Partial Characterization of Three Distinct Populations of Human 7-Crystallins

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Partial Characterization of Three Distinct Populations of Human 7-Crystallins J. 5. Zigler Jr,* P. Russell,* L J. Takemoto.t 5. J. Schwab,* J. S. Honsen.f J. Horwitz.J and J. H. Kinoshira* Certain low molecular weight crystallins may be involved preferentially in the process of human cataractogenesis. Three distinct populations of monomeric crystallins with molecular weights ranging from 19,000 to 24,000 have been demonstrated previously by electrophoresis in the presence of sodium dodecyl sulfate (SDS). The authors now report the chromatographic separation of these three components from young human lenses. Each of the three species has unique profiles on isoelectric focusing and native polyacrylamide gels as well as on SDS gels. All three species show similar patterns of age-related modification in their charge characteristics. These three populations of polypeptides all appear to be 7-crystallins. The relative amounts of the three vary with age, which accounts for the greater ease of their separation in extracts from young lenses as compared with adult lenses. Invest Ophthalmol Vis Sci 26:525-531, 1985 It has been reported by a number of investigators for over at least 20 years that there is a loss of watersoluble low molecular weight crystallins during human cataract formation. 1 Recently this observation has been confirmed by studies in which the crystallin composition of opaque and normally transparent regions of microdissected human cataracts have been compared. 2 In view of these findings, we have undertaken a collaborative effort to more fully characterize the low molecular weight proteins of the normal human lens, to clarify the age-related changes that these proteins undergo, and ultimately to determine their fate during the process of cataractogenesis. While the 7-crystallin from calf lens has been studied extensively, including the determination of the complete primary sequence 3 and the three-dimensional crystal structure 4 of the primary polypeptide, the human low molecular weight proteins remain relatively poorly characterized. It has become clear that they are more heterogeneous than the 7-crystallin from other species studied. Kabasawa et al 5 were the first to attempt to fractionate the human low molecular weight crystallins. They identified two primary peaks from Sephadex G75 chromatography as 7 H, From the Laboratory of Vision Research, National Eye Institute, National Institutes of Health, Bethesda, Maryland,* the Division of Biology, Kansas State University, Manhattan, Kansas,f and the Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California.:): Supported in part by grants from the National Eye Institute to L.J.T. and to J.H. Submitted for publication: June 29, 1984. Reprint requests: J. Samuel Zigler Jr., Ph.D., Bldg. 6, Rm. 235, National Institutes of Health, Bethesda, MD 20205. consisting of polypeptides of 24,000 daltons, and y L, consisting of polypeptides of 20,000 and 11,000 daltons. Subsequently Zigler et al 6 reported similar chromatographic patterns in which three distinct populations of polypeptides could be distinguished. The major peak in adult lenses (equivalent to "7 H " above) was shown to contain two SDS polypeptides (apparent mw = 24,000 and 21,000), a second peak yielded a single SDS polypeptide of mw 19,000, and a final poorly resolved minor peak contained components of about 10,000 daltons. The 10,000 dalton species are apparently breakdown products of a-crystallin 7 and/or 7-crystallin 8-9 and are absent in young lenses. This group of polypeptides will not be considered in the present study. The 19,000-dalton species was identified immunochemically and physicochemically as 7-crystallin, but separation of the 24,000 and 21,000 dalton species was not accomplished and the identification of these components was uncertain. This study and others 10 " 12 clearly have demonstrated that the heterogeneity of these low molecular weight proteins is much less in young human lenses than in the adult lens. Consequently, in the present study we have compared these polypeptides from lenses of various age. We have found that in very young lenses the 24,000- and 21,000-dalton species can be separated chromatographically. The data indicate that these two species as well as the 19,000-dalton population are 7-crystallins and that all three undergo similar modification in charge characteristics during aging. Materials and Methods Normal human lenses were stored at -70 C until use. Pairs of lenses were homogenized in 4.0 ml 525

