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Differential synthesis and degradation of protein in the hereditary Philly mouse cataract
Exp. Eye Res. (1980)
30, 69-78
Differential Synthesis and Degradation of Protein in the Hereditary Philly Mouse Cataract JORAM
Xectiox
on
National
Cellular Institute
PIATIGORSKY
Differentiation, Laboratory of Molecular Genetics, of Child Health and Hum.an Development
PETER F. KADOR AXD Xection
on
Biochemistry, National
JIN H. KINOSHITA
Lahoratory of Vision Resecxrch, Xational Eye Institute, of Henlth, Bethesda, Md 20205, U.S.A.
Institutes
(Received 19 June 1979, New York) Lens protein metabolism was investigated in the Philly mouse between the third and eighth postnatal week. As demonstrated in an accompanying article, the Philly mouse develops a hereditary, osmotic, cataract associated with influx of P;a+ and loss of K+ during this time interval. The contents of ,8- and y-crystallin were strikingly reduced in the Philly lens, as gel electrophoresis ancl by judged b;V sodium dodecyl sulfate (SDS)- urea-polyacrylamide immunodiffusion. This appeared to be due to proteolysis, since there were negligible amounts of crystallins found in the medium of cultured Philly lenses. x-Crystallin remained in the Philly lens but apparently accumulated discrete polypeptide cleavage products. The incorporation of [W]methionine into p- and y-crystallin polypeptides was markedly reduced in the Philly lens. By contrast,, the incorporation of [3SS]methionine into the cr-crystallin and the higher molecular weight non-crystallin polypeptides was as great, if not greater, in the Philly lens than in the normal lens. The non-crystallin polypeptides were associated with t,he 10 OOOxg pellet of the homogenate. The present data extend the correlation between alterations in protein metabolism and electrolyte concentrations to t’his hereditary cataract, and support the idea that selective degradation of crystallins and differential reduction in the synthesis of crystallins are primary causes for the lowered amounts of soluble proteinespecially p- and y-crystallin-found in cataracts associated with ionic imbalances. Key lords: hereditary cataract; Philly mouse lens; protein synthesis; protein degradation; ions; P\‘a+, I(+; lens crystallins.
1. Introduction Recently,
it has been shown that protein metabolism may be altered in osmotic (Piatigorsky, Fukui and Kinoshita, 1978; Kador: Zigler and Kinoshita, 1979). Experiments with cultured embryonic chick lenses have correlated selective alterations in crystallin synthesis with changes in ion concentration (particularly Na+ and K+) (Shinohara and Piatigorsky, 1977), suggesting that changes in electrolyte concentration may be resppnsible for the alterations in crystallin synthesis during cataractogenesis (Piatigorsky, 1979). I n addition to the differential changes in crystallin synthesis, differential degradation and leak-out of crystallins have also been correlated with ion imbalances in cataractous lenses (Piatigorsky et al., 1978; Kador et al., 1979). We explore here the composition, synthesis and leakage of lens proteins in the newly-bred Philly mouse described in the accompanying article, which develops a hereditary cataract associated with Na+ influx and K+ efflux (Kador, Fukui, Fukushi, Jernigan and Kinoshita, 1980). The results extend the correlation between alterations in ion concentrations and protein metabolism to t,his osmotic cataract. cat)arects
0014-4S35/80/010069+10
$01.00/O
0 1980 Academic 69
Press Inc. (London)
Limited
70
J. PIATIGORSKY,
Y. P. KADOR
2. Materials
ASD
J. H. KIYOSHITA
and Methods
Normal Swiss-Webster mice were obtained from the National Institutes Philly mice were of Swiss-Webster derivation (Kador et al., 1950). Lnheliny
of Hea1t.h.
of lenses
Lenses were removed surgically from normal or Phillp mice at the specified times after birth and placed into 0.7 ml of TC-199 medium made in bicarbonate bufEer containing 30 Inn%-fructose (Obazawa, Merola and Kinoshita, 1974) ; 500 ,uCi of [YQnethionine (New England Nuclear Corp, 581 Ci/mmol) was added to the medium. The lenses were incubated in groups of four in 16 mm diameter wells (tissue culture cluster dishes, Costar) for 5 hr at 37°C in a humidified environment of 5% CO, and 95% air. The labelled lenses and media were stored at -20°C before analysis.
Two lenses from each time point were homogenized in 1 ml of 0.01 &r-Tris, pH 7.8. Aliquots of 0.25 ml of the homogena.tes were mixed with an equal volume of 2% sodium dodecyl sulfate (SDS), 2% ,&nercaptoethanol, 20% glycerol, 0.25% phenol red, 8 &f-urea (Schwarz/Mann, ultrapure) and 1.2% Tris-HCl, pH 6.8, and heated to 100°C for 2 min. The remainder of the homogenate was centrifuged at 10 OOOxg for 10 min at 4°C. The supernatant factions were saved for immunological analysis. The pellet’s were washed three times with 1 ml of 0.01 x-sodium phosphate, pH 6.4, by centrifugation at 10 000 x g and dissolved in 0.1 ml of 1% SDS, 1% /3-mercaptoethanol, 10% glycerol, O.lq/, phenol red, 4 M-urea and 0.6% Tris-HCl, pH 6.8; by heating at 100°C for approximately 5 min.
Double immunodiffusion tests were performed in agar gels (Hyland, Travenol Laboratories, Inc. and Miles Laboratories, Inc.) overnight at 4°C. The gels were washed for 3 to 6 days at room temperature with 0.15 al-NaCl, stained with 0.01% Coomassie Brilliant Blue R for 1 hr and destained wit,11 5% acetic acid and 5% methanol. Rabbit antisera were against mouse a- and y-crystallin and against calf /3-crystallin. The crystallins were purified by gel filtration (Sephadex G-200) before being used as antigens. The anti-ccand y-crystallin antisera were a gift of MS D. A. Carper and the anti-@rystallin antiserum was a gift from Dr P. Russell. Immunoprecipitation in solution proceeded at 4°C over several days. The precipitates were washed by centrifugation three times either with 0.01 M-sodium phosphate, pH 6.4, or with Dulbecco’s salt solution containing 2% Triton X-100, and dissolved in 0.1 ml of 1% /3-mercaptoethanol, lo”/, glycerol, 4 x-urea, 0.1% phenol red and 0.6% Tris-HCl, pH 6.8, by heating to 100°C for 2-3 min. The immunoprecipitates were analyzed by SDSAn example of Dhe specificity of the immunourea-polyacrylamide gel electrophoresis. precipitation reactions is shown in Fig. 1. Electrophoresis
of proteins
Proteins (1 to 1Opg) were analyzed by discontinuous electrophoresis in 10% polyacrylamide gel slabs at pH 8.8 which contained O.lo/, SDS and 8 M-urea. A 4% polyacryla,mide stacking gel at pH 6.8 in 0.1% SDS and 8 If-urea overlay the separat,ing gel. Electrophoresis was performed at room temperature for 4-5 hr at loo-125 V. The gels were stained with 0.01% Coomassie Brilliant Blue R, dried under vacuum onto Whatman filter paper and autoradiographed with Kodak SB5 X-ray film. Purther details can be found elsewhere (Reszelbach, Xhinohara and Piatigorsky, 1977).
