Amino acid transport and crystallin synthesis in the bovine lens

Bovine lenses were incubated in a defined, bicarbonate-free culture medium (EMEM) and the kinetics of amino acid uptake and protein synthesis investigated. The kinetics were interpreted in terms of a simple multi-compartment model. [¹⁴C]tyrosine was found to be totally exchangeable in the incubated lens and the rate constant for the exponential increase in activity was 0·0175 hr^?1. The rate of influx was markedly reduced by incubating in the presence of ouabain (10^?5m), which also caused a concomitant disturbance of the normal sodium and potassium distributions.The soluble proteins from the ineubated lenses were fractionated on Sephadex G-200 and the rate of incorporation into the crystallins was shown to fall into two classes. The rate of synthesis of α and β_L crystallin was relatively rapid (rate constants approximately equal to 0·004 hr^?1), while the synthesis rates of β_H and $\text{β}{}_{\mathrm{<mtext>S</mtext>}}\text{γ}$ were both much slower (0·001 hr^?1).The efflux kinetics of [¹⁴C]tyrosine were determined and the rate of decrease of the free amino acid pool was identical to the rate of increase determined from an influx experiment. Hence the lenses are in a steady state with respect to free tyrosine throughout the incubation period (up to 160 hr). All classes of proteins continued to be synthesized during efflux experiments and there was no evidence for a breakdown of α or β_L crystallin during the time-course of these experiments.Ouabain slowed the rate of loss of tyrosine from the free amino acid pool, and this was interpreted in terms of an ouabain-induced decrease in synthesis rate rather than as a decrease in efflux rate from the lens. There was in fact a very marked decrease in the incorporation of [¹⁴C]tyrosine into the α and β_L crystallins on exposure to ouabain, and this decrease was apparent before and change in activity in the amino acid pool.     

Urea formation in rat,bovine, and human lens

Lenses produce both ammonia and urea, and a previous report suggested that bovine lenses contain a complete urea cycle capable of synthesizing urea from bicarbonate and ammonia. To determine whether lenses produce urea by a complete urea cycle or by arginase alone, intact lenses were cultured with [guanido-¹⁴C]-arginine or [¹⁴C]-bicarbonate. The [¹⁴C]-urea was volatilized to [¹⁴C]-CO₂ by urease and collected in KOH. The cultured rat, bovine and human lenses produced [¹⁴C]-urea from [¹⁴C]-arginine; therefore lens arginase activity was also examined in homogenates of rat and human lenses. Rat lens homogenates had constant arginase activity for at least 2 hr at 37°C, and activity increased linearly with the concentration of lens homogenate. Rat lens arginase had an apparent V_max of approximately 13 nmol/hr/mg lens wet weight in lens homogenates and produced 4–6 nmol urea/hr/mg at 25 mm arginine. Human lens homogenates produced 1–5 nmol/hr/mg. In contrast, neither bovine nor rat lenses cultured with [¹⁴C]-bicarbonate produced detectable [¹⁴C]-urea, although label was incorporated into unidentified nonvolatile products. These products were shown by ion exchange chromatography and enzymatic assay to contain no detectable arginine or urea. It was concluded that although arginase activity is present, neither rat nor bovine lenses contain significant urea cycle activity. However, it is possible that arginase serves as a source of lens ornithine.     

In vitro incubation paralleling changes occurring during mouse cataract formation

Certain biochemical and morphological changes involved in the formation of osmotic cataracts in the Nakano mouse and galactosemic rat lenses also occur in vitro. Incubation of young, normal, albino mouse lenses in glucose-free medium for 48 hr resulted in a gain in wet weight due to dramatic hydration. The glucose deprived lenses showed a marked decrease in the soluble protein content as is noted in cataract formation in vivo. Chromatography of the soluble protein demonstrated a substantial increase in heavy molecular weight material in the lenses incubated without glucose in comparison to the fresh lenses. A decrease in β_H-crystallin was also striking and might be correlated with the disappearance of a 31 000 mol. wt polypeptide band seen by SDS-PAGE gel. Interestingly, the membrane polypeptides in the in vitro incubated lenses mimicked changes observed during cataract formation in vivo. A 23 000 mol. wt polypeptide band became prominent in the membrane fraction of these lenses and there was a concomitant decrease in the 26 000 mol. wt component. Messenger RNAs were present in the glucose deprived lenses as shown by cell-free translation in vitro, although no [³⁵S]methionine incorporation into crystallin was noted. Both the hexokinase and glyceraldehyde phosphate dehydrogenase activities in the glucose deprived lenses fell rapidly and were virtually absent at the end of 48-hr incubation period. The data in this report suggest that the glucose deprived incubated mouse lens may serve as an in vitro model to the study of some biochemical and morphological changes occurring in the development of osmotic cataract in vivo.     

