Young Rat Lens Research Articles

One-shot LC-MS/MS analysis of post-translational modifications including oxidation and deamidation of rat lens α- and β-crystallins induced by γ-irradiation.

The eye lens is a transparent organ that functions to focus light and images on the retina. The transparency and high refraction of the lens are maintained by the function of α-, β-, and γ-crystallins. These long-lived proteins are subject to various post-translational modifications, such as oxidation, deamidation, truncation and isomerization, which occur gradually during the aging process. Such modifications, which are generated by UV light and oxidative stress, decrease crystallin solubility and lens transparency, and ultimately lead to the development of age-related cataracts. Here, we irradiated young rat lenses with γ-rays (5-500Gy) and extracted the water-soluble (WS) and water-insoluble (WI) protein fractions. The WS and WI lens proteins were digested with trypsin, and the resulting peptides were analyzed by one-shot LC-MS/MS to determine the specific sites of oxidation of methionine and tryptophan, deamidation sites of asparagine and glutamine, and isomerization of aspartyl in rat α- and β-crystallins in the WS and WI fractions. Oxidation and deamidation occurred in several crystallins after irradiation at more than, respectively, 50 and 5Gy; however, isomerization did not occur in any crystallin even after exposure to 500Gy of irradiation. The number of oxidation and deamidation sites was much higher in the WI than in the WS fraction. Furthermore, the oxidation and deamidation sites in rat crystallins resemble those reported in crystallins from human age-related cataracts. Thus, this study on post-translational modifications of crystallins induced by ionizing irradiation may provide useful information relevant to the formation of human age-related cataracts.

Site specific oxidation of amino acid residues in rat lens γ-crystallin induced by low-dose γ-irradiation

Although cataracts are a well-known age-related disease, the mechanism of their formation is not well understood. It is currently thought that eye lens proteins become abnormally aggregated, initially causing clumping that scatters the light and interferes with focusing on the retina, and ultimately resulting in a cataract. The abnormal aggregation of lens proteins is considered to be triggered by various post-translational modifications, such as oxidation, deamidation, truncation and isomerization, that occur during the aging process. Such modifications, which are also generated by free radical and reactive oxygen species derived from γ-irradiation, decrease crystallin solubility and lens transparency, and ultimately lead to the development of a cataract. In this study, we irradiated young rat lenses with low-dose γ-rays and extracted the water-soluble and insoluble protein fractions. The water-soluble and water-insoluble lens proteins were digested with trypsin, and the resulting peptides were analyzed by LC-MS. Specific oxidation sites of methionine, cysteine and tryptophan in rat water-soluble and -insoluble γE and γF-crystallin were determined by one-shot analysis. The oxidation sites in rat γE and γF-crystallin resemble those previously identified in γC and γD-crystallin from human age-related cataracts. Our study on modifications of crystallins induced by ionizing irradiation may provide useful information relevant to human senile cataract formation.

Mass Measurements of C-terminally Truncated α-Crystallins from Two-dimensional Gels Identify Lp82 as a Major Endopeptidase in Rat Lens

Molecular chaperone activity of lens alpha-crystallins is reduced by loss of the C terminus. The purpose of this experiment was to 1) determine the cleavage sites produced in vitro by ubiquitous m-calpain and lens-specific Lp82 on alpha-crystallins, 2) identify alpha-crystallin cleavage sites produced in vivo during maturation and cataract formation in rat lens, and 3) estimate the relative activities of Lp82 and m-calpain by appearance of protease-specific cleavage products in vivo. Total soluble protein from young rat lens was incubated with recombinant m-calpain or Lp82 and 2 mM Ca2+. Resulting fragmented alpha-crystallins were separated by two-dimensional gel electrophoresis. Eluted alpha-crystallin spots were analyzed by mass spectrometry. Cleavage sites on insoluble alpha-crystallins were determined similarly in mature rat lens nucleus and in cataractous rat lens nucleus induced by selenite. In vitro proteolysis of alphaA-crystallin by Lp82 and m-calpain produced unique cleavage sites by removing 5 and 11 residues, respectively, from the C terminus. In vivo, the protease-specific truncations removing 5 and 11 residues from alphaA were both found in maturing lens, whereas only the truncation removing 5 residues was found in cataractous lens. Other truncation sites, common to both calpain isoforms, resulted from the removal of 8, 10, 16, 17, and 22 residues from the C terminus of alphaA. Using uniquely truncated alphaA-crystallins as in vivo markers, Lp82 and m-calpain were both found to be active during normal maturation of rat lens, whereas Lp82 seemed especially active during selenite cataract formation. These C-terminal truncations decrease chaperone activity of alpha-crystallins, possibly leading to the observed increases in insoluble proteins during aging and cataract. The methodology that allowed accurate mass measurements of proteins eluted from 2D gels should be useful to examine rapidly other post-translational modifications.