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Total
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c[
FIG. 1. Immunoprecipitation of [3jS]~~lethionine-labeled crystallins from the 13-day-old mouse lens. The immunoprecipitates were analyzed by electrophoresis in a polyacrylamide gel followed by autoradiography. Total represents an aliquot of the lens homogenate.
3. Results We have examined the composition and synthesis of lens proteins in normal and Philly mice between the ends of the third and seventh postnatal weeks. During this time interval the lenses progressfrom an initial faint anterior subcapsular opacity, as seenby slit lamp, to a densenuclear cataract (Kador et al., 1980). Protein cornpositiorh As described in the accompanying article, the Philly mouse lensesweighed eonsiderably lessthan the normal lensesat all stagesexamined. The avera.gewet weights of the lensesused in the present experiments were 4~0mg (21 days), 4.1 mg (28 days), 5.2 mg (42 days) and 6.0 mg (49 clays) for the normal mice, and 2.4 mg (19 clays), 2.9 mg (30 days), 3.9 mg (43 days) and 3.3 mg (50 days) for the Philly m.ice. To investigate the possible basis for the reduced wet weights of the Philly lenses,the proteins of the normal and the Philly lenseswere analyzed by immunodiffusion and by SDS-urea-polyacrylamide gel electrophoresis. Although immunodiffusion is more qualitative than quantitative, inspection of the results suggestedthat the amount of soluble cc-crystallin was not appreciably different in the 49-da,y-old normal and the 50-day-old Philly lens, as judged by the apparent, staining intensities of the immunoprecipitin bands [Fig. 2(a), (b)]. In contrast to the results with a-crystallin, the Philly lens appeared to have much less soluble /3- and y-crystallin tha,n the normal lens. The staining intensities of the P-crystallin bands
72
J. PIATIGORSKY,
P. F. KADOR
AND
J. H. KINOSHITA
FIG. 2. Immunodiffusion of a-, j5’- and y-crystallin of normal [(a), (c)] and Phiily [(b), jd)l lens homogenates. (a), (b) Equal volumes (22 ~1) of antiserum and soluble protein were placed in the wells. The center wells contained the soluble protein and the outside wells the antisera. (c), (d) Equal volumes (8 ~1) of anti-y-crystallin antiserum and dilutions of soluble protein were used. The center well contained the anti-y-crystallin antiserum and t.he outside wells the soluble protein. The dilutions are indicated on the photograph.
were much cgreater in the normal than in the Philly lens extract when equal volumes of material were compared. This immunodiffusion test was performed with an antiserum to calf fin-crystallin, which immunoprecipitates both fin- and /3,-crystallins (Zigler and Sidbury, 1976). In addition to the reduced amount of precipitation with anti-/3,-crystallin antiserum, the immunoprecipitin bands derived from the Philly lens extract were more diffuse than those derived from the normal lens extract, suggesting that the remaining P-crystallins were partially degraded in the Philly lens. The immunodiffusion experiments also suggested a marked decrease in y-crystallin in t’he Philly lens. There was no visible, stained y-crystallin immunopreeipitin band in the Philly lens extract in this test [Fig. 2(b)], while the normal lens extract showed a diffuse immunoprecipitin band for y-erystallin near the well containing the anti-ycrystallin antiserum. Another test for y-crystallin was performed on a smaller agar gel using dilutions of the original extracts. A ma,rked y-crystallin immunoprecipitin band wa#s obtained with the normal lens extract with all the dilutions utilized [Pig. 2(c)]. Maximum immunoprecipitation took place in the normal lens extract which was diluted 1 : 32. A fuzzy immunoprecipitin band was present with the undiluted Philly lens extract, although its position in the gel makes it unclear whether or not this represented y-crystallin [Fig. 2(d)]. Th ere was no reaction with the diluted Philly lens extract. Xaximum reactions for cc- and P-crystallin occurred in the undiluted (data not shown). Apart from extracts for both the Philly and normal lens extracts the differences in ,& and y-crystallin, Fig. 2 shows that the normal lens extracts had greater amounts of protein which remained near the well of the washed gels than did the Philly lens extracts, indicating further differences between normal and Philly lens proteins. Similar results were obtained when comparing the soluble proteins of 43-day-old Philly lenses with those of 42-day old normal lenses; except that the differences in the relative amounts of p- and y-crystallin in the Philly and normal lens were less than that observed in the older mice (data not shown).
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Analysis of total homogenates by SDS-urea-polyacrylamide gel electrophoresis also showed a reduction in ,8- and y-crystallin polypeptides in. the Philly lens [Fig. 3(a)]. Particularly striking was the absence of the higher molecular weight P-crystallin polypeptides. These belong to the &r-crystallins (Herbdnk and Bloemendal, 19i4; Zigler and Sidbury, 19’76). The deficiency in the P-crystallin components was already evident in the 19-day-old Philly lens, which was still clear. By 43 days, the higher Normal 2ld
28d
42d
Philly 49d
193
30d
43d
50d
FIG. 3. SDS-urea-polyacrylalllide gel electrophoresis of [35S]methionine labeled proteins in normal and Philly lenses. (a) Etaining pattern. (b) Autoradiogram. The lenses were labeled for 6 hr with [WImethionine before analysis. Two lenses from each age were homogenized in 1 ml of buffer and 20 ~1 of each homogenate was subjected to electrophoresis. Kate the extra bands of protein in the Philly lens; these appear to be degradation products since they are not radioactively labeled. The three arrows in (b) point to the labeled crystallin bands in the Philly lens which are cc-crystallin polypeptides as judged by immunoprecipitation (see Fig. 4).
