of cells in the equatorial and cortical fiber zones, as observed by F-actin staining. Culturing the lenses in the presence of the SFK inhibitor prevented these lens cell aberrations as well as the development of lens opacity. CONCLUSIONS: The formation of cataract can involve activation of SFK-mediated pathway(s) leading to disorganization of developing lens fiber cells, and inhibiting these tyrosine kinases blocks cataract progression.

Endogenous casein kinase I catalyzes the phosphorylation of the lens fiber cell connexin49.

The lens fiber cell-specific gap junction protein connexin49 is a substrate for a membrane-associated Ser/Thr protein kinase that can be extracted from lens cell membranes by 0.6 M KCl. However, the identity of this protein kinase has not been defined. In this report, evidence is presented indicating that it is casein kinase I. Thus, connexin49 was shown to be a substrate for purified casein kinase I but not for casein kinase II; the endogenous connexin49 protein kinase activity extracted from lens membranes with KCl was inhibited by the casein kinase I-specific inhibitor, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CKI-7); the connexin49 protein kinase activity in the lens membrane KCl extract, which could be partially purified by gel filtration and affinity purification with a casein-Sepharose 4B column, copurified with casein kinase activity; phosphopeptide analysis showed that casein kinase I and the connexin49 protein kinase activity in the lens membrane KCl extract probably share the same phosphorylation sites in connexin49. Reverse transcription-PCR using total ovine lens RNA and casein kinase I isoform-specific oligonucleotide primers resulted in the amplification of cDNAs encoding casein kinase I-alpha and -gamma, while an in-gel casein kinase assay indicated casein kinase activity in the lens membrane KCl extract was associated with a major 39.2-kDa species, which is consistent with the 36 to 40-kDa size of casein kinase I-alpha in other animal species. These results demonstrate that the protein kinase activity present in the lens membrane 0.6 M KCl extract that catalyzes the phosphorylation of connexin49 is casein kinase I, probably the alpha isoform.

On the use of Zn2+ to discriminate endonucleases activated during apoptosis.

One approach to discriminate among specific DNases in apoptosis is to use inhibitors specific for each endonuclease. Zn2+ is known to inhibit Ca(2+)- and Mg(2+)-dependent endonuclease enzymatic activities during apoptosis. Acidic DNases were thought to be insensitive to Zn2+. In this paper, we analyse the effects of Zn2+ on activity of DNase II, either purified or in nuclei from lens fiber cells. These cells follow a physiological nuclear degeneration with DNase II accumulation in their nuclei. We show that Zn2+ is able to inhibit also this acidic endonuclease at a concentration of 1-6 mM. At a higher concentration of Zn2+, DNA is extensively degraded during the assay, masking the inhibition of the enzyme. This DNA degradation in the presence of Zn2+ has led to an overestimation of the activity of DNase II in studies of apoptosis. Hence, Zn2+ cannot be used to specifically identify one endonuclease among the different DNases involved in nuclear degradation during programmed cell death.

Xanthurenic acid derivative formation in the lens is responsible for senile cataract in humans.

BACKGROUND: The tryptophan degradation pathway leads to NAD production via 3-hydroxykynurenine. Kynurenine aminotransferase (KAT) transforms 3-hydroxykynurenine into xanthurenic acid. In this study, we measured the activity of KAT in human lenses and studied the consequences of xanthurenic acid formation METHODS: KAT activity was determined by the method of Tobes. Fluorescence spectroscopy and SDS-PAGE were used for the protein studies. Thin-layer chromatography and infrared and fast atom bombardment spectrometry were used for substance characterization. RESULTS: The KAT activity was detected in senile cataratous lenses, but was absent in the young lenses. Xanthurenic acid at physiological pH exists in equilibrium with its tautomeric form reported by us as oxo-xanthurenic acid (OXA), which is oxidized to di-oxoxanthurenic acid (DOXA), a naphthoquinone-like substance. The incubation of DOXA with crystallins in a solution of physiological pH led to crystallin crosslinking and formation of conjugates with glutathione. CONCLUSIONS: Xanthurenic acid is formed in human lenses. Its tautomerization and oxidation leads to a naphthoquinone-like substance, DOXA. DOXA provoked formation of conjugates with glutathione and crosslinking of crystallins. Thus, KAT activity seems to be the initial event in senile cataract formation in humans.