Calpain-induced light scattering in young rat lenses is enhanced by UV-B.

The purpose of this study is to determine if UV-B enhances light scattering after proteolysis of crystallins by calpains, and to determine if lens-specific calpain Lp82 is involved, along with m-calpain, in the mechanism of in vitro precipitation. Lens soluble proteins from young rats were hydrolyzed for 24 hr by endogenous lens calpains, and the proteins were further incubated for up to 7 days with periodic irradiation by UV-B. Light scattering was measured daily at 405 nm. SDS-PAGE and immunoblotting assessed proteolysis of crystallins, activation of calpains, and formation of high molecular weight aggregations. Appreciable light scattering occurred in lens soluble proteins after proteolysis of crystallins by m-calpain and Lp82. UV-B markedly enhanced this light scattering and the formation of higher molecular weight aggregates consisting of proteolyzed alpha- and beta- and intact gamma-crystallins. Calpain inhibitor E64 and antioxidants DTE or GSH prevented the light scattering. These results show that calpain-induced light scattering is enhanced by the natural oxidant UV-B. Activation of Lp82, along with m-calpain, contributed to the light scattering. The linkage between proteolysis and oxidation is important because both oxidation and truncation of crystallins are found in aged human lenses, which are constantly exposed to UV irradiation.

Calpain inhibitor, SJA6017, reduces the rate of formation of selenite cataract in rats

Purposes. 1) To measure the amount of calpain inhibitor SJA6017 taken up by lenses of young rats after administration; and 2) To test efficacy of SJA6017 against selenite cataract in regard to amelioration of proteolysis of lens protein and prevention of lens nuclear opacity. Methods. Selenite nuclear cataracts were produced by subcutaneous injection of an overdose of sodium selenite to 16-day-old rats. SJA6017 was administered daily using intraperitoneal injections at 100 mg/kg body weight/day for 4 days. Lenses were observed and photographed by slit lamp biomicroscopy, and scored into one of three stages. Enucleated lenses were also scored into one of four stages and lens opacities in the nuclear region were quantified by image analysis. Proteolysis of crystallins was detected by SDS-PAGE. The amount of SJA6017 taken up by the lens was detected with a column switching HPLC system. Results. Nuclear cataracts were visible in 31% of the animals receiving only selenite, while the frequency of nuclear cataract in the Se+SJA6017 group was reduced to only 16%. This effect of SJA6017 was confirmed by densitometric analysis as a reduction in the density of the nucleus. Similar proteolytic changes of crystallins occurred at all stages of selenite cataract formation. The amount of SJA6017 in the lens was detected at the level of 0.03 µM. Conclusions. Systemic SJA6017 was taken up by the lens, and SJA6017 ameliorated in vivo selenite cataract formation. These studies are important because they partially validate the biochemical rationale for developing non-surgical, drug treatments for cataract prevention in man.

Lp85 calpain is an enzymatically active rodent-specific isozyme of lens Lp82

Purposes. To clone and sequence the cDNA for Lp85 calpain from young rat lens, and to test for Lp85 protein expression and proteolytic activity. Methods. RT-PCR and molecular cloning were performed on total RNA from 12 day-old rats. Lp85 protein expression was visualized by immunoblotting using a specific antibody developed to the unique peptide sequence in Lp85. Proteolytic activity was assessed by casein zymography. Transient expression of Lp85 and previously characterized lens-specific calpain Lp82 were separately performed in mammalian COS-7 cells. Results. The 2410-bp cDNA for rat lens Lp85 encoded a protein of 737 amino acid residues with a calculated molecular weight of 85.0 kDa and a predicted pI of 5.67. The amino acid sequence of Lp85 was identical to Lp82 except for an insert region of 28 amino acids in domain IV of the calcium-binding region. mRNA and protein for Lp85 were present only in rat and mouse lenses and not in other tissues or species. Lp85 protein concentrations were highest in the nuclear region, most concentrated in the insoluble fraction, disappeared with lens maturation, and Lp85 exhibited migration similar to Lp82 on native PAGE gels. Lp85 was enzymatically active when expressed in COS-7 cells. Conclusions. Lp85 is a newly classified, lens- and rodent-specific, enzymatically active, member of the AX1 (alternative exon 1) subclass of calpains. In conjunction with Lp82 and m-calpain in lens, Lp85 may be responsible for proteolysis during normal lens development and maturation or during cataract formation in young rodents.