74
J. PIATIGORSKY,
P. F. KADOR Normal
AKD
J. H. KINOSHITA
Philly
i FIG. i. SDS-area-polyscrylamide protein of the 4%clay-old normal and with [%]methionine before analysis. aud white bands on a black background protein bands which were not labeled
gels of a-crystallin immunopreoipitates derived from the soluble the &day-old Philly mouse lens. The lenses were labeled for 5 hr The bla.ck bands on a white background a.re the staiuing pattern are the autoradiogram. The arrows depict the immunoprecipitated and are assumed to represent degradation products of a-crystallin.
molecular weight /%crystallin polypeptides were not visible and only trace amounts, if any, of the y-crystallin polypeptides were present in the stained gels of the Philly lens proteins. At 43 days, the Philly lens had a nuclear opacity. In addition to protein loss, inspection of the gels of the total homogenates suggested that the 43- and 50-day-old Philly lenses accumulated several bands of protein in the crystallin region which were not present in the normal lenses [Pig. 3(a)]. These Philly bands were more easily visualized after precipitation with anti-cc-crystallin antiserum (Fig. 4). The fact that these bands inmnmoprecipitated with anti-oc-crystallin antiserum suggests that the were cleavage products of cc-crystallin. Further work is recpnred in order to establish the nature of these Philly polypeptides. Lens protein qnthesis The pattern of protein synthesis was also different in the lenses of normal and Philly mice. Incorporation of [3%]methionine into the higher molecular weight, noncrystallin polypeptides was unaffected, if not accentuated, in the Philly lens [Fig. 3(b)]. In a separate test, these non-crystallin polypeptides were shown to be associated with the 10 000 x g pellet of the homogenate (Fig. 5). In cont’rast to the incorporation of [a%]methionine into the noncrystallin pofypeptides, incorporation into the lower molecular weight erystallin polypeptides was generally less in the Philly lens than in the normal lens. There was, however, some [35S]methionine incorporationinto crystallin polypeptides of the Philly lens (see below). The amounts of radioactivity present in
PROTEIN
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the crystallin and non-crystallin polypeptides of the normal and Philly lens were not diminished after a 24 hr chase in non-radioactive medium, suggesting that the lens proteins were not turning-over rapidly during the labeling time (5 hr) in these experiments. Thus; the differences in labeling appear to be due to differences in synthesis.
FIG. 5. Autoradiogram of an SDS-urea-polyacrylamide gel of [35S]methionine supernatant fraction and the pellet of a 49-day-old Philly lens homogenate 10 000 x g for 10 min. The pellet represents the total proteins from the pellet of represents the proteins from 5% of the soluble protein of a lens. Similar results normal lens (not shown).
labeled proteins in the after centrifugation at one lens: the supernate vere obtained with the
Most of the [35S]methionine incorporated into the crystallins of the Philly lens was into three bands denoted by arrows in Pig. 3(b). These were a-crystallin polypeptides, as judged by an immunoprecipitation test (Fig. 4). Interestingly, in one test with the 42-day-old lens and two tests with the 49-day-old lens there was approximately twice as much [35S]methionine associated with the m.-crystallin bands which immunoprecipitated with anti-u.-crystallin antiserum in the Philly lens than in the normal lens of the same age. By contrast, only trace amounts of radioactivity precipitated with anti-p- or anti-y-crystallin antiserum in tests conducted with the Philly lens (data not shown).
76
J. PIATIGORSKY,
P. F. KADOR
AND
J. H. KINOSHITA
Finally, it should be noted that several of the Philly bands which immunoprecipitated with anti-or-crystallin antiserum were unlabeled (Fig. 4, arrows). This is. consistent with the interpretation (see above) that these bands contain partially degraded crystallin polypeptides which were synthesized before the lens were la,beled with [35S]n~etllionine. Some unlabeled polypeptides present in this region of the gel also precipitated with anti-/3-crystallin antiserum (data not shown). It is unclear whether these are cleavage products since there is little synthesis of P-crysta,llin in the Philly lens. Lens proteirl
leakage
The medium of cultured normal and Philly lensesfrom each of the different a,ges examined was assayedby SDS-urea-polyacrylamide gel electrophoresis for the presence of proteins which m.ay have escapedfrom the lens. Negligible amounts of crystallins were found in the medium after 5 or 24 hr of culture (data not shown). Sutoradiography of the gel revealed, however. that someradioactively labeled polypeptides with higher molecular weights than the crystallin polypeptides leaked from the normal and the Philly lensesunder these culture conditions, as has been reported elsewhere(Piatigorsky et al., 1978). 4. Discussion The present results show that protein synthesis and degradation are markedly affected in the Philly cataract. Since the concentration of NaT increasesand that of KT decreasesin the Philly lens (Kador et al., 1980), this hereditary cataract provides another example of a correlated change in protein metabolism and electrolyte concentration. Other reports in which such a correlation has been made include the cultured, vitreous-free (Piatigorsky and Shinohara, 1977)and ouabain-treated (Shinohara. and Piatigorsky, 1977) embryonic chick lens, the Nakano and ouabain-treated mouse lens (Piatigorsky et al.: 1978), the galactose-fed rat lens (Kador et al., 1980) and the U18666A-treated (cholesterol biosynthesis inhibited) rat lens (Cenedella and Bierkamper, 1979). None of these experiments, including the present ones,has established that Na+ and Kf are causally or directly related to the alterations in protein metabolism in these lenses,or that the samefactors are responsiblefor the changesin protein synthesis in the different lenses. In addition to Naf and K+, experiments suggest that Cl- may be involved in the protein synthesis alterations in lenses containing ionic imbalances. Cl- inhibits mRNA translation in several eukaryotic cell-free systems (Weber, Hickey, Maroney and Baglioni, 1977). Cl- and acetate, as well as Na+ and K+, also both affect the ratio of the &crystallin polypeptides synthesized in a rabbit reticulocyte cell-free system prim.ed with purified 6-crystallin mRNA from the embryonic chick lens (Piatigorsky et al., 1979; Shinohara and Piatigorsky, in preparation). Moreover, Cl- has been shown to increase in lensesof rabbits (Kinoshita, 1974) and embryonic chicks (Shinohara and Piatigorsky, in preparation) treated with ouabain, and in cataractous lensesof rats fed with galactose (Kinoshita and Merola, 1964; Kinsey and Hightower, 1978). Comparison of the changesin protein metabolism which take place in Philly lenses with those that occw in other cataracts associatedwith ionic imbalances showsboth common features and individual differences. In general, cataracts with altered ionic compositions have reduced contents of soluble protein (see Dische, 1966; Barber, 1973; Harding and Dilley, 1976; Piatigorsky, 1979). The greatest reductions are in
PROTEIN