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.

A high phosphotyrosine phosphatase activity is present in differentiating bovine lens cells.

Activity of phosphotyrosine-protein phosphatases (PTPases) has been investigated in the different cellular regions of bovine eye lens. PTPases were tested in cellular detergent extracts using phospholabelled synthetic peptides and p-nitrophenyl phosphate. We show that a high PTPase activity is only present in cells which undergo differentiation, namely the equatorial epithelium and cortex fiber cells. Since this activity is found to be severely inhibited by a specific inhibitor of receptor-type PTPases, it can be suggested that one or more members of this class of PTPases might play an important role in the lens differentiation process.

Soluble ascorbate free radical reductase in the human lens.

A major and a minor ascorbate free radical (AFR) reductase were separated from the soluble fraction in the human lens cortex by DEAE-cellulose ion-exchange column chromatography. These AFR reductases also exhibited diaphorase activity using dichlorophenolindophenol and ferricyanide as electron acceptors. The major AFR reductase was partially purified by 5'AMP-Sepharose 4B affinity column chromatography. This partially purified AFR reductase showed a single band of diaphorase activity in native polyacrylamide disc gel electrophoresis. This activity band corresponded to the major protein observed in protein staining by Coomassie Brilliant Blue. However, the protein staining by Coomassie Brilliant Blue showed this activity band surrounded by diffused staining. Molecular weight of the partially purified AFR reductase was determined to be 32 kDa by gel filtration, and the apparent Km value for AFR was about 15 microM. This major lens AFR reductase could be distinguished from soluble Neurospora, Euglena and cucumber AFR reductases, and from two ubiquitous enzymes with reduction activity of AFR and/or foreign compounds, ie, NADH-cytochrome b5 reductase and DT-diaphorase, by their molecular weights, Km values and/or ion-exchange chromatographic behaviors.

Low and high Km forms of dinitrophenylglutathione-stimulated ATPase in bovine lens.

Dinitrophenyl S-glutathione (Dnp-SG) ATPase which catalyses the hydrolysis of ATP in the presence of GSH-conjugates has been implicated previously in the transport of these conjugates. In the present studies we demonstrate that Dnp-SG ATPase is present in bovine lens epithelium and cortex. The specific activity per mg membrane protein was found to be 75-fold higher in the epithelium as compared to the cortex. No enzyme was detected in the nuclear region of the lens. Dnp-SG ATPase was purified from bovine lens epithelium and cortex using Dnp-SG-Sepharose 6MB affinity chromatography. The partially purified Dnp-SG ATPase had two distinct Km values, 120 microM and 1.0 mM. The antibodies raised against human erythrocyte Dnp-SG ATPase cross-reacted with the bovine lens epithelium Dnp-SG ATPase which was identified by Western blot as a band corresponding to an approximate M(r) value of 80,000 Da.

Distribution of lens sodium-potassium-adenosine triphosphatase.

