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The development of the eye lens in the prenatal phase is accompanied by a gradual change in the polypeptide composition of α-crystallin. As the lens matures from that of a 6-week-old embryo to that of a new-born calf, there is a successive appearance of different polypeptides although two obviously principal polypeptides, A2 and B2, are present at every stage of development. The youngest embryonic lenses studied contain four polypeptides. Two of them differ from the well-known polypeptides as found in cortical bovine α-crystallin.

To investigate the possible role of glycation in the aging of lens proteins, we used bovine lenses as a model. We studied crystallins isolated from prenatal bovine lenses, calf lenses and lenses from mature animals (up to 20 years old). The experiments show an increase in glycation levels with age in all crystallin fractions. Regarding the lysine content of the different crystallins, gamma-crystallin showed relatively high levels of early glycation products. The results also revealed high levels of early glycation products for the HM material (containing mainly alpha-crystallin). In alpha-crystallin, alpha A-subunits were glycated to a higher extent compared with the alpha B-subunits. There is an age-related increase in advanced glycation products, measured as specific fluorescence (excitation/emission wavelengths 370/440 nm), mainly present in the HM and alpha-crystallin fraction.

To investigate the possible role of glycation in the onset of diabetic cataract we used calf lens crystallins as a model. After incubation with reducing sugars, the proteins were investigated by high-pressure gel permeation chromatography, SDS-PAGE and analytical ultracentrifugation. Glucose-6-phosphate incubation resulted in an increase in mean molecular weight of all crystallin fractions and the occurrence of high-molecular weight material, partly formed by disulphide bonds. The glycated crystallins showed a decrease of tryptophan fluorescence and an increase of a specific non-tryptophan fluorescence. This fluorescence was, however, not exclusively associated with the high molecular weight protein, but was present in all protein fractions.

Wegen der besonderen Art ihrer Entwicklung und ihres Wachstums lassen sich an der Augenlinse viele Altersveranderungen auf zellularer und molekularer Ebene verfolgen. Ausgepragte postsynthetische Veranderungen wie Aggregation, Phosphorylierung, Desamidierung, Bildung kovalenter Vernetzungen und Racemisierung wurden vor allem an den Strukturproteinen der Linse, den Kristallinen, eingehend untersucht. Die zukunftige Forschung wird zeigen mussen, inwiefern die bisherigen Erkenntnisse allgemeine Bedeutung fur die Prozesse der biologischen Alterung haben.

Water-soluble crystallins were obtained from clear human lenses of different age (4-81-year-olds) and lenses of individuals showing senile or diabetic cataracts. Levels of early glycation products were high in the high molecular weight material (HM) and the alpha-crystallin fractions, compared with beta- and gamma-crystallins. This difference becomes more prominent upon aging. The content of total early glycation products in HM and alpha-crystallin increases clearly with age, whereas levels remain relatively constant in the beta- and gamma-crystallins. There is an elevation of early products in cataractous lenses from diabetic individuals compared with those suffering from senile cataract. Specific non-tryptophan fluorescence (excitation/emission wavelengths 370/440 nm), used as an indicator for late glycation products, increased dramatically with age and was 2-fold higher in the diabetic subjects. Levels of fluorescence decreased in the order HM > alpha- > beta- > gamma-crystallins. The results suggest an increase in glycation rate in alpha-crystallin as a result of aging and diabetes, while the rate of glycation of beta- and gamma-crystallins remains almost constant.

Glycation is a non-enzymatic modification of proteins by sugars, probably responsible for the initiation of complications in diabetes patients and aging individuals. Our in vitro experiments show an inhibition of sugar attachment in the presence of Diclofenac. The levels of advanced glycation products, measured as specific fluorescent groups, were also lowered due to Diclofenac. These results were compared with inhibition by Aspirin (acetylsalicylic acid), a known inhibitor of the glycation process. The protection by Diclofenac is based on a non-covalent interaction of the drug with serum albumin. There is evidence that Diclofenac specifically blocks at least one of the major glycation sites of human serum albumin.