Calpains in the lens and cataractogenesis.

Many types of cataracts in the lens of the eye show elevated concentrations of calcium, which could activate calpains, and thus this chapter presents the techniques specialized for measuring calpain activity in lens. Ubiquitous calpain has been assayed in lenses from man, cow, pig, rat, sheep, mouse, guinea pig, and rabbit (). Further, a lens-specific calpain, termed Lp82, was recently discovered in young rat lens (). Cloning and sequencing of the cDNA for Lp82 calpain showed that Lp82 is actually a splice variant of muscle-type calpain p94 missing several exons and containing a new exon 1. Examples of calpain-induced proteolysis are found in rodent cataracts induced by selenite, buthionine sulfoximine, calcium ionophore A23187, hydrogen peroxide, diamide, xylose, galactose, and streptozotocin; and in several animal models, Nakano mice, UPL hereditary rat cataract, Shumiya cataract rat (SCR), and transgenic mice expressing human immunodeficiency virus (HIV) protease (). Calpain-induced proteolysis of lens crystallins is also a common feature of maturing rodent lenses ().

Intense Myristoylation of a Single Protein in the Ocular Lens

A single protein of the ocular lens was intensely myristoylated following short term incubation of cultured bovine lens epithelial cells and intact rat lenses with3H-myristic acid. It was acidic (pI <5), about 19 kDa and present exclusively in the cytosol of both cultured epithelial cells and the epithelium of the young rat lens. Fiber cell proteins were not labeled. The myristoylated protein was not seen in the epithelium of the adult rat. Essentially no protein mass was evident in the 19-20 kDa range when samples of the labeled-soluble protein were fractionated by either HPLC coupled with SDS–PAGE or 2D-electrophoresis. These findings suggest that the myristoylated-soluble protein of 19 kDa in lens (p19L) is a rapidly-turning over minor protein likely associated with lens growth. The absence of any apparent membrane association for a myristoylated protein appears unusual. The trace nature of p19Lhas frustrated attempts at its identification by MALDI-MS.

Oxidation enhances calpain-induced turbidity in young rat lenses

Purpose. To determine if oxidation enhances turbidity after proteolysis of rat lens crystallins by the calcium-activated protease calpain (EC 3.4.22.17). Methods. Total soluble proteins from young rat lens were hydrolyzed for 24 hr by endogenous lens calpain, and the proteins were further incubated with the oxidant diamide for up to 7 days. Turbidity was measured daily at 405 nm. To measure proteolysis and turbidity in cultured lenses, rat lenses were cultured for 6 days in low calcium medium and diamide. The lenses were then photographed to assess transmission of light. SDS-PAGE and immunoblotting assessed proteolysis of crystallins, a-spectrin, and activation of calpain. Results. Appreciable in vitro turbidity occurred in soluble proteins from young rat lenses after proteolysis of crystallins by endogenous calpain. Calpain inhibitor E64, or anti-oxidants DTE and GSH, inhibited this turbidity. On the other hand, the oxidant diamide markedly enhanced calpain-induced turbidity. Cultured rat lenses showed elevated intra-lenticular calcium and proteolysis of crystallins by calpain, but no nuclear cataract. Addition of diamide to the culture medium caused development of nuclear cataract. Conclusions. Diamide enhanced turbidity only when crystallins were proteolyzed. Oxidation may be one of the factors promoting light scatter and insolubilization after proteolysis. These data are consistent with the hypothesis that proteolysis of crystallins from young rat lens may expose cysteine residues, which are then oxidized, become insoluble and scatter light.