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the /l- and y-crystallins. A partial explanation for the lowered amount of soluble protein appears to be the differential inhibition of crystallin synthesis which correlates with the alterations in ion concentrations. In Nakano mouse (Piatigorsky et al., 1978) and galactose-fed rat (Kador et al., 1980) cataracts, c(-, p- and y-crystallin synthesis are nearly arrested. By contrast, in hypocholesteremic rat cataracts (Cendella and Bierkamper, 1979), y-crystallin synthesis is selectively reduced, and in Philly mouse cataracts (this report) /3- and y-crystallin synthesis are differentially inhibited. The reason for these discrepancies is not known. One possibility is that the changes in ion concentration are compartmentalized within different regions of the different lenses. The onset of the differential reductions in protein synthesis is a,lso not the same in each case. For example, the loss of crystallin synthesis occurs later in the Nakano cataract than it does in the galactose or Philly cataracts. Leakage and degradation of crystallins also contribute to the reduction in soluble protein in cataracts with altered concentrations of electrolytes. Considerable amounts of y-crystallin, some /2-crystallin and traces of a-crystallin were found in the medium of cultured lenses from Nakano mice (Piatigorsky et al., 1.978) and galactose-fed rats {Kador et al., 1980). Radioimmunoassay of the aqueous humor in r\‘akano mice has established that y-crystallin escapes from the catara,ctous lens in vivo (Russell et al.; 1979), indicating that leak-out is an avenue of protein loss in this cataract. Approximately 90% of the y-erystallin is lost from the Nakano lens (Russell et al., 1978). Surprisingly, crystallin leakage was not detected in the cultured Philly lens in the present investigation, suggesting a fundamental difference in the state of the cell surface membranes between the Philly and the Nakano or galactose cataracts. It remains possible that erystallins do leak from the Philly lens along with proteases and, consequently, that the crystallins are degraded in the medium. This seems unlikely, however, since the labeled higher molecular weight polypeptides in the medium did not appear degraded. The apparent absence of crystallin leaka,ge in the Philly lens suggests that degradation is a major pathway of crystallin loss in this cataract. The reduction in P-crystallins is particularly striking in the Philly cataract a,nd exceeds that observed in the Nakano (Piatigorsky et al., 1978) and galactose (Kador et al., 1980) cataract. Partial cleavage products of crystallin polypeptides have been noted in Nakano (Piatigorsky et al., 1978) and Philly (this report) lenses. These appear to be derived from CI- and possibly /3crystallin polypeptides. Experiments with ouabain have suggested that the partial degradation of c(- and /3-crystallin may be related to the changes in ion concentrations within the lens. Further discussion of the changes in protein metabolism in cataractous lenses can be found elsewhere (Piatigorsky, 1979). In conclusion, then, the present results extend the correlation between ionic imbalances and alterations in protein metabolism to the Philly cataract, and indicate that the reduced content of soluble protein in this hereditary cataract is due both to the differential reduction of synthesis and to degradation of ,& and y-crystallin. We do not know if the changes in protein content and metabolism described here are related to opacification in the Philly lens, but we assume that they must lead to deterioration of the lens and the development of an irreversible cataract. ACKNOWLEDGMENTS
We thank Dr J. S. Zigler, Jr. and MS D. A. Carper for a gift of the crystallin antisera, and MS T. Broderick for expert typing.
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REFERENCES Barber, G. W. (1973). Human cataractogenesis: A review. Exp. Eye Res. 16, 85-94. Cenedella, R. J. and Bierkamper, G. B. (1979). Mechanism of cataract production by 3-p (2diethylaminoethoxy) androst-5-en-17-one hydrochloride, U18666A: An inhibitor of cholesterol biosynthesis. Exp. Eye Res. 28, 673-88. Dische, Z. (1966). Alterations of lens proteins as etiology in cataracts. In Biochemistry of the Eye. Symp. Tutzing Castle, pp. 413-28. Karger, Basel, New York. Fukui, H. N., Merola, L. 0. and Kinoshita, J. H. (1978). A possible cataractogenic factor in the NTakano mouse lens. Exp. Eye Res. 26,477-85. Harding, J. J. and Dilley, K. J. (1976). Structural proteins of the mammalian lens: A review with emphasis on changes in development, aging, and cataract. Exp. Eye Res. 22, l-73. Herbrink, P. and Bloemendal, H. (1974). Studies on/?-crystallin. I. Isolation and part’ial characterization of the principal polypeptide chain. Biochim. Biophys. Acta 336, 370-82. Kador, P. F., Fukui, H. N., Fukushi, S., Jernigan, H. M. and Kinoshita, J. H. (1980). Philly mouse: A new model of hereditary cataract. Exp. Eye Res. 30, 59-68. Kador, P. F., Zigler, J. S., Jr. and Kinoshita, J. H. (1979). Alteration of protein synthesis in galactosemic rats. Invest. Opthalmol. Vis. Xci. 18, 696-702. Kinoshita, J. H. (1974). Mechanisms initiating cataract formation. Invest. Ophthabnol. 13,713-24. Kinoshita, J. H. and Merola, L. 0. (1964). Hydration of the lens during the development of galactose cataract. Invest. Ophthalmol. 3, 577-84. Kinsey, V. E. and Hightower, K. R. (1978). Studies on the crystalline lens. XXVII. Kinetic and bioelectric measurements of galactose cataracts in rats. Exp. Eye Res. 26, 521-8. Obazawa, H., Merola. L. 0. and Kinoshita, J. H. (1974). The effects of xylose on the isolated lens. Invest. Ophthalmol. 13, 204-9. Piatigorsky, J. (1979). Intracellular ions, protein metabolism and cataract formation. In Current Topics of Eye Research, Vol. 3 (Eds Zadunaisky, J. A. and Davson, H.). Academic Press, Inc., New York and London (in press). Piatigorsky, J., Fukui, H. N. and Kinoshita, J. H. (1978). Differential metabolism and leakage of protein in an inherited cataract and a normal lens cultured with ouabain. hlature (Lordox) 274,558-62. Piatigorsky, J. and Shinohara, T. (1977). Lens cataract formation and reversible alteration in crystallin synthesis in cultured lenses. Xcielzce 196, 1345-7. Piatigorsky, J., Shinohara, T., Bhat, S. P., Reszelbach, R., Jones, R. L. and Sullivan, M. A. (1979). Correlated changes in &crystallin synthesis and ion concentrations in the embryonic chick lens: Summary, current experiments and speculations. Ann. N. Y. Acad. if&. (in press). Reszelbach, R., Shinohara, T. and Piatigorsky. J. (1977). Resolution of two distinct embryonic gel electrophoresis in ‘the presence of sodium chick 6crystallin bands by polyacrylamide dodecyl sulfate and urea. Exp. Eye Res. 25, 583-93. Russell, P., Carper, D. and Kinoshita, J. H. (1978). The development and application of a radioimmunoassay to lens crystallins. Exp. Eye Res. 27, 673-80. Russell, P., Smith, S. G., Carper, D. A. a,nd Kinoshita, J. H. (1979). Age and cataract-related changes in the heavy molecular weight proteins and gamma crystallin composition of the mouse lens. Exp. Eye Res. 29, 245-55. Shinohara, T. and Piatigorsky, J. (1977). Regulation of protein synthesis, intracellular electrolytes and cataract formation in vitro. Nature (London) 270,406-11. Weber, L. A., Hickey, E. D., Maroney, P. A. and Baglioni, C. (1977). Inhibition of protein synthesis by Cl-. J. Bid. Chem. 252,4007-10. Zigler, J. S., Jr. and Sidbury, J. B., Jr. (1976). A Comparative study of /%cryst,allin from six mammals. Camp. Biochem. Physiol. 53B, 349-55.