526 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1985 Vol. 26 20 30 40 50 60 FRACTION NUMBER 95 Years 31 Years 3 Years 4 Months Fig. 1. Direct UV monitor recordings of the absorbance at 280 nm for Sephadex G75 superfine fractionations of four pairs of normal human lenses. Fractions of 75 drops (~5.0 ml) were collected. The low molecular weight peaks elute after fraction 50 and are indicated by numbers on the bottom panel. buffer (0.05 M Tris, ph 7.4, 0.1 M KC1, 1 mm EDTA, 10 mm 2-mercaptoethanol, and 0.02% NaN 3 ) and the homogenates centrifuged at 27,000 X g for 20 min. The supernatants were applied to a column of Sephadex G75 superfine (Pharmacia, Piscataway, NJ) (2.6 X 85 cm) and eluted with the same buffer. Five-milliliter fractions were collected, and the eluate was monitored at 280 nm by a Uvicord II monitor (LKB Instruments, Gaithersburg, MD). To date, more than a dozen pairs of Eye Bank lenses (aged 2 months to 95 yr) have been fractionated and analyzed. Data are presented on individual lens pairs encompassing the entire age range. These data are representative of the entire group. SDS-polyacrylamide disc gels were run using the procedure of Weber and Osborn 13 on gels 5 mm in diameter and 100 mm long. Samples were made 1% in SDS and 1 % in 2-mercaptoethanol and were placed in a boiling water bath for 2 min prior to application to the gels. Running time was about 3'/2 hr at 75 V (constant voltage). Gels were stained with Coomassie blue R250. 70 Native polyacrylamide electrophoresis was by the method of Laemmli 14 but without the presence of SDS in either the gel or the chamber buffers. A 2- mm-thick slab gel of 10% acrylamide was used. Running time was about 4'/2 hr at 100 V constant voltage. Staining and destaining was by the same procedure used for SDS gels. Isoelectric focusing was performed on LKB Pagplates with ph range 3.5-9.5, using the LKB Multiphor system. Desalted samples were applied and run by the protocol recommended by LKB. Gels were stained with Coomassie blue R-250. Characterization of radioiodinated tryptic peptides was done as previously described. 15 Briefly, this technique involved radioiodination of purified polypeptides in the presence of SDS, resolution of the radioiodinated polypeptides using SDS-polyacrylamide gel electrophoresis, digestion of the radioiodinated polypeptides when immobilized in polyacrylamide gel, and resolution of the resultant peptides in two dimensions using electrophoresis and chromatography. Antisera were prepared in rabbits against purified calf a, js, and 7-crystallins as previously described. 16 Immunodiffusion was performed using commercially prepared agarose plates (Hyland Diagnostics, Malvern, PA). Transfer of proteins from native electrophoretic gels to nitrocellulose paper was accomplished in a Trans Blot Cell (Bio Rad Laboratories) using 1% acetic acid at 100 ma constant current for 16 hr. The nitrocellulose paper subsequently was visualized by the immunblot technique 17 essentially according to the Bio Rad Laboratories protocol. Peroxidase labeled goat antirabbit IgG was from Kirkegaard and Perry Laboratories (Gaithersburg, MD). Results Figure 1 gives the elution patterns obtained from four pairs of normal lenses of varying age from a Sephadex G75 superfine column. Unlike the previous study, 6 when larger numbers of lenses were pooled and fractionated first on Sephadex G200, in this study the whole lens homogenates were fractionated directly on G75 superfine. The material eluting after fraction 50 represents the protein that would elute in the final protein peak from a G200 column. The patterns obtained from the two older pairs of lenses are similar to those reported by Kabasawa et al 5 and in our previous article for adult lenses with essentially two peaks present, whereas the younger lenses clearly have a third component eluting between the two peaks seen in the adult lens. In the adult lens, peak 1 (see bottom panel for numbering scheme) contained SDS polypeptides of 24,000 and 21,000 daltons, as previously reported, 6 while peak 3 contained the 19,000-dalton polypeptide. In Figure 2 we have com-

No. 4 THREE DISTINCT POPULATIONS OF HUMAN 7-CRYSTALLINS / Zigler er ol. 527 pared the SDS gel patterns for the top fractions from each of the three peaks present in the 4-month-old lenses (Fig. 1). Peak 1 gives a single major band with apparent MW = 24000, peak 2 a single primary band with apparent MW = 21,000-22,000, and peak 3 a single band of ~ 19,000. The apparent molecular weights of peaks 1, 2, and 3 do not change with age, based on either G-75 elution patterns or SDS gel data. To further characterize these components, we have fractionated them by gel electrophoresis in the absence of SDS or other denaturant (Fig. 3). The patterns obtained for the preparations from 4-month-old lenses are shown in lanes 1-3. Peaks 2 and 3 each consist essentially of a single slowly migrating band, although in each case a second faint band is present, even in preparations from very young lenses. The band from peak 2 migrates slightly faster than that of peak 3. For peak 1 the major component migrates considerably faster than the bands seen in peaks 2 and 3, but there is also a second weaker band that migrates only slightly ahead of the peak 2 band. Comparison of these patterns with those obtained using material from adult lenses revealed marked differences. Peaks 2 and 3 from 32-year-old lenses appeared to retain 94K 68K 43K 30K 20K 14K Fig. 2. SDS-polyacrylamide gel patterns for the low molecular weight peaks obtained from the 4-month human lenses. I. Molecular weight standards (Pharmacia); 2. Peak 1; 3. Peak 2; 4. Peak 3; 5. Mixture of the samples applied to gels 2-4. 1 2 3 4 5 6 7 8 9 Fig. 3. Native 10% polyacrylamide gel patterns for low molecular weight peaks from human lens preparations. 