The structural bovine eye lens protein alpha-crystallin was dissociated in 7 M urea and its four subunits, A1, A2, B1, and B2, were separated by means of ion-exchange chromatography. Homopolymeric reaggregates of these subunits were prepared by removal of the denaturant via dialysis. It was found that subunits were exchanged upon incubation of mixtures of two homopolymers under native conditions. New hybrid species were formed within 24 h as demonstrated by isoelectric focusing. Moreover, native alpha-crystallin molecules also exchanged subunits when incubated with homopolymeric aggregates of B2 subunits. Subunit exchange between native alpha-crystallin molecules is postulated, and a "dynamic quaternary structure" is presented that allows the polydisperse protein to adapt to changes in cytoplasmic conditions upon aging of the lens tissue.

In November 1980 a workshop for the classification and characterization of lenses and lens proteins was held in Louvain-La-Neuve, Belgium. Some essential results are summarized in this paper.

Calf lens nuclear α-crystallin was separated into the three known molecular weight populations on Ultrogel AcA22. Electron microscopy illustrates that the two higher molecular weight classes, which are formed upon aging of the lens, are chain-like oligomers and polymers of the spherical monomeric α-crystallin molecules of 15 nm average diameter and 1·1 × 106 average molecular weight which form the lowest molecular weight class. Our combined sedimentation and electron microscopy data make the alleged non-equilibrium state within these populations questionable. From the polypeptide chain composition, determined by isoelectric focusing, it can be inferred that the total amount of C-terminal shortened chains increases progressively from 33 to 53% from monomeric to polymeric α-crystallin. Reassociation of different types of isolated shortened A-chains in various mixtures indicates a tendency for formation of higher molecular weight reaggregates with increasing relative amount of degraded chains. The involvement of degraded chains in polymerization is postulated although it is not likely to be the only factor causing aggregation.

Human sera were subjected to analytical ultracentrifugation and "high-pressure" gel-permeation chromatography on a system of combined TSK Gel G5000 PW and G3000 SW columns. The chromatographic method produced remarkably superior resolution of the proteins, especially those exceeding 100 000 Da. We calculated the molecular masses of eluted fractions on the basis of their detection by low-angle laser light scattering and their differential refractive index. We discuss the results in relation to the clinical data.

The stability of the native quaternary structure of bovine alpha-crystallin was studied, by sedimentation analysis and electron microscopy, as a function of pH (7--11), ionic strength (0.01--0.5), temperature (6--60 degrees C) and calcium ion concentration (0 and 10 mM). Three successive transitions are distinguished at 20 degrees C. Firstly, a slow transconformation step, which is independent of pH, ionic strength or calcium ions. Secondly, an irreversible primary dissociation step, favoured by increasing pH above 8 and/or a lower ionic strength, with formation of 'alkali-modified alpha-crystallin', which is spherically shaped like the native protein but has a smaller average diameter, sedimentation coefficient and molecular weight. Thirdly, with further increase of pH above 9, a rapidly reversible dissociation of alkali-modified alpha-crystallin characterized by a single reaction boundary in sedimentation velocity analysis. In the presence of calcium ions the quaternary structure is stabilized to the extent that no dissociation is observed up to at least pH 10.3. Upon increase of temperature, at pH 7.3, a slow irreversible dissociation and swelling run parallel until a limit is reached around 37 degrees C with formation of 'temperature-modified alpha-crystallin', which is indistinguishable from the native protein by electron microscopy, but has a higher relative viscosity and lower sedimentation coefficient and molecular weight. Calcium ions have little or no effect on this transition. Above 37 degrees C a reversal of this transition or aggregation is indicated. These findings, together with previous structural data on microheterogeneity, reassociation from urea, and aging of alpha-crystallin in vivo, are incorporated into a hypothetical scheme of transitions, based on a three-layer model for the quaternary structure.

Racemization of aspartyl residues in proteins is a post-translational process, related to ageing. A method is presented for the detection of aspartic acid enantiomers in protein hydrolysates, based on chiral capillary gas chromatography. It is fast, easy and preferable to the usual diastereomeric dipeptide technique. We present evidence that traces of metals that are extracted from the glassware during acidic hydrolysis are the main cause for high background racemization, which often troubles accurate measurements. Effective ways to reduce this background and its standard deviation to acceptable levels are discussed, and a mathematical approach to correct for background racemization is given. Hydrolysates of aged human eye lens proteins were used to demonstrate the enantiomeric separation.