30, 69-78
Differential Synthesis and Degradation of Protein in the Hereditary Philly Mouse Cataract JORAM
Xectiox
on
National
Cellular Institute
PIATIGORSKY
Differentiation, Laboratory of Molecular Genetics, of Child Health and Hum.an Development
PETER F. KADOR AXD Xection
on
Biochemistry, National
JIN H. KINOSHITA
Lahoratory of Vision Resecxrch, Xational Eye Institute, of Henlth, Bethesda, Md 20205, U.S.A.
Institutes
(Received 19 June 1979, New York) Lens protein metabolism was investigated in the Philly mouse between the third and eighth postnatal week. As demonstrated in an accompanying article, the Philly mouse develops a hereditary, osmotic, cataract associated with influx of P;a+ and loss of K+ during this time interval. The contents of ,8- and y-crystallin were strikingly reduced in the Philly lens, as gel electrophoresis ancl by judged b;V sodium dodecyl sulfate (SDS)- urea-polyacrylamide immunodiffusion. This appeared to be due to proteolysis, since there were negligible amounts of crystallins found in the medium of cultured Philly lenses. x-Crystallin remained in the Philly lens but apparently accumulated discrete polypeptide cleavage products. The incorporation of [W]methionine into p- and y-crystallin polypeptides was markedly reduced in the Philly lens. By contrast,, the incorporation of [3SS]methionine into the cr-crystallin and the higher molecular weight non-crystallin polypeptides was as great, if not greater, in the Philly lens than in the normal lens. The non-crystallin polypeptides were associated with t,he 10 OOOxg pellet of the homogenate. The present data extend the correlation between alterations in protein metabolism and electrolyte concentrations to t’his hereditary cataract, and support the idea that selective degradation of crystallins and differential reduction in the synthesis of crystallins are primary causes for the lowered amounts of soluble proteinespecially p- and y-crystallin-found in cataracts associated with ionic imbalances. Key lords: hereditary cataract; Philly mouse lens; protein synthesis; protein degradation; ions; P\‘a+, I(+; lens crystallins.
1. Introduction Recently,
it has been shown that protein metabolism may be altered in osmotic (Piatigorsky, Fukui and Kinoshita, 1978; Kador: Zigler and Kinoshita, 1979). Experiments with cultured embryonic chick lenses have correlated selective alterations in crystallin synthesis with changes in ion concentration (particularly Na+ and K+) (Shinohara and Piatigorsky, 1977), suggesting that changes in electrolyte concentration may be resppnsible for the alterations in crystallin synthesis during cataractogenesis (Piatigorsky, 1979). I n addition to the differential changes in crystallin synthesis, differential degradation and leak-out of crystallins have also been correlated with ion imbalances in cataractous lenses (Piatigorsky et al., 1978; Kador et al., 1979). We explore here the composition, synthesis and leakage of lens proteins in the newly-bred Philly mouse described in the accompanying article, which develops a hereditary cataract associated with Na+ influx and K+ efflux (Kador, Fukui, Fukushi, Jernigan and Kinoshita, 1980). The results extend the correlation between alterations in ion concentrations and protein metabolism to t,his osmotic cataract. cat)arects
0014-4S35/80/010069+10
$01.00/O
0 1980 Academic 69
Press Inc. (London)
Limited
70
J. PIATIGORSKY,
Y. P. KADOR
2. Materials
ASD
J. H. KIYOSHITA
and Methods
Normal Swiss-Webster mice were obtained from the National Institutes Philly mice were of Swiss-Webster derivation (Kador et al., 1950). Lnheliny
of Hea1t.h.
of lenses
Lenses were removed surgically from normal or Phillp mice at the specified times after birth and placed into 0.7 ml of TC-199 medium made in bicarbonate bufEer containing 30 Inn%-fructose (Obazawa, Merola and Kinoshita, 1974) ; 500 ,uCi of [YQnethionine (New England Nuclear Corp, 581 Ci/mmol) was added to the medium. The lenses were incubated in groups of four in 16 mm diameter wells (tissue culture cluster dishes, Costar) for 5 hr at 37°C in a humidified environment of 5% CO, and 95% air. The labelled lenses and media were stored at -20°C before analysis.
Two lenses from each time point were homogenized in 1 ml of 0.01 &r-Tris, pH 7.8. Aliquots of 0.25 ml of the homogena.tes were mixed with an equal volume of 2% sodium dodecyl sulfate (SDS), 2% ,&nercaptoethanol, 20% glycerol, 0.25% phenol red, 8 &f-urea (Schwarz/Mann, ultrapure) and 1.2% Tris-HCl, pH 6.8, and heated to 100°C for 2 min. The remainder of the homogenate was centrifuged at 10 OOOxg for 10 min at 4°C. The supernatant factions were saved for immunological analysis. The pellet’s were washed three times with 1 ml of 0.01 x-sodium phosphate, pH 6.4, by centrifugation at 10 000 x g and dissolved in 0.1 ml of 1% SDS, 1% /3-mercaptoethanol, 10% glycerol, O.lq/, phenol red, 4 M-urea and 0.6% Tris-HCl, pH 6.8; by heating at 100°C for approximately 5 min.
Double immunodiffusion tests were performed in agar gels (Hyland, Travenol Laboratories, Inc. and Miles Laboratories, Inc.) overnight at 4°C. The gels were washed for 3 to 6 days at room temperature with 0.15 al-NaCl, stained with 0.01% Coomassie Brilliant Blue R for 1 hr and destained wit,11 5% acetic acid and 5% methanol. Rabbit antisera were against mouse a- and y-crystallin and against calf /3-crystallin. The crystallins were purified by gel filtration (Sephadex G-200) before being used as antigens. The anti-ccand y-crystallin antisera were a gift of MS D. A. Carper and the anti-@rystallin antiserum was a gift from Dr P. Russell. Immunoprecipitation in solution proceeded at 4°C over several days. The precipitates were washed by centrifugation three times either with 0.01 M-sodium phosphate, pH 6.4, or with Dulbecco’s salt solution containing 2% Triton X-100, and dissolved in 0.1 ml of 1% /3-mercaptoethanol, lo”/, glycerol, 4 x-urea, 0.1% phenol red and 0.6% Tris-HCl, pH 6.8, by heating to 100°C for 2-3 min. The immunoprecipitates were analyzed by SDSAn example of Dhe specificity of the immunourea-polyacrylamide gel electrophoresis. precipitation reactions is shown in Fig. 1. Electrophoresis
of proteins
Proteins (1 to 1Opg) were analyzed by discontinuous electrophoresis in 10% polyacrylamide gel slabs at pH 8.8 which contained O.lo/, SDS and 8 M-urea. A 4% polyacryla,mide stacking gel at pH 6.8 in 0.1% SDS and 8 If-urea overlay the separat,ing gel. Electrophoresis was performed at room temperature for 4-5 hr at loo-125 V. The gels were stained with 0.01% Coomassie Brilliant Blue R, dried under vacuum onto Whatman filter paper and autoradiographed with Kodak SB5 X-ray film. Purther details can be found elsewhere (Reszelbach, Xhinohara and Piatigorsky, 1977).