1. Peak I from 4- month lenses; 2. Peak 2 from 4-month lenses; 3. Peak 3 from 4- month lenses; 4. Peak 1 from 32-year lenses; 5. Peak 2 from 32- year lenses; 6. Peak 3 from 32-year lenses; 7. Peak 1 from 66-year lenses; 8. Peak 2 from 66-year lenses; 9. Peak 3 from 66-year lenses. the bands present in the young lenses, although the bands were much more diffuse, and in'addition four or five new bands of higher mobility were present. Peak 1 also retained the major band present in the 4-month lenses and had developed three or four new faster migrating species. The slower migrating minor band present in peak 1 from the 4-month lenses was no longer evident. The regularity of the interval between the bands of all three peaks in the adult lenses was striking. In addition, it appeared that with increasing age the relative distribution of the various components in each peak shifted progressively toward the faster migrating (more acidic) species. This clearly is demonstrated by comparison of peak 3 from 66- year-old lenses (lane 9) with the corresponding material from the 32-year-old lenses (lane 6). The very marked difference in heterogeneity between young and old lenses with respect to these proteins is demonstrated further by isoelectric focusing (Fig. 4). The individual peaks from the 4-month-old lenses each have relatively simple patterns consisting of one or two primary components with isoelectric points generally >7. In contrast, in the adult lens the patterns are much more heterogeneous and much more acidic, with isoelectric points ranging as low as

528 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / April 1985 Vol. 26 1 2 3 4 5 6 ph 5 ph 6 ph 7 ph 8 Fig. 4. Isoelectric focusing patterns on LKB Pagplate (ph 3.5-9.5) for low molecular weight peaks from human lens preparations. 1. Peak 1 from 4-month lenses; 2. Peak 2 from 4-month lenses; 3. Peak 3 from 4-month lenses; 4. Peak 1 from 66-year lenses; 5. Peak 2 from 66-year lenses; 6. Peak 3 from 66-year lenses. 5. Similar findings have been reported by others for the total -y-crystallins from prenatal and adult human lenses. 12 The accumulated evidence suggests that similar processes are occurring in all three components during aging. To investigate the possible relationship of these species, we have compared the three low molecular weight crystallin peaks immunochemically using antisera specific for calf a, j3, and 7 crystallins, respectively. Figure 5 demonstrates that by immunodiffusion all three low molecular weight peaks from 2-monthold lenses crossreact strongly (although not with complete identity) with antiserum to calf 7-crystallin. Furthermore, none of the peaks are reactive with either anti-a or anti-/? serum. Similar results were obtained with low molecular weight preparations from older lenses as well. To determine the reactivity with anti-7 serum of the multiple species present in older lenses, peaks 1-3 isolated from a pooled preparation of older lenses (aged 55-60 yr) were run on native gel, blotted to nitrocellulose, and then reacted with specific antisera. Figure 6 demonstrates that in fact all of the bands visible in the stained pattern (panel A) for peaks I, 2 and 3 from both infant and adult lenses are reactive with the antiserum to 7- crystallin as seen in the peroxidase-labeled immunblot (panel B). Thus, the more acidic species that accumulate progressively with aging can be identified as 7-crystallins. Note that the antiserum used has no demonstrable reactivity with /5-crystallin present in lane 7. Additional evidence demonstrating a structural relationship among the three peaks was obtained from tryptic peptide maps using samples from 4-monthold lenses (Fig. 7). These data suggest that peaks 2 and 3 are most closely related, based on the number of shared iodinated peptides. Peak 1 shares several peptides with both 2 and 3; it also shares two major peptides with peak 2, which are not seen in peak 3. Discussion Horwitz et al 2 recently have reported that in microdissected sections from human lens nucleus there is a marked decrease in a soluble low molecular weight fraction in cataracts relative to age-matched normal lenses. Electrophoretic analysis of this fraction indicated that it was the "heavy low molecular weight" peak that we previously reported 6 to contain two SDS polypeptides with apparent molecular weights of 24,000 and 21,000. In view of the selective loss of this peak from the HPLC profile of cataracts, we have attempted to better characterize this material using Sephadex G75 superfine chromatography. This chromatographic medium, which has been used previously to study human low molecular weight crystallins, 5 ' 6 has greater resolving power than the HPLC A Pk 1 Pk 1 Pk 3 Pk 2 Pk 3 Pk^-2 I Pk 3. Hk 2 Pk 1 Fig. 5. Immunodiffusion studies on the low molecular weight peaks from 2-monthold human lenses. Center wells contain antisera specific for calf a-crystallin (A), /Scrystallin (B), and 7-crystallin (C). Peripheral wells contain bovine a, js, or 7-crystallin preparations or human low molecular weight peaks 1, 2, or 3, as indicated. Antigen concentrations were approximately 0.5 mg/ml in phosphate-buffered saline.