Protein for Lp82 Calpain Is Expressed and Enzymatically Active in Young Rat Lens

mRNA for a newly discovered isoform of calpain, termed Lp82, was recently discovered in young rat lens. The purpose of the present experiments was to test for expression of Lp82 protein. Casein zymography after incubation with calcium was used to detect Lp82 proteolytic activity in regions of lenses from young rats. Lp82 protein was detected by immunoblotting or by ELISA after DEAE-5PW chromatography using a polyclonal antibody generated to a peptide sequence in Lp82. Northern blot analysis assessed expression of Lp82 mRNA.Four results demonstrated expression of Lp82 protein; (1) immunoblot reactivity at the predicted molecular mass of 82 kDa, (2) a unique band of calcium-activated lysis in casein zymograms, (3) partial purification and retention of activity from a single Lp82 peak on DEAE-5PW chromatography, and (4) positive immunoblotting and Northern blot analysis only in lens and not in other rat tissues. These results showed that Lp82 protein is lens-preferred, relatively abundant in young rats (especially nucleus), and enzymatically active. Proteolysis of crystallins in the nucleus of young rat lens during normal maturation and cataract formation, formerly attributed solely to m-calpain, may in fact be due to concerted action of both lens Lp82 and ubiquitous m-calpain.

Probing cataractogenesis associated with mevalonic aciduria

PURPOSE. Mevalonic aciduria in humans results from a genetic deficiency of mevalonate kinase and is characterized by very high plasma mevalonic acid levels, developmental malformations and cataracts. This study tested the possibility that the cataracts could result from direct toxicity of the accumulated mevalonate. METHODS. Young rat lenses were cultured for up to 4 days in medium TC199 containing 1 to 5 mM mevalonic acid. Changes in the water, sodium and potassium content of the lens were followed; electrolytes were measured by atomic absorption spectroscopy. The identities of proteins leaked from the lens were determined by sodium dodecylsulfate polyacrylamide electrophoresis and isoelectric focusing. Changes in cation flux were measured by 86 Rb uptake. Lens concentrations of mevalonic acid were measured from uptake of 3 H-mevalonolactone. RESULTS. Culture of young rat lenses with 3 to 5 mM mevalonic acid produced lens opacification and nuclear cataracts starting within 1 to 2 days of culture. Mevalonic acid did not concentrate in the lens. Treated lenses accumulated water and sodium and lost potassium and soluble gamma crystallin proteins. These changes were preceded by a loss of the lens's capacity to concentrate 86 Rb, a potassium analogue. The loss of 86 Rb uptake might have been due to a slow poisoning of the cation pump, direct effects on membrane integrity or both. CONCLUSIONS. The results show that chronic exposure of the lens to mevalonic acid can induce cataracts, which appear caused by a progressive increase in the permeability of lens cell membranes. The cataracts associated with mevalonic aciduria could be due to toxicity from mevalonic acid.

Changes in Calpain II mRNA in Young Rat Lens during Maturation and Cataract Formation

The purpose of these experiments was to describe the expression of mRNA for calpain II proteolytic enzyme (EC 3.4.22.17) during normal maturation of rat lens and in cataract formation. Quantitative RT-PCR indicated that the concentration of mRNA for calpain II in whole lens was 3–24 times higher than in age-matched rat liver, kidney, lung and brain, and it was at least five times higher than in young human lens. mRNA levels for calpain II were highest in the outer regions of young rat lens at 5×106copies μg−1total RNA. Early-stage experimental cataract caused increased calpain II mRNA, while mature nuclear cataract showed a 64% loss. In contrast, mRNA levels for GAPDH, β-actin, and lens-specific structural protein βA4 remained constant during experimental cataract formation. Unlike the lower and constant levels in rat liver, kidney and lung; calpain II mRNA levels in whole rat lens decreased with age.These data help explain the high enzymatic activity of calpain II in young rat lens, susceptibility of young rat lens to a variety of cataracts showing increased calcium and calpain-induced proteolysis, and low calpain enzyme activity in human lens. Since the up-regulation of calpain II mRNA was more dynamic than either the amounts of calpain II enzyme or proteolysis of crystallins in cortex, resulting proteolytic activity against the bulk of lens proteins seems to be regulated by post-translational factors, such as increased calcium. The precise role of the up-regulation of calpain II mRNA is unknown, but we hypothesize that it may be associated with the initial cataractogenic response in the epithelial cells or peripheral cortical fibers.

In vitro precipitation of rat lens crystallins by calpain I--a calpain requiring low amounts of calcium for activation.