Analysis of lens protein synthesis in a cataractous mutant mouse: The Cat Fraser

The cataract produced by the dominant Cat Fraser gene in mouse is associated with quantitative changes in lens proteins (crystallin) and with capsule abnormalities. We have analyzed and compared the protein synthesis in control and mutant lenses using [³H]leucine and [³H]proline incorporation. The specific activities of free [³H]leucine in the intracellular pools of the two mouse strains were identical, while the incorporation of both labelled amino acids in proteins was largely increased in Cat Fraser lens. These data indicate that the higher labelling of Cat Fraser lens proteins reflects a true change in the cellular synthesis activity by Cat Fraser lens cells. Despite the enhanced type IV collagen synthesis by Cat Fraser epithelial cells, the amount of type IV collagen in Cat Fraser capsule is lower than in control. This altered type-IV collagen metabolism may disturb the structure of Cat Fraser capsule which becomes thicker.     

Differential synthesis and degradation of protein in the hereditary Philly mouse cataract

Lens protein metabolism was investigated in the Philly mouse between the third and eighth postnatal week. As demonstrated in an accompanying article, the Philly mouse develops a hereditary, osmotic, cataract associated with influx of Na⁺ and loss of K⁺ during this time interval. The contents of β- and γ-crystallin were strikingly reduced in the Philly lens, as judged by sodium dodecyl sulfate (SDS)-urea-polyacrylamide gel electrophoresis and by immunodiffusion. This appeared to be due to proteolysis, since there were negligible amounts of crystallins found in the medium of cultured Philly lenses. α-Crystallin remained in the Philly lens but apparently accumulated discrete polypeptide cleavage products. The incorporation of [³⁵S]methionine into β- and γ-crystallin polypeptides was markedly reduced in the Philly lens. By contrast, the incorporation of [³⁵S]methionine into the α-crystallin and the higher molecular weight non-crystallin polypeptides was as great if not greater, in the Philly lens than in the normal lens. The non-crystallin polypeptides were associated with the 10 000 × g pellet of the homogenate. The present data extend the correlation between alterations in protein metabolism and electrolyte concentrations to this hereditary cataract, and support the idea that selective degradation of crystallins and differential reduction in the synthesis of crystallins are primary causes for the lowered amounts of soluble protein—especially β- and γ-crystallin—found in cataracts associated with ionic imbalances.     

Glutathione synthesis and glutathione redox pathways in naphthalene cataract of the rat

This investigation examined many parameters during the course of early development of naphthalene-induced cataract in a time span of 0 to 79 days of treatment. Feeding naphthalene daily to Black-Hooded rats resulted in gradual progressive development of cataract. The first faint opacities were detectable after 7 days. Free soluble total glutathione (oxidized and reduced) of these lenses was shown to gradually decrease to a maximum loss of about 20%, a value reached by day 30 of treatment. No activity loss of either enzyme required for glutathione synthesis (gamma-glutamylcysteine synthetase or glutathione synthetase) was observed in homogenates of naphthalene versus control lenses. There was also neither impairment of [35S]-L-cystine uptake nor of [35S]-glutathione synthetic capacity in lenses cultured from rats after 12, 24 or 36 days of naphthalene feeding when compared to control lenses. Hence, glutathione loss cannot be explained by a damaged glutathione synthesis system. Progressive activity loss of glutathione peroxidase and glutathione reductase was observed. The loss of glutathione peroxidase activity was especially remarkable. Thus, the defense system against oxidative damage is impaired and may be a significant factor in naphthalene-induced cataract of the rat.     

Effect of osmotic stress on phosphorylcholine efflux and turnover in rat lenses.