No. 4 THREE DISTINCT POPULATIONS OF HUMAN 7-CRYSTALLINS / Zigler er ol. 529 A B Fig. 6. Native acrylamide gel staining patterns (A) and immunblot data from a duplicate set of samples (B). Samples were as follows: 1. 2-month peak 1; 2. 2-month peak 2; 3. 2-month peak 3; 4. Adult peak 1; 5. Adult peak 2; 6. Adult peak 3; 7. calf js-crystallin. The adult preparations were from pooled lenses aged 55-60 years. * 1 2 3 4 5 6 7 1 2 3 4 5 6 7 gel filtration columns in the molecular weight range of interest and also can be loaded with sufficient protein to facilitate subsequent analysis of individual components. The elution patterns obtained for older adult lenses were similar to those reported previously by Kabasawa et al 5 and by our group. 6 However, when we looked at very young lenses we were able to resolve three low molecular weight peaks instead of the two present in the adult lenses. To our knowledge, this is the first report of such a finding. The data (Fig. 1) suggest that peak 2 is a major low molecular weight component in the young lens, but with increasing age it becomes quantitatively much less prominent, while peak 1 becomes much more prominent. By 20-30 years of age peak 2 is variably apparent as a shoulder on peak 1 and in older lenses is no longer seen in the elution pattern. Electrophoretic analysis of the three peaks from young lens revealed that peaks 1 and 2 correspond to the ~ 24000 and ~ 21,000 dalton SDS polypeptides, respectively, which previously were reported to constitute the "heavy low molecular weight" peak from adult lens, while peak 3 corresponded to the 19,000 dalton y-crystallin species. 6 In the adult lenses, a 10,000-dalton population of polypeptides elutes with and following peak 3, but, as reported by others, was absent from the young lenses. 16 Since the 21,000 dalton polypeptide still is detectable in older lenses by electrophoresis, it appears that its loss from the elution pattern in older adult lenses is simply the result of its being "swamped out" by the increase in peak 1 with age. In addition to the definite age-related changes in the composition of the low molecular weight peaks that are evident chromatographically, isoelectric focusing reveals marked modification of polypeptide charge with age. As we reported previously for the 19,000-dalton component, all three chromatographic fractions have quite simple isoelectric focusing patterns in very young lenses. In contrast, even young adult lenses show extremely heterogeneous patterns of bands with lower isoelectric points, probably resulting from posttranslational modifications. These data reinforce the findings of Ringens et al, 11 who found that agerelated modifications to lens crystallins begin to occur even in adolescent human lenses. It appears, therefore, that the use of very young human lens tissue will be necessary in order to avoid the difficulties associated with the heterogeneity present after such modification has occurred. Among the most intriguing findings in this study are the patterns obtained on native polyacrylamide gels. Kabasawa et al 5 published photographs of gels showing similar patterns for the primary component of "YH" (equivalent to "heavy low molecular weight") and for y L (equivalent to 19,000 dalton 7) from a 54-year-old lens. Their data are consistent with our results for peaks 1 and 3 from adult lenses (see Fig. 3). The present data extend these results substantially by comparing such patterns with those from very

500 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / April 1985 Vol. 26 I Fig. 7. Resolution of radioiodinated tryptic peptides from the three low molecular weight peaks from 4-month human lenses, a. Peak 1; b. Peak 2; c. Peak 3; Peptides common to all three peaks are designated by ; those common to peaks 1 and 2 by :>?; and those common to peaks 2 and 3 by. young lens. It appears that initially peaks 2 and 3 each consist of a single band on this system; peak 1 has two bands but it is possible that at earlier age only a single band would be found and that the second band represents already modified polypeptides. With increasing age, all three peaks show a similar pattern of appearance of new, more acidic polypeptides and a progressive quantitative increase of the more acidic species at the expense of the less acidic ones. The very regular interval present between the bands on the native gel for all three peaks suggests the possibility that each succeeding band may represent a population of polypeptides with a discrete charge difference from the preceding, less acidic band. Such a situation might arise, for instance, via deamidation, which is well known to occur in crystallins. 