Evaluation of sulfhydryl data derived from the nucleus and cortex of individual human lenses, ranging in age from 40 to 90 years, revealed the following: (a) no significant correlation between the age and the sulfhydryl content of normal and nuclear-cataractous lenses; (b) a significant relationship between the distribution of non-protein-bound sulfhydryl compounds among the lens parts and both age and cataractous state; (c) no significant difference in sulfhydryl data from the cortex between groups of cataractous lenses with increasing nuclear color; and (d) a significant difference in nuclear protein sulfhydryl content between the groups of lenses, varying in cataractous state. If lens parts are separated on the basis of cataract localization, sulfhydryl oxidation in nuclear cataractogenesis seems to be confined to the colored nuclear region. It is suggested that nuclear cataract develops from the inner nucleus towards the cortex of the lens.

Electron micrographs of high (40-S) and very-high-molecular-weight (240S) alpha-crystallin preparations show large aggregates with a random arrangement of individual low-molecular-weight alpha-crystallin molecules. The electron microscopic data are completed with viscosimetric and light-scattering experiments. All data obtained with higher-molecular-weight alpha-crystallins point to aggregates with non-spherical structures.

Bovine cortical lens extracts were fractionated on Bio-Gel A-5m or Sephacryl S-200 and revealed three and four size classes for the β -crystallins, respectively. The greatest aggregates, β H , turned out to be subject to reversible, concentration-dependent dissociation into smaller aggregates. It was inferred that the association-dissociation equilibrium is mainly affected by hydrophilic interactions. On Sephacryl S-200 a β -crystallin size class with mol. wt 71 000, determined at sedimentation equilibrium, was isolated. Moreover, a β L -crystallin fraction (mol. wt 48000) and β S -crystallin (mol. wt 28 500) could be isolated. The polypeptide compositions of all β -crystallin aggregates have been determined by sodium dodecylsulfate (SDS) gel electrophoresis and isoelectric focusing in urea. On SDS gels β S -crystallin revealed a molecular weight of 22 000 contrasting sharply with the value determined at sedimentation equilibrium.

The native water-soluble proteins of equator, anterior cortex, posterior cortex and nucleus from bovine lenses in the age range 0·3–33·7 years were analyzed by high-pressure gel-permeation chromatography and high-pressure ion-exchange chromatography. Unlike the equator and cortices, the nucleus shows a gradual decrease in α-crystallin proportion with age which is not compensated for by an increase in HM-crystallin. The β H 6 -crystallin species, almost the only β H -component in the youngest lens, is largely replaced by at least four fractions with higher and lower molecular weights in the older lenses. In the nucleus a β L -component (39 000 MW) increasingly seems to replace the major β L -crystallin ( β L 2 , 50 000 MW). Moreover, a switch in the synthesis of monomeric crystallins is demonstrated. This study clearly reveals an age-related increase in the size heterogeneity of the native soluble crystallins with age.

The concentration of the free aromatic amino acids tryptophan and tyrosine, the antioxidant uric acid and some unidentified compounds have been determined in the water-soluble extracts of parts from clear and nuclear-cataractous human lenses. With age (29-90 years) and cataract formation, associated with increasing nuclear pigmentation, no significant changes were observed in content of tryptophan, tyrosine and uric acid. Furthermore, these compounds did not show variation in distribution within the lens. An unidentified fluorophore, excitation and emission maxima of 345 and 425 nm, respectively, increases significantly in content with nuclear cataract formation, especially in the nucleus. Another unidentified, presumably aromatic compound shows a striking age-related decrease.

Yellow protein derived from European and Pakistani human nuclear cataractous lenses was digested. Anthranilic acid was found in 50 times greater amount in Pakistani than in European material. Treating

Oxidative processes are suggested to be a main cause of the covalent cross-linking of lenticular proteins. For a major part these cross-links are disulfides (cystine). With progression of nuclear cataract, cystine may be oxidized further to cysteic acid. Another oxidative degradation product of cystine is the symmetric thioether lanthionine. In this study, we clearly demonstrate that lanthionine is a protein cross-link of cataractous human lenses. Possible mechanisms of its formation are discussed.