The concentration of the phospholipid precursor, phosphorylcholine (P-choline), is greater than 10 mM in rat lenses. Cataractogenic osmotic or oxidative stress affects lenticular choline uptake and metabolism and decreases the P-choline concentration. To study the mechanism(s) of the decrease in P-choline concentration induced by the cataractogenic sugars, xylose, galactose or glucose, rat lenses were first incubated in TC-199 medium containing [3H]choline, and then the metabolism of the resulting lenticular P-[3H]choline was followed in culture. Lenses which were osmotically stressed by incubation in TC-199 medium with 30 mM xylose lost more than 50% of their P-[3H]choline within 48 hr. Most of the P-[3H]choline lost from the stressed lenses was recovered in the incubation medium as P-[3H]choline, indicating that leakage of P-choline from the osmotically damaged lenses was the principal factor contributing to the decrease. Leakage of P-[3H]choline from lenses in the xylose medium was about three-fold greater than in the control medium, which contained 30 mM fructose. The turnover of the P-choline pool in rat lenses was also studied, and the concentration of P-choline was found to be a balance between hydrolysis and synthesis. The hydrolysis of lenticular P-choline was similar in xylose or control medium. In contrast, P-choline synthesis is slower in osmotically stressed lenses, resulting in a net conversion of P-[3H]choline to [3H]choline in the stressed lenses. Because some of the [3H]choline derived from P-choline hydrolysis was lost to the surrounding culture medium, this mechanism also contributed to the decreased P-choline in lenses incubated with xylose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anomeric preference in uptake of D-glucose and of D-galactose by rat lenses

To determine the anomeric preference of uptake of D-glucose and of D-galactose by rat lenses, we crystallized alpha-, beta-D-[U-14C]glucose (720 microCi/mmol) and alpha-, beta-D-[U-14C]galactose (180 microCi/mmol) by our method and incubated them separately with rat lenses for 1 min, because of the short half-life of mutarotation of alpha-D-glucose (9.6 min) and of alpha-D-galactose (4.6 min) in HEPES medium at 30 degrees C. During aerobic incubation of rat lenses in HEPES medium containing radioactive alpha or beta anomer of D-glucose, there was no significant difference in the rate of uptake between alpha and beta anomers of D-glucose by rat lenses. However, 1.59 times greater incorporation of alpha-D-galactose was observed over that of beta-D-galactose under the same conditions.     

The effect of cycloheximide on rubidium fluxes in the cultured bovine lens

Background : Changes in lens electrolyte balance and hydration are considered to play a significant role in the aetiology of cataract. However, the reasons for these changes are not understood. The purpose of this study was to examine the requirement of protein synthesis for maintenance of electrolyte balance in the lens. Methods : Whole adult bovine lenses were incubated for up to five days in the presence and absence of 0.4 mM cycloheximide. The uptake and subsequent efflux of [86]Rb was measured before and after exposure of the lenses to ouabain. Results : Incubation with cycloheximide for 23 hours did not affect [86] Rb uptake in the cultured bovine lens. However, exposure to cycloheximide for five days reduced the ability of the lens to accumulate [86] Rb by 69 per cent. This is similar to the 65 to 75 per cent reduction, which was obtained when lenses maintained in culture for zero or five days were incubated with ouabain. Ouabain and cycloheximide did not affect efflux rates for the [86] Rb after 23 hours. Exposure to cycloheximide for five days resulted in a three-fold increase in efflux rate relative to the control lenses. Subsequent exposure of the five-day control lenses to ouabain produced a similar increase. Conclusions : The data are consistent with the notion that continued protein synthesis is required for the maintenance of normal electrolyte balance in the lens.     

Identification of the preferentially targeted proteins by carbamylation during whole lens incubation by using radio-labelled potassium cyanate and mass spectrometry