10 It is interesting to note that Slingsby and Miller, 19 in a study of the low molecular weight crystallins from bovine and rabbit lenses of various ages, reported that some 7-crystallin fractions could be induced to form similar multiple-band electrophoretic patterns in vitro by long-term storage in solution or by modification of buffer and ph. Based on the IEF gels, it would appear that the native gel is not sufficiently sensitive to demonstrate the full extent of microheterogeneity present in the older lenses, although the more diffuse appearance of the native gel bands for the adult preparations may reflect such microheterogeneity. The structural basis for the regular patterns seen on the native gel and correlation of these patterns with the IEF patterns are subjects currently under investigation by our group. An important question concerns the relationship among these three low molecular weight crystallin components. In our previous study, we were able to identify conclusively only the 19,000 dalton species (peak 3) as 7-crystallin. The major component that contained both the 24,000 and 21,000 dalton species appeared to be slightly larger, much more acidic, and to have quite different tertiary structure than expected for a true 7-crystallin. Furthermore, it reacted only very weakly to anti-7 crystallin serum. Thus, we elected simply to call these species low molecular weight proteins rather than 7-crystallins. 6 Our present data, however, indicate that all three peaks represent 7-crystallins, although peaks 1 and 2 are somewhat different in certain characteristics (apparent size, tertiary structure) from bovine 7-crystallin. This conclusion is consistent with the similarities present in the tryptic peptide maps for the three components, the finding that all three have rather high isoelectric points initially (in young lens), the similar patterns of age-related change that all three components undergo, and, most importantly, immunochemical data derived from new antisera to bovine 7- and /?- crystallins. This anti-7 crystallin serum reacts well with all three species from the 2-month lens, whereas the anti-js crystallin serum shows no reactivity on immunodiffusion (Fig. 5). That the additional species that appear on the native gel patterns from older lenses are 7-crystallins is demonstrated by the immunblotting technique (Fig. 6). Further evidence that the more acidic species that appear with increasing age represent postsynthetically modified 7-crystallin has been provided by the development of a monoclonal antibody specific for peak 1, which reacts well with both the peak 1 from young lens and with the multiple native gel species present in peak 1 samples from older lenses on immunblots. 20 Neither a- nor /3-crystallins compete for binding with this antibody. It is unclear why the previous 7-crystallin antiserum failed to react clearly with the 24,000- and 21,000- dalton species, however, Ringens et al 12 also found anticalf 7 crystallin to react only weakly with the

No. 4 THREE DISTINCT POPULATIONS OF HUMAN 7-CRYSTALLINS / Zigler er ol. 531 total low molecular weight fraction from the adult human lens, while it reacted strongly with fetal human 7-crystallin. Additionally, recent studies have indicated markedly decreased binding of monospecific anti-7- crystallin serum on Western blots by 7-crystallin from opaque human lens nucleus when compared with similar preparations from age-matched normal lens nucleus. 21 Thus, the changes that occur in the human 7-crystallins with age and cataractogenesis may lead to loss or alteration of some antigenic determinants and thus decreased affinity for some antibody molecules. The recent report 12 that the low molecular weight crystallins from adult human lenses have only a minor amount of N-terminal glycine is also of interest in this regard, since 7-crystallins typically have free amino terminal glycine, while a- and js-crystallins have blocked amino termini. Further investigation is needed to determine whether all human 7-crystallins have free amino terminal glycine and, if so, whether it may become blocked during aging. In summary, we