The overall goal of this study was to provide data on the function and physiologic significance of lens calpain I, a cysteine protease requiring low amounts of calcium for activation. Reverse-transcriptase polymerase chain reaction was used to detect mRNAs for calpains I and II in young rat lenses. An in vitro model of crystallin precipitation was used to assess the ability of calpain I to induce hydrolysis and precipitation of crystallins. We found that incubation of crystallins with purified calpain I was indeed a powerful inducer of crystallin precipitation. However, mRNA levels for calpain I in whole lens appeared to be lower compared to calpain-II mRNA. Participation of calpain I in crystallin precipitation during normal maturation of rodent lenses or cataract formation is thus theoretically possible, but unlikely, because of the low level of expression of calpain I.

The further metabolism of sorbitol-3-phosphate and fructose-3-phosphate in the mature rat lens.

Glucose and phosphorus metabolism in mature (8-month-old) rat lenses were examined with NMR spectroscopy. Nondiabetic mature lenses contained sorbitol-3-phosphate (S3P) and fructose-3-phosphate (F3P) which were absent from young (1- to 2-month-old) normal rat lenses. The concentrations of these two phosphates can be changed through (1) diabetes induction with streptozotocin - this results in a dramatic increase in both compounds; and (2) oral dosing with a drug known to prevent sorbitol production - both metabolites disappeared. When normal mature lenses were incubated in 35.5 mM 13C1-glucose, both 13C1-lactate and 13C3-lactate were produced. Preservation of the 13C label at C1 is likely through the formation of 13C1-S3P and -F3P, which were then split through an aldolase-like mechanism into two 3-carbon compounds, one an unlabeled glycerol and the other 13C1-alpha-glycerophosphate (from S3P) and 13C1-dihydroxyacetone phosphate (from F3P). These reactions can contribute to the increase in alpha-glycerophosphate observed in both the streptozotocin-induced diabetic lenses and lenses incubated in high glucose.

Investigations into the loss of glutathione from lenses in organ culture.

To investigate possible causes and implications of the decrease in glutathione concentration in rat lenses during organ culture. Freshly excised lenses were incubated in modified TC-199 medium. Ellman's Reagent or the GSH-400 assay were used to assay glutathione levels in lenses cultured for different times and under a variety of altered culture conditions. In lenses from young rats the glutathione decrease was not ameliorated by reduction of oxygen tension in the incubator, nor by supplementation of the culture medium with various antioxidants or sulfhydryl compounds, nor with the amino acid precursors of glutathione. Addition of 2-mercaptoethanol stimulated cysteine transport into the lens but had only a modest effect in maintaining the level of glutathione. The decrease in glutathione concentration was less in cultured lenses from older rats. Lenses from rhesus monkeys exhibited no decrease in glutathione levels when maintained in organ culture for up to 48 h. The basis for the decreased glutathione in cultured young rat lenses is still uncertain. The data from the present study indicate a definite relationship between glutathione loss and age for cultured rat lenses, with young lenses being much more susceptible. The resistance of cultured monkey lenses to loss of glutathione demonstrates species differences in this property which may be relevant to previously reported differences in susceptibility to oxidative damage.

Precipitation of crystallins from young rat lens by endogenous calpain

The purpose of these experiments was to study the mechanism for precipitation of lens crystallins in cataract. An in vitro model was developed to activate the endogenous protease calpain II in the soluble proteins from young rat lens by addition of calcium in the presence of 120 mM KCl. Light-scattering, insoluble proteins were produced approximately 4-6 days after calpain II activation. Results showed that proteolysis was caused by activation of lens calpain II, proteolysis preceded precipitation by several days, and alpha-crystallin acted as a molecular chaperone against precipitation of crystallins caused by proteolysis. These data supported our hypothesis that calpain-induced proteolysis of the N-terminal arms of beta-crystallin polypeptides leads to a loss of normal oligomerization of beta-crystallin polypeptides and formation of abnormal insoluble aggregates, possibly stabilized by hydrophobic interactions.

Calcium cataract: a model for optical anisotropy fluctuations

Young rat lenses were incubated in organ culture media enriched with 20 mM calcium. Lenses in the calcium rich medium developed cataracts and were characterized by the absence of vimentin in the urea soluble protein fractions. Sections from the same lenses were studied by polarized light scattering. The I+/Iparallel scattering intensity ratios were higher from the lenses in calcium-rich media than from the control lenses. This indicated an increase in the optical anisotropy fluctuations during cataractogenesis. The turbidity that developed due to these fluctuations was caused partly by the disappearance of vimentin and which in turn caused the enhancement of birefringence of the lens.