AIM: To attempt to identify the primary targets of carbamylation in bovine lenses incubated under physiological condition. METHODS: Fresh intact bovine lenses were incubated with [14C]-labelled potassium cyanate for seven days. The water-soluble proteins (WSP) of both cortex and nucleus lens were isolated by size-exclusion chromatography on a Sephacryl S-300HR column. The higher radioactive fractions were pooled and freeze-dried, and separated further by loading on an Affinity Blue column to separate some enzymes. In addition, WSP from cortex was separated directly by affinity chromatography. The most reactive fractions with higher radioactivity from [14C]-cyanate were further analyzed by SDS-gels and mass spectrometry. RESULTS: The majority of protein incorporating [14C]-labelled potassium cyanate was in the water-soluble fractions, and much more in the cortex than in the nucleus. Chromatography results demonstrated that the major incorporated [14C]-carbamylated crystallins were fractions corresponding to α-crystallin, β-crystallin and ξ-crystallin in the cortex, but β-crystallin and γ-crystallin in the nucleus. The SDS gels showed that bound fractions of cortex crystallins after Affi-Gel Blue separation were abundant with 20 and 35kDa proteins. However, the bound fractions of nucleus crystallins mainly showed 20kDa proteins. Mass spectrometry analysis of these higher radioactivity fractions and a database search revealed that the proteins were originated from bovine α-crystallin A and B chains and ξ-crystallin in the cortex; βA1 and αB-crystallins with a little γB-crystallin in the nucleus respectively. Further analysis suggested the location of this carbamylation of αB-crystallin in the nucleus to be at Lys 92 and 103. CONCLUSION: α-and ξ-crystallin from cortex can be prefe- rentially targeted by carbamylation during whole lens incubations. Carbamylation of these crystallins at the earlier stage may result in further unfolding and misfolding of lens proteins, leading to aggregation of crystallins and eventually to cataract formation.     

Metabolism of 15N-labeled amino acids in rat lens

Rat lenses were cultured 4–24 hr at 37°C in balanced salt medium containing 5 mm [¹⁵N]-glutamate, [¹⁵N]-alanine, or amido-[¹⁵N]-glutamine. Free amino acids were extracted with 6% trichloracetic acid containing α-aminoisobutyrate as an internal standard, and trifluoroacetyl-n-butyl (TAB) derivatives were prepared. Amino acids were quantified by gas chromatography, and ¹⁵N enrichment in amino groups of several free amino acids was determined by mass spectrometry. Culture of lenses with either [¹⁵N]-glutamate or [¹⁵N]-alanine resulted in [¹⁵N]-labeling of the glutamate, aspartate, alanine, proline, serine and glycine pools. No detectable amino-¹⁵N (< 5%) was observed in amino acids when lenses were cultured with amido-labeled [¹⁵N]-glutamine or with [¹⁵N]-ammonium chloride. It is concluded that the α-amino groups of several amino acids are actively involved in lens metabolism. In contrast, although glutamine may serve as the major source of glutamate for lens, the amido nitrogen of glutamine plays a minor role, if any, as a source of α-amino groups for amino acid metbolism.     

Calcium-induced opacification and loss of protein in the organ-cultured bovine lens

A long-term system of organ culture for bovine lenses was used to investigate the effect of osmotic stress on lens opacification and crystallin loss. Lenses were pre-incubated in control medium containing L-[U-14C]tyrosine so that labelled crystallins were produced. The fate of these crystallins was studied in relation to two forms of osmotic stress. The addition of either ouabain or EGTA to the medium induced severe osmotic swelling and disturbance of the lens monovalent cation balance, but only the former treatment was followed by an increase in lens calcium. The changes due to osmotic stress were accompanied by loss of transparency and protein only in the lenses with increased calcium. Both opacification and increased calcium were found largely to be confined to the outer cortical fibres. Protein loss increased with time as lens calcium continued to increase. The protein recovered from the incubation medium was characterized by gel filtration and immunological techniques. The first protein detected was beta L-crystallin, and this formed the major part of the lost protein throughout, although alpha- and gamma-crystallins were detected at a later stage. Increased calcium also resulted in a change in the susceptibility of the crystallins to aggregation, since there was an increase in [14C]tyrosine incorporated into the lens high-molecular-weight (HM) fraction after exposure to ouabain, but not after exposure to EGTA. The relevance of these findings to human cataract is discussed.     

Biosynthesis of proteoglycans by lens epithelial cells of cataractous mouse (Nakano strain)

The capsules (with epithelial cells attached) of lenses from normal and cataractous mice (Nakano strain) were biosynthetically labeled in vitro with radioactive precursors. The labeled macromolecules were chromatographed on a Sepharose CL-4B column and analyzed by specific enzyme digestion. The incorporation of [3H]-proline and [3H]-glucosamine into macromolecules was comparable in the cataract and normal capsules, while that of [35S]-sulfate was reduced by 60% in the cataract capsules, indicating that the proteoglycan synthesis was specifically decreased in the cataract lens. Glycosaminoglycan analyses showed an increased synthesis of hyaluronic acid and decreased synthesis of heparan sulfate in the cataract capsules. It is possible that the alterations in the synthetic level and glycosaminoglycan components of proteoglycan affect the permeabilities of macromolecules to lens capsule and lead to cataract in Nakano mouse lens.     