have shown that the three SDS polypeptides previously described as comprising the human low molecular weight crystallins 6 all can be separated chromatographically from young lenses. With increasing age, the relative amounts of the species change such that the two heavier ones no longer are readily separable. All three species show a marked increase in heterogeneity and become progressively more acidic as older lenses are investigated. The three species all are immunochemically identifiable as 7-crystallins. Further studies are underway to better characterize this group of crystallins, which may be involved intimately in the process of human cataractogenesis. Key words: human lens, low molecular weight proteins, 7- crystallins, three distinct populations, separation, partial characterization, aging effects Acknowledgments Special thanks are given to the National Diabetes Research Interchange, from which normal human lenses were obtained, and to the Mid-South Eye Bank, Memphis, Tennessee, from which the pairs of 2-month and 4-month-old lenses were obtained via Dr. H. M. Jernigan, Jr. The clerical work of J. Mclntyre and F. Carpenter in preparing the manuscript gratefully is acknowledged. This work is a part of the Cooperative Cataract Reseach Group effort. References 1. Harding JJ: Changes in lens protein in cataract. In Molecular and Cellular Biology of the Eye Lens, Bloemendal H, editor. New York, Wiley-Interscience, 1981, pp. 327-366. 2. Horwitz J, Ding LL, and Cheung CC: The distribution of soluble crystallins in the nucleus of normal and cataractous human lenses. Lens Research 1:159, 1983. 3. Croft LR: The amino acid sequence of 7-crystallin (Fraction II) from calf lens. Biochem J 128:961, 1972. 4. Wistow G, Turnell B, Summers L, Slingsby C, Moss D, Miller L, Lindley P, and Blundell T: X-ray analysis of the eye lens protein 7-II crystallin at 1.9 A resolution. J Mol Biol 170:175, 1983. 5. Kabasawa I, Barber GW, and Kinoshita JH: Aging effect and some properties of the human lens low molecular weight proteins. Jpn J Ophthalmol 21:87, 1977. 6. Zigler JS Jr, Horwitz J, and Kinoshita JH: Studies of the low molecular weight proteins of human lens. Exp Eye Res 32:21, 1981. 7. Roy D and Spector A: Human insoluble lens protein II. Isolation and characterization of a 9600 dalton polypeptide. Exp Eye Res 26:445, 1978. 8. Garner WH, Garner MH, and Spector A: Comparison of the 10,000 and 43,000 dalton polypeptide populations isolated from the water soluble and insoluble fractions of human cataractous lenses. Exp Eye Res 29:257, 1979. 9. Takemoto LJ, Hansen JS, and Horwitz J: Biochemical analysis of microdissected sections from the normal and cataractous human lens. Curr Eye Res 2:443, 1983. 10. Hoenders HJ and Bloemendal H: Aging of lens proteins. In Molecular and Cellular Biology of the Eye Lens, Bloemendal H, editor. New York, Wiley-Interscience, 1981, pp. 279-326. 11. Ringens PJ, Hoenders HJ, and Bloemendal H: Effect of aging on the water-soluble and water-insoluble protein pattern in normal human lens. Exp Eye Res 34:204, 1982. 12. Ringens PJ, Hoenders HJ, and Bloemendal H: Protein distribution and characterization in the prenatal and postnatal human lens. Exp Eye Res 34:815, 1982. 13. Weber K and Osborn M: The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406, 1969. 14. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277: 680, 1970. 15. Takemoto LJ, Hansen JS, and Horwitz J: Interspecies conservation of the main intrinsic polypeptide (MIP) of the lens membrane. Comp Biochem Physiol 68B:101, 1981. 16. Zigler JS Jr, Horwitz J, and Kinoshita JH: Human ^-crystallin I. Comparative studies on the /3,, 0 2 and /J 3 -crystallins. Exp Eye Res 31:41, 1980. 17. Towbin H, Staehelin T, and Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76: 4350, 1979. 18. Garner WH and Spector A: A preliminary study of the dynamic aspects of age dependent changes in the abundances of human lens polypeptides. Doc Ophthalmol Proc Series 18: 91, 1979. 19. Slingsby C and Miller LR: Purification and crystallization of mammalian lens 7-crystallins. Exp Eye Res 37:517, 1983. 20. Russell P, Zigler JS Jr, and Reddy V: The development of a monoclonal antibody to a human 7-crystallin. Curr Eye Res 3:1329, 1984. 21. Takemoto LJ, Hansen JS, Zigler JS Jr, and Horwitz J: Characterization of polypeptides from human nuclear cataracts by Western blot analysis.. Exp Eye Res (In press)