The very acidic polypeptide chains found in aging human eye lenses are products of post-synthetic alterations known to occur in mammalian eye lens proteins. Isoelectric focusing in the presence of ure

High molecular weight (HM-)crystallin was isolated from lens cortex and lens nucleus of the rabbit by column chromatography on agarose. Electron microscope studies revealed the presence of relatively small aggregates in cortical HM-crystallin, obviously oligomers of α-crystallin. On the other hand, nuclear HM-crystallin showed a wide size distribution of large, apparently at random-aggregated and irregularly-shaped protein molecules. In addition, a considerable proportion of nuclear HM-crystallin, isolated by column chromatography, consisted of particles with a molecular weight above 50 000 000 Daltons. On immunoelectrophoresis it revealed two precipitin lines, i.e. one in the α-crystallin and one in the β-crystallin mobility range. On ion-exchange chromatography nuclear HM-crystallin was dissociated into essentially two fractions with sedimentation values of approximately 4 S and 20 S. The first fraction showed characteristics of β-crystallin, while the second fraction was identical to nuclear α-crystallin. Column chromatography on agarose of the nuclear lens extract of 3- to 4-month-old rabbits revealed a protein fraction with a sedimentation coefficient of about 18 S, composed of β-crystallin subunits. Possibilities concerning the architecture of nuclear HM-crystallin are discussed.

Fractionation of the soluble proteins of rabbit lens by gel chromatography on Sepharose 6B revealed six components, viz. HM-, α-, β 1 -, β 2 -, β 3 - and γ-crystallin. On isoelectric focusing α-crystallins revealed a diffuse region, whilst approximately 10 bands were found for the β-crystallins and 7–8 components for γ-crystallin. Immunoelectrophoretically HM-crystallin revealed two precipitin lines, one in the α- and one in the β-mobility range. Sedimentation coefficients of 18·0, 8·0, 3·7, 2·2 and 2·1 S were calculated for α-, β 1 -, β 2 -, β 3 - and γ-crystallin, respectively. By isoelectric focusing in the presence of 6 m -urea, 13 bands between pH 5·3 and 6·9 were found for HM-crystallin, approximately six bands between pH 5·6 and 6·9 for α-crystallin, about 13 bands between pH 5·9 and 7·0 for the β-crystallins and 7–8 major bands between pH 7·0 and 7·8 for γ-crystallin. Molecular weight estimation by SDS gel electrophoresis revealed values between 16 000 and 39 000 for HM-crystallin and the β-crystallins, whereas values of approximately 21 000 and 20 500 were found for α- and γ-crystallin.

In α-crystallin from the nuclear part of the lens, polypeptide chains were found, which were not present in the cortex of the lens. These additional subunits were fractionated by means of chromatography on DEAE-cellulose (DE-52) or CM-cellulose (CM-52). Characterization of two of them showed that they were identical to shorter αA-chains, and hence they were designated as αA21–151and αA11–151. By studying α-crystallin as function of fiber cell development it turned out that the subunit structure of α-crystallin is not constant. The polypeptide chains in the α-crystallin aggregates are subject to specific and limited degradation processes, causing an ever changing polypeptide chain composition.

alpha-Crystallin from calf eye lens is found to have 30 sulfhydryl groups per 800 000 daltons by modification with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 4,4'-dithiopyridine in 6 M urea. These -SH groups can be divided into three different classes in native alpha-crystallin from their reactivity with DTNB, 4,4'-dithiopyridine, iodoacetamide and ethylenimine. Results obtained with these reagents point to the presence of 7 +/- 1 thiol groups (Class I) which are likely to be surface exposed, with a concomitant 40--45% quenching of tryptophan fluorescence in DTNB-modified alpha-crystallin. Another 10 +/- 1 thiol groups (Class II) must be in a hydrophobic environment since they react only with the hydrophobic reagents, causing a further decrease in fluorescence intensity. Class III, 13 +/- 2 thiol groups, is inaccesible to any of these reagents. Introduction of up to 15 negatively charged thionitrobenzoate groups or seven positively charged aminoethyl groups in the alpha-crystallin molecule at pH 7.5--8.0 did not change the state of aggregation as judged from the sedimentation coefficients.

A highly sensitive method was used to determine d - and l -aspartyl residues in protein fractions from normal and cataractous human lenses. A linear relationship with age was found in all fractions from normal lenses. However, no correlation with cataract could be established. It is concluded that racemization of aspartyl residues is a continuous process in eye-lens proteins but plays no role in cataract formation.

Human lens epithelium is cultured and the epithelial origin of the cells is established ultrastructurally. The epithelial cells have a limited growth capacity in vitro; only in rare cases a confluent monolayer is obtained. When primary confluent cultures are split 1:1·5 the cells again form a monolayer; they do not form a monolayer when split at 1:2. The light microscopical and ultrastructural features of early (1 week) and late (older than 1 month) stage cultures are described. Moreover, the ultrastructure of human lens epithelium in situ is presented. Incubation of early and late stage cultures in a 35S-methionine containing medium revealed that the crystallin synthesis in younger cultures is reduced, whereas it has completely ceased in the older ones. These late stage cultures only synthesize cytoskeletal components (43 and 57 K daltons). These observations are confirmed by the indirect immunofluoresence study.