Differential synthesis of crystallins in the developing rat eye lens

The patterns of protein synthesis in rat lenses ranging in age from newborn to 4 months were compared. After incubation of lenses in [35S]methionine-containing medium it was possible to identify the de novo synthesized crystallins by two-dimensional gel electrophoresis and fluorography, in combination with peptide mapping and immunoblotting. It was found that the relative synthesis of alpha A and beta A3 stays fairly constant in rat lenses of all investigated ages. The relative synthesis of beta B2 and gamma s shows a pronounced increase with age in these post-natal lenses. A differential decrease can be observed in the relative synthesis of the other six gamma-crystallins (gamma A-gamma F). There appears to be a good correlation between the changes in relative synthesis of the various crystallins and previously reported alterations in mRNA levels, although certain mRNAs exhibit marked differences in translational efficiency.     

The metabolism of glutamine in the bovine lens: glutamine as a source of glutamate

The metabolism by the intact bovine lens of l-[U-¹⁴C]glutamine has been studied and compared with that of l-[U-¹⁴C]glutamate. [¹⁴C]Glutamine was taken up and oxidized to ¹⁴CO₂ at three times the rate of [¹⁴C]glutamate. [¹⁴C]Glutamine was largely converted to [¹⁴C]glutamate, which was found in the lens in the free form, in glutathione and in protein. Proline and three unidentified compounds were also labelled.The presence of unlabelled 2 mm-glutamine greatly reduced the production of ¹⁴CO₂ and labelled protein from 5 μm[¹⁴C]glutamate, whereas unlabelled 2 mm-glutamate had little effect on the metabolism of 5 μm [¹⁴C]glutamine. The concentration of free glutamate in the lens was greater after incubation with 2 mm-glutamine than with 2 mm-glutamate.Glutamine and glutamate were assayed in the lens and aqueous humour. The ratio of concentrations in lens water to that in the aqueous humour is about 10·7 for glutamate and 0·55 for glutamine.     

Mechanism of cataract production by 3-beta(2-diethylaminoethoxy) androst-5-en-17-one hydrochloride, U18666A: an inhibitor of cholesterol biosynthesis.

The present report describes a new model for cataract research induced by a compound (3-β(2-diethylaminoethoxy) androst-5-en-17-one hydrochloride; i.e. U18666A) which inhibits the enzymatic reduction of desmosterol to cholesterol and provides information on the possible mechanism of cataractogenesis. Treatment of the new born rat with U18666A results in nuclear cataracts appearing between 15 and 25 days of age. This cataract is apparently unrelated to accumulation of desmosterol, since this sterol was not detected in opaque lenses until long after their first appearance. Rather, development of this opacity could involve a selective reduction in the concentrations of γ-crystallin, the smallest of the four classes of water-soluble crystallins and the single major constituent of the rat lens after water. The observed lowering of γ-crystallin levels in opaque lens is apparently explained by a differential reduction in protein synthesis measured from [³H]leucine injected intraocularly. This differential effect could be explained by marked changes in the intracellular concentration of Na⁺ and K⁺ ions in opaque lens; Na⁺ levels greatly increased and K⁺ decreased. Recent reports indicate that decreasing the intracellular ratio of $\text{K}{}^{\mathrm{+}}\text{Na}{}^{\mathrm{+}}$ in lens results in a shift of protein synthesis from lower to higher molecular weight species. In view of this information and the results of the present study, we suggest that the development and/or progression of the cataracts induced by U18666A could be related to a selective block in formation of γ-crystallin secondary to alteration in the levels of Na⁺ and K⁺ ions in lens.     

Ascorbic acid effects on lens 86 rubidium transport

Rat lenses incubated with 20 mg% ascorbic acid without glucose for 1 hr demonstrate a 35% decrease in ⁸⁶Rb uptake. Incubation in the presence of 2 × 10^?5m cupric sulfate increases the magnitude of the ascorbic acid effect to (?) 63%. Incubation after the addition of either glucose or catalase to the medium without glucose completely prevents both of these ascorbic acid effects. Both hydrogen peroxide (1 mm) and dehydroascorbic acid (20 mg%) cause a significant decrease in lens ⁸⁶Rb uptake in the absence of glucose. However, the complete prevention of the ascorbic acid effect in the presence of catalase indicates that the observed decrease in lens ⁸⁶Rb uptake is due mainly, if not totally, to the hydrogen peroxide generated from the oxidation of ascorbic acid in the medium.     