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c[
FIG. 1. Immunoprecipitation of [3jS]~~lethionine-labeled crystallins from the 13-day-old mouse lens. The immunoprecipitates were analyzed by electrophoresis in a polyacrylamide gel followed by autoradiography. Total represents an aliquot of the lens homogenate.
3. Results We have examined the composition and synthesis of lens proteins in normal and Philly mice between the ends of the third and seventh postnatal weeks. During this time interval the lenses progressfrom an initial faint anterior subcapsular opacity, as seenby slit lamp, to a densenuclear cataract (Kador et al., 1980). Protein cornpositiorh As described in the accompanying article, the Philly mouse lensesweighed eonsiderably lessthan the normal lensesat all stagesexamined. The avera.gewet weights of the lensesused in the present experiments were 4~0mg (21 days), 4.1 mg (28 days), 5.2 mg (42 days) and 6.0 mg (49 clays) for the normal mice, and 2.4 mg (19 clays), 2.9 mg (30 days), 3.9 mg (43 days) and 3.3 mg (50 days) for the Philly m.ice. To investigate the possible basis for the reduced wet weights of the Philly lenses,the proteins of the normal and the Philly lenseswere analyzed by immunodiffusion and by SDS-urea-polyacrylamide gel electrophoresis. Although immunodiffusion is more qualitative than quantitative, inspection of the results suggestedthat the amount of soluble cc-crystallin was not appreciably different in the 49-da,y-old normal and the 50-day-old Philly lens, as judged by the apparent, staining intensities of the immunoprecipitin bands [Fig. 2(a), (b)]. In contrast to the results with a-crystallin, the Philly lens appeared to have much less soluble /3- and y-crystallin tha,n the normal lens. The staining intensities of the P-crystallin bands
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FIG. 2. Immunodiffusion of a-, j5’- and y-crystallin of normal [(a), (c)] and Phiily [(b), jd)l lens homogenates. (a), (b) Equal volumes (22 ~1) of antiserum and soluble protein were placed in the wells. The center wells contained the soluble protein and the outside wells the antisera. (c), (d) Equal volumes (8 ~1) of anti-y-crystallin antiserum and dilutions of soluble protein were used. The center well contained the anti-y-crystallin antiserum and t.he outside wells the soluble protein. The dilutions are indicated on the photograph.
were much cgreater in the normal than in the Philly lens extract when equal volumes of material were compared. This immunodiffusion test was performed with an antiserum to calf fin-crystallin, which immunoprecipitates both fin- and /3,-crystallins (Zigler and Sidbury, 1976). In addition to the reduced amount of precipitation with anti-/3,-crystallin antiserum, the immunoprecipitin bands derived from the Philly lens extract were more diffuse than those derived from the normal lens extract, suggesting that the remaining P-crystallins were partially degraded in the Philly lens. The immunodiffusion experiments also suggested a marked decrease in y-crystallin in t’he Philly lens. There was no visible, stained y-crystallin immunopreeipitin band in the Philly lens extract in this test [Fig. 2(b)], while the normal lens extract showed a diffuse immunoprecipitin band for y-erystallin near the well containing the anti-ycrystallin antiserum. Another test for y-crystallin was performed on a smaller agar gel using dilutions of the original extracts. A ma,rked y-crystallin immunoprecipitin band wa#s obtained with the normal lens extract with all the dilutions utilized [Pig. 2(c)]. Maximum immunoprecipitation took place in the normal lens extract which was diluted 1 : 32. A fuzzy immunoprecipitin band was present with the undiluted Philly lens extract, although its position in the gel makes it unclear whether or not this represented y-crystallin [Fig. 2(d)]. Th ere was no reaction with the diluted Philly lens extract. Xaximum reactions for cc- and P-crystallin occurred in the undiluted (data not shown). Apart from extracts for both the Philly and normal lens extracts the differences in ,& and y-crystallin, Fig. 2 shows that the normal lens extracts had greater amounts of protein which remained near the well of the washed gels than did the Philly lens extracts, indicating further differences between normal and Philly lens proteins. Similar results were obtained when comparing the soluble proteins of 43-day-old Philly lenses with those of 42-day old normal lenses; except that the differences in the relative amounts of p- and y-crystallin in the Philly and normal lens were less than that observed in the older mice (data not shown).
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Analysis of total homogenates by SDS-urea-polyacrylamide gel electrophoresis also showed a reduction in ,8- and y-crystallin polypeptides in. the Philly lens [Fig. 3(a)]. Particularly striking was the absence of the higher molecular weight P-crystallin polypeptides. These belong to the &r-crystallins (Herbdnk and Bloemendal, 19i4; Zigler and Sidbury, 19’76). The deficiency in the P-crystallin components was already evident in the 19-day-old Philly lens, which was still clear. By 43 days, the higher Normal 2ld
28d
42d
Philly 49d
193
30d
43d
50d
FIG. 3. SDS-urea-polyacrylalllide gel electrophoresis of [35S]methionine labeled proteins in normal and Philly lenses. (a) Etaining pattern. (b) Autoradiogram. The lenses were labeled for 6 hr with [WImethionine before analysis. Two lenses from each age were homogenized in 1 ml of buffer and 20 ~1 of each homogenate was subjected to electrophoresis. Kate the extra bands of protein in the Philly lens; these appear to be degradation products since they are not radioactively labeled. The three arrows in (b) point to the labeled crystallin bands in the Philly lens which are cc-crystallin polypeptides as judged by immunoprecipitation (see Fig. 4).