The isolated and purified polypeptide chains of calf lens α-crystallin were reaggregated in 11 different mixing ratios. The aggregates were studied by immunoelectrophoresis, and in the analytical ultracentrifuge. The deaggregating agent, 6 M urea, was removed by dialysis for 16, 40 and 100 h. In order to avoid the possible loss of low molecular weight material during dialysis, removal of the 6 M urea was also performed on a Sephadex G-10 column. All reaggregates showed the same sedimentation coefficient and their electrophoretic mobilities reflected only in part the mixing ratio of the polypeptide chains. The aggregates with a high quantity of acidic chains did not, or only partially, incorporate the basic chains.

A recently developed chiral gas chromatographic method and a diastereomeric high-performance liquid chromatographic method for the analysis of aspartic acid enantiomers in protein hydrolyzates have been evaluated. Although both techniques are fast and convenient, the latter is preferred because of its higher reproducibility and shorter analysis time. Furthermore, this method offers the possibility of on-line derivatization and analysis.

Steric exclusion liquid chromatography in the presence of intermediate urea concentrations with lowangle laser light scattering detection was used to investigate the stepwise dissociation of the multimeric bovine eye lens protein α-crystallin. The change in the quaternary structure of α-crystallin as a function of increasing urea concentration clearly resembled dissociation by increasing alkaline pH, urea or guanidine-HC1 concentrations when studied by sedimentation velocity analysis. Next to native and native-like threelayer aggregates (Mr 6.5 − 7.5 × 105), the first dissociation products (two-layer molecules Mr 4 − 5.5 × 105), the second dissociation products (core molecules Mr 2.5 − 3 × 105), and monomeric subunits (Mr 20 000) could be characterized. In the range from 2.6 to 4.4 M urea, we found a gradual decrease in the proportion of the remaining three-layer aggregates and an increase in that of monomeric subunits. The fluorescence emission maxima showed increasing solvent exposure of the tr...

Experimental cataracts were obtained by X-irradiation of one eye of 2-month-old rabbits with 1800 rad. In the cataractous lens high molecular weight (HM-)crystallin is already found in great amounts in the lens cortex, whereas in the unirradiated lens this is true only for the nucleus. In the cataractous cortex and nucleus the subunit structures of α-crystallin, HM-crystallin and US fraction (urea-soluble part of the albumoid) are very complex. Their compositions are similar to those of the corresponding preparations from the nucleus of the normal lens. They are composed of α- and β-crystallin polypeptide chains. In the case of US fraction of the cataractous lens also trace amounts of γ-crystallin were observed. On the other hand, relatively simple polypeptide chain compositions were found in the normal lens cortex. Electron microscopic studies of HM-crystallin from the cataractous lens cortex and nucleus revealed a wide size distribution of irregularly shaped giant aggregates. The same feature was observed for HM-crystallin of the normal nucleus, whereas only relatively small aggregates were seen in HM-crystallin of the normal cortex. The ultrastructure of the normal lens fibres exhibits an electron-dense appearance without any significant demonstrable organelles. On the other hand, the cataractous lens showed pathological changes, as demonstrated by a distinct granular content of the fibres. Indications for an interpretation of X-ray induced cataract in terms of an accelerated aging process are discussed.

Limited proteolysis of α-crystallin with 10 different endopeptidases in vitro shows that only 5–20 residues of the carboxy termini of A and B chains are accessible to these enzymes, without disruption of the quaternary structure. The A chains are more susceptible to proteolysis in vitro than B chains, which corresponds with the degradation in vivo upon aging of α-crystallin in the lens. Six proteases specifically cleave off the carboxyl-terminal pentnpcptide of all B chains, which indicates that the B (Thr170 -Ala171) bond, the only observed degradation site of B chains in vivo, is surface-exposed and proteolytically very labile. None of the endopeptidases split the A (Asp151 -Ala152) bond, the main degradation site of A chains in vivo, but a nearby segment A156–163 iS very accessible to several enzymes. We postulate that this A151–152 bond is close to the surface of α-crystallin and, although proteolytically stable in the native protein, it could become exposed as a result of age-related changes in secondary and/or tertiary structure. Two minor tryptic cleavage sites in A chains of native α-crystallin become more accessible in reassociated a-crystallin, and particularly in pure A-chain reaggregates, which indicates a change in secondary/tertiary structure upon dissociation-reassociation. Although the accessibility of B chains suggest that they are in equivalent positions in the α-crystallin quaternary structure, the A chains exhibit various rates of proteolysis and therefore occur in more than one orientation. The anomalously slow migration of B chain in dodecylsulfate gel electrophoresis is abolished after removal of its carboxyl-terminal pentapeptide, Ala-Ala-Pro-Lys-Lys.