Involvement of system A in the retina-to-blood transport of l-proline across the inner blood-retinal barrier

The purpose of the present study was to elucidate the mechanisms of retina-to-blood transport of l-proline across the blood-retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The vitreous humor/retina-to-blood transport of [³H]l-proline across the BRB was evaluated by microdialysis. Transport mechanisms of [³H]l-proline were investigated by cellular uptake using an in vitro model of the inner BRB (TR-iBRB2 cells). The mRNA level of system A was determined by quantitative real-time PCR analysis with specific primers. [³H]l-Proline and [¹⁴C]d-mannitol, which is a bulk flow marker, were bi-exponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [³H]l-proline during the terminal phase was 1.6-fold greater than that of [¹⁴C]d-mannitol. The terminal elimination rate constant difference between [³H]l-proline and [¹⁴C]d-mannitol was reduced in the retinal presence of 3 mM l-proline and 5 mM α-methylaminoisobutyric acid, suggesting that l-proline is transported via a carrier-mediated retina-to-blood transport process across the BRB. [³H]l-Proline uptake by TR-iBRB2 cells appeared to be mediated through a saturable and Na⁺-dependent process. The corresponding Michaelis-Menten constant was 392 μM. This process was reduced by substrates for system A, suggesting that system A is involved in l-proline uptake. Of the isoforms of system A, ATA1, ATA2, and ATA3, ATA2 mRNA is predominantly expressed in TR-iBRB2 cells and isolated rat retinal endothelial cells. In conclusion, system A, most likely ATA2, is responsible for the retina-to-blood transport of l-proline across the inner BRB and may play a role in maintaining the concentration of small neutral amino acids in the retina.     

Cytosol binding of retinyl palmitate and palmitic acid in pigment epithelium and retina

A 6S protein which binds [¹⁴C]retinyl palmitate is present in the soluble fraction (cytosol) of the pigment epithelium but not the retina. There is no binding of [¹⁴C]retinyl palmitate by the 2S retinol receptor in either the pigment epithelium or the retina. [¹⁴C]Palmitic acid is bound to a peak at 6S in pigment epithelial cytosol and at both 6S and 2S in retinal cytosol.     

Transport and metabolism of glutathione in the lens

Measurements were made of influx and efflux of reduced and oxidized glutathione (GSH and GSSG) in rabbit lens. Three times as much radioactivity was found in the lens when the anterior surface was exposed to [³⁵S]-GSH compared with the posterior side. Nonlabeled GSH added to the medium decreased the penetration of labeled GSH, primarily across the anterior surface. The entry of [³⁵S]-GSSG into the lens was approximately the same across both surfaces. In cultured lenses, the accumulation of [³⁵S]-GSH decreased significantly in the absence of glucose and at reduced temperature, but was unaffected by ouabain. The entry process for GSH was also shown to be a saturable mechanism. Of the three constituent amino acids in the tripeptide only cysteine was found to compete for the transport system of GSH, suggesting that GSH and cysteine may share a common carrier. The rate of entry of ³⁵S-GSSG in cultured lenses was unaffected by the presence of either nonlabeled cysteine or GSH.No significant efflux of GSH or GSSG occurs from intact lenses. The efflux is extremely rapid when both capsule and epithelium are removed from the lens. When capsule alone was removed with collagenase, leaving the epithelum intact, no detectable amount of GSH or GSSG was found to leave the lens. A significant amount of [³⁵S]-GSH that entered the lens was degraded, giving rise to labeled cysteine. Lenses cultured in the presence of ¹⁴C-labeled cysteine rapidly incorporated the amino acid into GSH, suggesting that GSH is totally degraded and resynthesized in the lens. From the time course of decrease in the specific activity of glutathione introduced into the lens, the coefficient of turnover of GSH was calculated to be 0·014 hr^?1, a value found to be in reasonable agreement with the turnover rate of GSH calculated previously from the rates of incorporation of constituent amino acids.