PURPOSE: The specific activity of sodium-potassium-adenosine triphosphatase (Na-K-ATPase) in lens fiber cells is lower than the specific activity in lens epithelium. To test whether there is a reduction in the expression of Na-K-ATPase molecules in lens fibers, a Western blot technique was used. METHODS: Membrane material was isolated from different regions of the rabbit lens. Na-K-ATPase (adenosine triphosphate hydrolysis) activity was measured in each membrane sample and Western blots were performed using an antibody to rabbit kidney Na-K-ATPase. RESULTS: By immunoblotting, Na-K-ATPase polypeptide was detected in all lens cells. In contrast, adenosine triphosphate hydrolysis by the Na-K-ATPase (Na-K-ATPase activity) was not detectable or was detectable only at very low levels in fiber membranes from the lens nucleus and in cortex. CONCLUSION: These findings suggest that plasma membrane adenosine triphosphatase enzyme responsible for sodium-potassium transport is expressed in newly formed lens fibers and the transport molecules are retained as the fibers age and are compressed toward the center of the lens. However, with fiber aging there is a loss of functional ability of the Na-K-ATPase to hydrolyze adenosine triphosphate.

Distribution and activity of glutathione-S-transferase in normal human lenses and in cataractous human epithelia.

The distribution of glutathione-S-transferase (GST) activity was determined in frozen normal human lenses. The highest activity of GST was found in the peripheral and equatorial regions, whereas the lowest activity was found in the nucleus. Western blot showed that both mu and pi isoenzymes of GST were present in human lenses. This result is similar to that found in rat lenses. In addition, GST activity was analyzed in 50 lens epithelia which were obtained during cataract surgery. Twenty-seven lens epithelia showed no activity. Statistically significant association was found between cortical and mixed cortical--nuclear cataract and loss of GST activity. No association was found between pure nuclear cataract and loss of epithelial GST activity.

Pravastatin and simvastatin differently inhibit cholesterol biosynthesis in human lens.

PURPOSE: In the current study, the hypocholesterolemic drugs pravastatin and simvastatin were compared for their influence on cholesterol biosynthesis in the human lens. METHODS: For measurements of cholesterol and fatty acid synthesis rates, human lenses were incubated for 20 hr in the presence of [14C]-acetate, and pravastatin or simvastatin. Radiolabeled [14C]-cholesterol and [14C]-fatty acids were determined. To avoid the influence of individual differences, one lens from each donor was incubated without drug (control) and the other lens was incubated in the presence of drug. For each lens pair, the percentage inhibition of the cholesterol synthesis caused by the drug was calculated. Fatty acid synthesis was not influenced by the drugs. By comparing the fatty acid synthesis rate of the drug-incubated with the control lens of a pair, a predefined exclusion criterion was used to eliminate lens pairs in which the lenses had no comparable biosynthetic capacities. RESULTS: Using various concentrations of the drugs, a dose-response curve was constructed for the inhibition of the cholesterol synthesis. The IC50 values (drug concentration give 50% inhibition) were 0.5 mumol/l and 0.004 mumol/l for pravastatin and simvastatin, respectively. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in microsomal membranes from human lens cortex was inhibited by simvastatin and pravastatin to the same extent. CONCLUSIONS: Under the conditions used in this study, cholesterol synthesis in human lenses is inhibited by simvastatin 100-fold more effectively than by pravastatin. This difference was likely due to differences in the intracellular exposure of the reductase to the drugs in intact human lenses.

Regional and subcellular distribution of ascorbate free radical reductase activity in the human lens.

This paper reports that the human lens is endowed with a characteristic regional and subcellular distribution of ascorbate free radical (AFR) reductase (EC 1.6.5.4) activity, and it is associated with the soluble fraction of the lens protein. AFR reductase activity in either immature senile cataractous or transparent lens was higher in the cortex, where the level of insoluble protein is lower, than in the nucleus. The high reductase activity and low insoluble protein content of the cortex were comparable between the assayed cataractous and transparent lenses. In the nucleus, in contrast, the reductase activity tended to decrease with an increase in the level of insoluble protein and with the development of nuclear coloration (sclerosis). As for the subcellular distribution of AFR reductase activity, 70-90% of the enzyme activity in the lens was located in the cytosol fraction. Based on the above results, it was proposed that cytosolic free radicals may be involved in oxidation, coloration and aggregation of lens protein in senile cataractogenesis and in lens aging.