Intumescent cataractous lenses were investigated as to their water content and protein composition. Nuclei were separated from cortices. Water-soluble, water-insoluble, urea-soluble and urea-insoluble portions were quantified and the subunit composition of the water-soluble crystallin fraction was looked at on SDS-gel electrophoresis. Compared to normal lenses it is observed that the water content in intumescent cataract is increased, water-soluble and urea-soluble fractions are diminished, whereas the urea-insoluble portions are augmented. No major changes are noted in the subunit composition of the soluble protein fractions. Particularly, the relative amount of gamma-crystallins is diminished in intumescent cataractous lenses.

Extracts of water-soluble proteins from cortex and nucleus of single human lenses have been analyzed by high-pressure gel permeation chromatography in combination with a size- and a concentration-sensitive detector. Using this rapid and accurate method, changes in molecular weight and proportion of the native crystallins upon aging and formation of nuclear cataract are observed. In the range from 30 to about 40 years the gamma-crystallin content of normal lenses increases and that of alpha-crystallin decreases; over 40 years the level of gamma-crystallin decreases and that of alpha-crystallin remains constant in the cortex (11%) as well as in the nucleus (1-2%). These changes are accompanied by an increase in the water-insoluble fraction. With progressing nuclear cataract an increase in beta 3- and a drastic decrease in gamma-crystallin content, especially in the nucleus, are accompanied by a steep increase in the water-insoluble fraction. The molecular weights of the crystallins do not change significantly, except that of alpha- and beta 1-crystallin which show an increase with age.

Total extracts of both water-soluble and water-insoluble portions are obtained from fetal and adult human lenses. In the water-soluble extracts of lenses older than 14 years of age little difference is seen between the cortical and nuclear preparations. In lenses older than 14 years a polypeptide chain of approximately 10 k appears. In fetal and newborn lenses a 43 k polypeptide is detected which becomes more diffuse upon aging. Concomitant with a decrease of a 29·5 k polypeptide, there is a relative increase of a 29 k chain, suggesting a situation comparable with the transition from calf βB1a to βB1b. In young lenses a major β-polypeptide co-migrating with calf βBp is seen. As for the water-insoluble portions, no difference between cortical and nuclear preparations is detectable after 14 years of age. In fetal lens there is a high amount of the 29·5 k polypeptide, which decreases with aging and finally disappears. Till 6 months four bands with molecular weights of 200 k, 100 k, 57 k and 43 k are detected, co-migrating with calf myosin, α-actinin, vimentin and actin, respectively.

Contrary to the elution pattern of bovine eye lens proteins chromatographed on a TSK GEL G5000 PW type column, an additional peak, representing 4.10 6 dalton aggregates, was observed on a combined G5000 PW–G4000 SW type system. This extra peak, found between the G5000 PW total exclusion fraction and α-crystallin (M r  1.1 · 10 6 ), appeared to correspond to the G4000 SW void volume. Simulation calculations substantiated that this peak is the result of coupling the two columns and does not originate from a distinct sub-population of molecules. Such artificial peaks may be expected whenever a broad molecular weight distribution extends beyond the fractionation limits of at least one of the constituents of a combined gel permeation system. It is inferred that the hypothetical exclusion limit for compact, globular, symmetrical proteins on TSK GEL G5000 PW is near 10 8 dalton.