74
J. PIATIGORSKY,
P. F. KADOR Normal
AKD
J. H. KINOSHITA
Philly
i FIG. i. SDS-area-polyscrylamide protein of the 4%clay-old normal and with [%]methionine before analysis. aud white bands on a black background protein bands which were not labeled
gels of a-crystallin immunopreoipitates derived from the soluble the &day-old Philly mouse lens. The lenses were labeled for 5 hr The bla.ck bands on a white background a.re the staiuing pattern are the autoradiogram. The arrows depict the immunoprecipitated and are assumed to represent degradation products of a-crystallin.
molecular weight /%crystallin polypeptides were not visible and only trace amounts, if any, of the y-crystallin polypeptides were present in the stained gels of the Philly lens proteins. At 43 days, the Philly lens had a nuclear opacity. In addition to protein loss, inspection of the gels of the total homogenates suggested that the 43- and 50-day-old Philly lenses accumulated several bands of protein in the crystallin region which were not present in the normal lenses [Pig. 3(a)]. These Philly bands were more easily visualized after precipitation with anti-cc-crystallin antiserum (Fig. 4). The fact that these bands inmnmoprecipitated with anti-oc-crystallin antiserum suggests that the were cleavage products of cc-crystallin. Further work is recpnred in order to establish the nature of these Philly polypeptides. Lens protein qnthesis The pattern of protein synthesis was also different in the lenses of normal and Philly mice. Incorporation of [3%]methionine into the higher molecular weight, noncrystallin polypeptides was unaffected, if not accentuated, in the Philly lens [Fig. 3(b)]. In a separate test, these non-crystallin polypeptides were shown to be associated with the 10 000 x g pellet of the homogenate (Fig. 5). In cont’rast to the incorporation of [a%]methionine into the noncrystallin pofypeptides, incorporation into the lower molecular weight erystallin polypeptides was generally less in the Philly lens than in the normal lens. There was, however, some [35S]methionine incorporationinto crystallin polypeptides of the Philly lens (see below). The amounts of radioactivity present in
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the crystallin and non-crystallin polypeptides of the normal and Philly lens were not diminished after a 24 hr chase in non-radioactive medium, suggesting that the lens proteins were not turning-over rapidly during the labeling time (5 hr) in these experiments. Thus; the differences in labeling appear to be due to differences in synthesis.
FIG. 5. Autoradiogram of an SDS-urea-polyacrylamide gel of [35S]methionine supernatant fraction and the pellet of a 49-day-old Philly lens homogenate 10 000 x g for 10 min. The pellet represents the total proteins from the pellet of represents the proteins from 5% of the soluble protein of a lens. Similar results normal lens (not shown).
labeled proteins in the after centrifugation at one lens: the supernate vere obtained with the
Most of the [35S]methionine incorporated into the crystallins of the Philly lens was into three bands denoted by arrows in Pig. 3(b). These were a-crystallin polypeptides, as judged by an immunoprecipitation test (Fig. 4). Interestingly, in one test with the 42-day-old lens and two tests with the 49-day-old lens there was approximately twice as much [35S]methionine associated with the m.-crystallin bands which immunoprecipitated with anti-u.-crystallin antiserum in the Philly lens than in the normal lens of the same age. By contrast, only trace amounts of radioactivity precipitated with anti-p- or anti-y-crystallin antiserum in tests conducted with the Philly lens (data not shown).
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Finally, it should be noted that several of the Philly bands which immunoprecipitated with anti-or-crystallin antiserum were unlabeled (Fig. 4, arrows). This is. consistent with the interpretation (see above) that these bands contain partially degraded crystallin polypeptides which were synthesized before the lens were la,beled with [35S]n~etllionine. Some unlabeled polypeptides present in this region of the gel also precipitated with anti-/3-crystallin antiserum (data not shown). It is unclear whether these are cleavage products since there is little synthesis of P-crysta,llin in the Philly lens. Lens proteirl
leakage
The medium of cultured normal and Philly lensesfrom each of the different a,ges examined was assayedby SDS-urea-polyacrylamide gel electrophoresis for the presence of proteins which m.ay have escapedfrom the lens. Negligible amounts of crystallins were found in the medium after 5 or 24 hr of culture (data not shown). Sutoradiography of the gel revealed, however. that someradioactively labeled polypeptides with higher molecular weights than the crystallin polypeptides leaked from the normal and the Philly lensesunder these culture conditions, as has been reported elsewhere(Piatigorsky et al., 1978). 4. Discussion The present results show that protein synthesis and degradation are markedly affected in the Philly cataract. Since the concentration of NaT increasesand that of KT decreasesin the Philly lens (Kador et al., 1980), this hereditary cataract provides another example of a correlated change in protein metabolism and electrolyte concentration. Other reports in which such a correlation has been made include the cultured, vitreous-free (Piatigorsky and Shinohara, 1977)and ouabain-treated (Shinohara. and Piatigorsky, 1977) embryonic chick lens, the Nakano and ouabain-treated mouse lens (Piatigorsky et al.: 1978), the galactose-fed rat lens (Kador et al., 1980) and the U18666A-treated (cholesterol biosynthesis inhibited) rat lens (Cenedella and Bierkamper, 1979). None of these experiments, including the present ones,has established that Na+ and Kf are causally or directly related to the alterations in protein metabolism in these lenses,or that the samefactors are responsiblefor the changesin protein synthesis in the different lenses. In addition to Naf and K+, experiments suggest that Cl- may be involved in the protein synthesis alterations in lenses containing ionic imbalances. Cl- inhibits mRNA translation in several eukaryotic cell-free systems (Weber, Hickey, Maroney and Baglioni, 1977). Cl- and acetate, as well as Na+ and K+, also both affect the ratio of the &crystallin polypeptides synthesized in a rabbit reticulocyte cell-free system prim.ed with purified 6-crystallin mRNA from the embryonic chick lens (Piatigorsky et al., 1979; Shinohara and Piatigorsky, in preparation). Moreover, Cl- has been shown to increase in lensesof rabbits (Kinoshita, 1974) and embryonic chicks (Shinohara and Piatigorsky, in preparation) treated with ouabain, and in cataractous lensesof rats fed with galactose (Kinoshita and Merola, 1964; Kinsey and Hightower, 1978). Comparison of the changesin protein metabolism which take place in Philly lenses with those that occw in other cataracts associatedwith ionic imbalances showsboth common features and individual differences. In general, cataracts with altered ionic compositions have reduced contents of soluble protein (see Dische, 1966; Barber, 1973; Harding and Dilley, 1976; Piatigorsky, 1979). The greatest reductions are in