Spatial distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase messenger RNA in the ocular lens: relationship to cholesterologenesis.

This study probes the regulation of cholesterol biosynthesis in the ocular lens by estimating the concentration and distribution of the messenger RNA for the rate-controlling enzyme for sterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Because the lens is dependent on biosynthesis for cholesterol, HMGR activity is crucial for the life-long growth of this organ. Young rat lenses were serially divided into several fractions by dissolution in an SDS-containing buffer and each fraction was equated to a percent of the lens radius based upon its protein content. HMGR enzyme activity and cholesterol synthesis has been shown to disappear from the lens cortex over a narrow arc of radius due to loss of enzyme protein. Using a published competitive reverse transcriptase-polymerase chain reaction method for amplifying HMGR mRNA (Powell, E. E., and P. A. Kroon. 1992. J. Lipid Res. 33: 609-614), an average of about 46,000 copies of this mRNA was estimated per lens at all rat ages examined (5-day-old to adult). However, copies/microgram total RNA decreased with aging. The distribution of HMGR mRNA across 95-60% of the lens radius was essentially uniform at 2000-3000 copies/mm3 tissue. But the very superficial cortex contained 5- to 7-times this concentration and accounted for about 35% of the total copies/lens. We estimated that cells in this region each contained 1 to 2 copies of message, a value similar to the estimated copy number of HMGR message in human lymphocytes (Powell and Kroon, ibid). This suggests that the translational efficiency and stability of lens HMGR mRNA must be very high.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and protein mass in the ocular lens.

We have attempted to map the regional distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme activity and protein mass along the radial axis of the ocular lens. Because lens plasma membrane is extremely rich in cholesterol and the lens must synthesize this cholesterol, the activity of HMGR could be a key factor controlling lens membrane formation. Lenses of young rats were divided into discrete fractions by gradual dissolution in a tergitol-containing buffer; each fraction was then equated to a specific arc of the radius based upon its protein content. Aliquots of each fraction were assayed for HMGR enzyme activity and protein mass. Relative protein mass was quantitatively estimated by Western blotting using a monoclonal antibody to HMGR with immunoreactivity detected by enhanced chemiluminescence. Lens HMGR possessed a molecular mass of about 97 kDa and localized in the cell's insoluble fraction. Peak levels of both HMGR enzyme activity and protein mass were found in the outer 5% of the lens radius; levels of both decreased precipitously from there to the outer 10% radius mark. This distribution paralleled synthesis of the membrane's cholesterol, phospholipid, and intrinsic protein. We conclude that the abrupt cessation of plasma membrane synthesis in the ocular lens could involve loss of HMGR activity over a narrow arc of the lens radius, and that this activity loss is due to disappearance of enzyme protein.

Alpha-Crystallin chaperone activity is reduced by calpain II in vitro and in selenite cataract.

This study reports the first demonstration of a marked reduction in alpha-crystallin chaperone activity in an experimental model of cataract, and the study implicates activation of the cysteine protease calpain II (EC 3.4.22.17) as the in vivo protease responsible for decreased chaperone activity. Chaperone activity of normal alpha-crystallin from lenses of young rats was assayed by measuring attenuation of heat-induced aggregation and scattering of beta L-crystallin. alpha-Crystallin from the nucleus of lenses with selenite cataract showed specific selective proteolysis, and chaperone activity was diminished. Proteolysis of alpha-crystallin from selenite cataract lenses was mimicked by incubating normal alpha-crystallin with calpain II, and this also resulted in loss of chaperone activity. Two-dimensional gel electrophoresis and peptide mapping were used to identify four partially degraded alpha A- and alpha B-crystallin polypeptides following incubation of normal alpha-crystallin with calpain. Similar partially degraded alpha A and alpha B polypeptides were found in selenite cataract. Previous experiments indicated that alpha-crystallin chaperone activity decreases because of removal of the COOH terminus. Our experiments support this observation and suggest that calpain proteolysis of alpha-crystallin at the COOH terminus may result in a loss of chaperone activity in selenite cataract.