Cortial alpha-crystallin was size-fractionated by gel filtration on Ultrogel AcA22 and charge-fractionated by anion-exchange chromatography on DE-52 DEAE-cellulose using gradient elution. Electron microscopy demonstrates that both native and reassociated alpha-crystallin are heterogeneous populations of spherical or slightly ellipsoidal molecules with diameters of 13.5--16.0 nm (maximum at 14.0--15.0 nm) for native alpha-crystallin and 8.5--12.5 nm (maximum at 10.0--10.5 nm) for reassociated alpha-crystallin. An enormous charge heterogeneity of native alpha-crystallin was detected, which is shown to arise from variations in the stoichiometry of the 5 main types of subunits. The molar ratio of acidic chains (A2, A1 and A1/2-151) to basic chains (B2 and B1) varies from 70/30--80/20 (averaging about 3/1) and the amount of deamidated chains (A1 and B1) varies from 7--37%. Recombination of the subunits, after dissociation in 6 M urea, leads to a charge heterogeneity of reassociated alpha-crystallin very similar to that of native alpha-crystallin. Therefore, specific formation of pure A or B chain aggregates is not preferred. Instead, random combination of subunits is theoretically shown to be sufficient to describe the observed charge microheterogeneity of both reassociated and native alpha-crystallin. No obvious relationship exists between size and charge heterogeneity. Within these ranges of molecular weight and subunit composition there are more than 1000 different combinations of A2, A1, A1/2-151, B2 and B1 conceivable.

Intact fetal human lenses are cultured for 20 hr in a ( 35 S)-methionine containing medium. The water-soluble proteins are isolated and separated by means of gel chromatography. Newly synthesized α - and γ -crystallin are found; in addition, all β -crystallin species are newly synthesized. After SDS-PAGE and isoelectric focusing in the presence of 6 m -urea the samples are autoradiographed. The presence of radioactivity in the various bands on both patterns is discussed.

Knowledge of the quaternary structure of α-crystallin is required to understand the age-dependent superaggregation processes, eventually leading to lens opacification. Different approaches, e.g. sulfhydryl modification, chemical cross-linking, limited proteolysis and dissociation studies revealed new information, providing a basis for further studies of aging and higher-order structures. A model for the architecture of native α-crystallin from calf lens cortex, featuring subunit arrangement and surface exposure, will be presented.

The molecular weight of bovine alpha-crystallin, isolated at 37 degrees C, was studied and found to be about 800,000. This contrasts with the results of Thomson and Augusteyn (Thomson, J. A., and Augusteyn, R. C. (1983) Exp. Eye Res. 37, 367-377) who isolated a species of about half this molecular weight. We show here that this form of alpha-crystallin can only be isolated under unphysiological conditions with regard to buffer pH and ionic strength.

Carrier-free continuous electrophoresis was applied for the separation of α-crystallin on a preparative scale. Every 24 hr 1g of the purified protein can be obtained. Purity was verified by means of polyacrylamide gel electrophoresis and sedimentation analysis. If necessary, leucine aminopeptidase activity may be removed from the α-crystallin preparation by gel chromatography on Sepharose 6-B.

Extracts from epithelial cells, seven successive concentric layers of the cortex, and the nucleus of calf lenses were submitted to gel chromatography on agarose Bio-Gel A-5m. In the mentioned order, corresponding to increasing age of the relevant lens cells, the content of α-crystallin in these extracts decreased significantly whereas the content of a very high-molecular weight fraction, resembling an aggregate composed of the polypeptide chains of α- and β-crystallin, increased markedly. The sedimentation coefficients of the α-crystallin preparations at concentrations of 0·8 mg/ml ranged from 17 to 21 S and passed through a minimum. Gel electrophoresis in polyacrylamide containing 6 m urea revealed significant changes in polypeptide composition. Nevertheless, no clear relation between sedimentation coefficients and polypeptide composition was found. Evidence has been obtained for the interaction of α-crystallin with constituents of β-crystallin which may lead to the formation of very high-molecular, finally insoluble aggregates.

KNOWLEDGE of the intracellular processes of ageing and breakdown of proteins is still very limited1,2, but the extremely slow turnover of lens proteins in the eye provides a unique opportunity to analyse such processes. Throughout life fibre cells are formed from epithelial cells at the periphery of the lens, being successively covered by younger cells. Protein synthesis ceases soon after the differentiation into fibre cells is completed3. As a result the nucleus of the lens of an adult still contains the proteins formed during foetal life. Here we demonstrate that differences in polypeptide structure observed between the cortex and nucleus4–6 of the lens are due to an ageing process. Our data provide the first example of progressive degradation and deamidation of a protein, probably caused by non-enzymatic mechanisms.