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the /l- and y-crystallins. A partial explanation for the lowered amount of soluble protein appears to be the differential inhibition of crystallin synthesis which correlates with the alterations in ion concentrations. In Nakano mouse (Piatigorsky et al., 1978) and galactose-fed rat (Kador et al., 1980) cataracts, c(-, p- and y-crystallin synthesis are nearly arrested. By contrast, in hypocholesteremic rat cataracts (Cendella and Bierkamper, 1979), y-crystallin synthesis is selectively reduced, and in Philly mouse cataracts (this report) /3- and y-crystallin synthesis are differentially inhibited. The reason for these discrepancies is not known. One possibility is that the changes in ion concentration are compartmentalized within different regions of the different lenses. The onset of the differential reductions in protein synthesis is a,lso not the same in each case. For example, the loss of crystallin synthesis occurs later in the Nakano cataract than it does in the galactose or Philly cataracts. Leakage and degradation of crystallins also contribute to the reduction in soluble protein in cataracts with altered concentrations of electrolytes. Considerable amounts of y-crystallin, some /2-crystallin and traces of a-crystallin were found in the medium of cultured lenses from Nakano mice (Piatigorsky et al., 1.978) and galactose-fed rats {Kador et al., 1980). Radioimmunoassay of the aqueous humor in r\‘akano mice has established that y-crystallin escapes from the catara,ctous lens in vivo (Russell et al.; 1979), indicating that leak-out is an avenue of protein loss in this cataract. Approximately 90% of the y-erystallin is lost from the Nakano lens (Russell et al., 1978). Surprisingly, crystallin leakage was not detected in the cultured Philly lens in the present investigation, suggesting a fundamental difference in the state of the cell surface membranes between the Philly and the Nakano or galactose cataracts. It remains possible that erystallins do leak from the Philly lens along with proteases and, consequently, that the crystallins are degraded in the medium. This seems unlikely, however, since the labeled higher molecular weight polypeptides in the medium did not appear degraded. The apparent absence of crystallin leaka,ge in the Philly lens suggests that degradation is a major pathway of crystallin loss in this cataract. The reduction in P-crystallins is particularly striking in the Philly cataract a,nd exceeds that observed in the Nakano (Piatigorsky et al., 1978) and galactose (Kador et al., 1980) cataract. Partial cleavage products of crystallin polypeptides have been noted in Nakano (Piatigorsky et al., 1978) and Philly (this report) lenses. These appear to be derived from CI- and possibly /3crystallin polypeptides. Experiments with ouabain have suggested that the partial degradation of c(- and /3-crystallin may be related to the changes in ion concentrations within the lens. Further discussion of the changes in protein metabolism in cataractous lenses can be found elsewhere (Piatigorsky, 1979). In conclusion, then, the present results extend the correlation between ionic imbalances and alterations in protein metabolism to the Philly cataract, and indicate that the reduced content of soluble protein in this hereditary cataract is due both to the differential reduction of synthesis and to degradation of ,& and y-crystallin. We do not know if the changes in protein content and metabolism described here are related to opacification in the Philly lens, but we assume that they must lead to deterioration of the lens and the development of an irreversible cataract. ACKNOWLEDGMENTS
We thank Dr J. S. Zigler, Jr. and MS D. A. Carper for a gift of the crystallin antisera, and MS T. Broderick for expert typing.
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REFERENCES Barber, G. W. (1973). Human cataractogenesis: A review. Exp. Eye Res. 16, 85-94. Cenedella, R. J. and Bierkamper, G. B. (1979). Mechanism of cataract production by 3-p (2diethylaminoethoxy) androst-5-en-17-one hydrochloride, U18666A: An inhibitor of cholesterol biosynthesis. Exp. Eye Res. 28, 673-88. Dische, Z. (1966). Alterations of lens proteins as etiology in cataracts. In Biochemistry of the Eye. Symp. Tutzing Castle, pp. 413-28. Karger, Basel, New York. Fukui, H. N., Merola, L. 0. and Kinoshita, J. H. (1978). A possible cataractogenic factor in the NTakano mouse lens. Exp. Eye Res. 26,477-85. Harding, J. J. and Dilley, K. J. (1976). Structural proteins of the mammalian lens: A review with emphasis on changes in development, aging, and cataract. Exp. Eye Res. 22, l-73. Herbrink, P. and Bloemendal, H. (1974). Studies on/?-crystallin. I. Isolation and part’ial characterization of the principal polypeptide chain. Biochim. Biophys. Acta 336, 370-82. Kador, P. F., Fukui, H. N., Fukushi, S., Jernigan, H. M. and Kinoshita, J. H. (1980). Philly mouse: A new model of hereditary cataract. Exp. Eye Res. 30, 59-68. Kador, P. F., Zigler, J. S., Jr. and Kinoshita, J. H. (1979). Alteration of protein synthesis in galactosemic rats. Invest. Opthalmol. Vis. Xci. 18, 696-702. Kinoshita, J. H. (1974). Mechanisms initiating cataract formation. Invest. Ophthabnol. 13,713-24. Kinoshita, J. H. and Merola, L. 0. (1964). Hydration of the lens during the development of galactose cataract. Invest. Ophthalmol. 3, 577-84. Kinsey, V. E. and Hightower, K. R. (1978). Studies on the crystalline lens. XXVII. Kinetic and bioelectric measurements of galactose cataracts in rats. Exp. Eye Res. 26, 521-8. Obazawa, H., Merola. L. 0. and Kinoshita, J. H. (1974). The effects of xylose on the isolated lens. Invest. Ophthalmol. 13, 204-9. Piatigorsky, J. (1979). Intracellular ions, protein metabolism and cataract formation. In Current Topics of Eye Research, Vol. 3 (Eds Zadunaisky, J. A. and Davson, H.). Academic Press, Inc., New York and London (in press). Piatigorsky, J., Fukui, H. N. and Kinoshita, J. H. (1978). Differential metabolism and leakage of protein in an inherited cataract and a normal lens cultured with ouabain. hlature (Lordox) 274,558-62. Piatigorsky, J. and Shinohara, T. (1977). Lens cataract formation and reversible alteration in crystallin synthesis in cultured lenses. Xcielzce 196, 1345-7. Piatigorsky, J., Shinohara, T., Bhat, S. P., Reszelbach, R., Jones, R. L. and Sullivan, M. A. (1979). Correlated changes in &crystallin synthesis and ion concentrations in the embryonic chick lens: Summary, current experiments and speculations. Ann. N. Y. Acad. if&. (in press). Reszelbach, R., Shinohara, T. and Piatigorsky. J. (1977). Resolution of two distinct embryonic gel electrophoresis in ‘the presence of sodium chick 6crystallin bands by polyacrylamide dodecyl sulfate and urea. Exp. Eye Res. 25, 583-93. Russell, P., Carper, D. and Kinoshita, J. H. (1978). The development and application of a radioimmunoassay to lens crystallins. Exp. Eye Res. 27, 673-80. Russell, P., Smith, S. G., Carper, D. A. a,nd Kinoshita, J. H. (1979). Age and cataract-related changes in the heavy molecular weight proteins and gamma crystallin composition of the mouse lens. Exp. Eye Res. 29, 245-55. Shinohara, T. and Piatigorsky, J. (1977). Regulation of protein synthesis, intracellular electrolytes and cataract formation in vitro. Nature (London) 270,406-11. Weber, L. A., Hickey, E. D., Maroney, P. A. and Baglioni, C. (1977). Inhibition of protein synthesis by Cl-. J. Bid. Chem. 252,4007-10. Zigler, J. S., Jr. and Sidbury, J. B., Jr. (1976). A Comparative study of /%cryst,allin from six mammals. Camp. Biochem. Physiol. 53B, 349-55.