Proc. Natl. Acad. Sci. USA Vol. 82, pp. 4712-4716, July 1985 Cell Biology cAMP-dependent phosphorylation of bovine lens cv-crystallin* (AX, A2, B1, and B2 chains/post-translational modification/protein kinase) ABRAHAM SPECTOR, RAJL CHIESA, JANET SREDYt, AND WILLIAM GARNER Biochemistry and Molecular Biology Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 Communicated by Karl Meyer, March 18, 1985

ABSTRACT This communication reports that the Al and A few of the major cAMP-dependent phosphorylated pro- B1 chains of bovine lens a-crystallin are phosphorylated. The teins found in the lens have been identified. They include the conclusion is based on the following evidence: (i) When soluble major fiber membrane protein MP26 (4, 5), vimentin (6), actin preparations from lens cortex are incubated with [y-32P]ATP, (6), and fodrin (7). a cAMP-dependent labeling of a high molecular weight protein Of particular interest is the preliminary finding of a major is obtained. (it) After NaDodSO4/PAGE, the label is found in phosphorylated polypeptide with Mr in the 20,000 range, two bands with Mr 22,000 and 20,000, corresponding to the B which appears in the outer fiber layers (the outer cortex) but and A chains of a-crystallin, respectively. (Wil) Isoelectric not in the epithelium (3). This report presents evidence which focusing indicates that the radioactivity is almost exclusively in suggests that this phosphorylated fraction consists of the Al bands with pI values of 5.58 and 6.70, corresponding to the Al and B1 chains ofa-crystallin, one of the major proteins of the and B1 chains, respectively. (iv) Similar results are obtained in fiber cell. The phosphorylation may be involved in the experiments of [32P]orthophosphate incorporation in lens or- process ofterminal differentiation and the organization ofthe gan culture. (v) Analyses of the digested protein indicate the fiber cell. Thus, the major lens proteins, previously thought label is exclusively in phosphoserine. (VI) 31P NMR analyses of to only contribute to the uniform refractive index of the native, proteolytically digested, and urea-treated a-crystallin tissue, may have other functions and be under stringent gives a chemical shift of 4.6 ppm relative to 85% H3P04 at pH metabolic control. 7.4, suggesting that the phosphate is covalently bound to a serine in the protein. An abundance of approximately one MATERIALS AND METHODS phosphate per four or five monomer units was found. (vii) Similar results were obtained by chemical analyses of indepen- A soluble fraction from calf lens outer cortex (LOCSF) was dently prepared a-crystallin samples. The results are consist- prepared with bovine eyes from approximately 15-week-old ent with the view that the Al and B1 chains arise as result of the calves obtained from a local slaughterhouse. The lenses phosphorylation of directly synthesized A2 and B2 polypep- (1.2-1.6 g) were removed from the eyes and decapsulated by tides. It is suggested that this metabolically controlled phos- dissection. Outer cortex fiber cells (10-20% by weight) were phorylation may be associated with the terminal differentiation obtained by placing the decapsulated lenses in a buffer of the lens epithelial cell and the intracellular organization of containing 10 mM Hepes, and 50 mM 2-mercaptoethanol, pH the lens fiber cell. 7.4, and stirring vigorously for 5-10 min on ice. The suspen- sion of fiber cells was homogenized by using a Because protein phosphorylation is an important reaction Potter-Elvehjem homogenizer and centrifuged at 105,000 x involved in the control of a number of diverse biological g for 45 min. The supernatant, containing 30-50 mg ofprotein processes such as cell differentiation and gene expression (1), per ml, was collected, divided into aliquots, quick frozen investigation of phosphorylation reactions in the lens of the under liquid nitrogen, and stored at -800C until used. eye was initiated. The lens is a particularly inviting tissue for In vitro phosphorylation of the LOCSF preparation was such study. It contains a single layer of epithelial cells, which performed in 200 ,ul of reaction mixture containing 20 mM in the equatorial region are constantly going into terminal imidazole, 1 mM MgCl2, 0.02 mM cAMP, 4 mM NaF, 2 mM differentiation (2). In this process, the cells increase dramat- theophylline, 0.3 tLM [y-32P]ATP (3000 Ci mmol-1, obtained ically in volume, extending to both the anterior and posterior from New England Nuclear; 1 Ci = 37 GBq), and 4 mg of side of the tissue. The cells lose their nuclei and gradually protein. The reaction, started by the addition of [y32P]ATP, their ability to synthesize protein. During terminal differen- was carried out at 370C for 10 min and stopped by addition of tiation, an entirely new population of cytosol and membrane ice-cooled trichloroacetic acid to bring a final concentration proteins is produced. The new fibers are constantly displac- of 10%. The protein precipitate was collected by centrifuga- ing older fibers in towards the center of the tissue. This tion at 12,000 x g for 15 min and washed five times with 95% process results in the production of an age-dependent gradi- (vol/vol) ethanol and five more times with diethyl ether. In ent with the oldest, metabolically inactive cells in the center certain experiments, the phosphorylation reaction was ofthe tissue and the youngest fibers in the outer perimeter of stopped by injection of the incubation mixture in an HPLC the tissue in contact with the epithelium. system to be immediately fractionated. Previous studies on protein phosphorylation in the lens (3) [32P]Orthophosphate incorporation in lens organ culture indicate that there is little phosphorylation in the inner was carried out in lenses removed from the eye by dissection, regions of the tissue. The protein phosphorylation observed in the outer fiber cells of the tissue gives a distinctly different Abbreviations: IEF, isoelectric focusing; LOCSF, lens outer cortex pattern from that observed in the epithelial cell layer. Most soluble fraction. ofthe observed protein phosphorylation is cAMP dependent. *Preliminary reports were presented at the Seventh International Congress of Eye Research, Alicante, Spain, October 1-7, 1984, and at the Association for Research in Vision and Ophthalmology The publication costs of this article were defrayed in part by page charge Meeting, Sarasota, FL, May 6-10, 1985. payment. This article must therefore be hereby marked "advertisement" tPresent address: Ayerst Laboratories Research, Inc., Princeton, NJ in accordance with 18 U.S.C. §1734 solely to indicate this fact. 08540.

4712 Downloaded by guest on October 2, 2021 Cell Biology: Spector et al. Proc. Natl. Acad. Sci. USA 82 (1985) 4713 leaving a layer of vitreous approximately 4 mm thick on the RESULTS posterior surface. They were immediately placed in 10 ml of a medium containing 1.8 mM CaC12, 5.36 mM KCl, 137 mM The cAMP-dependent phosphorylation ofproteins in LOCSF NaCl, 0.8 mM MgSO4, 5.6 mM glucose, 25 mM Hepes at pH preparations from 1-year-old steer lenses incubated in the 7.4, and [32P]orthophosphate at 0.1 mCi/ml (1 Ci mmol-1), presence of [y-32PJATP has been previously studied by adjusted to 310 milliosmolar with NaCl. After incubation at NaDodSO4/PAGE (3). One of the major cAMP-dependent 370C for 5 hr, the capsule was removed and a soluble fraction phosphorylated products was found with Mr in the 20,000 from the lens outer cortex was prepared as described above range. Working with LOCSF preparations from 4-month-old and analyzed by NaDodSO4/PAGE. calf lenses, under the conditions of this experiment, we Phosphorylated LOCSF preparations were fractionated by observed a similar NaDodSO4/PAGE pattern of cAMP- gel filtration HPLC using a 0.75 x 60 cm TSK G 4000 SW dependent phosphorylated proteins. However, two compo- column (Kratos Analytical Instruments, Ramsey, NJ) oper- nents were resolved in the Mr 20,000 range instead of one ating at a flow rate of 1 ml/min with a buffer containing 20mM (Fig. 1). The Coomassie blue-stained gel profile and its sodium phosphates, 100 mM sodium sulfate, and 50 mM respective radioautograph show the cAMP-dependent 2-mercaptoethanol, pH 6.9. Four milligrams of protein was [32P]phosphate incorporation in two major Coomassie blue- applied in an injection volume of 200 pkl. The elution was stained components with Mr 20,000 and 22,000. monitored with an UV detector at 280 nm. Fractions (0.33 ml) These results suggested that both the A and B chains of were collected and subjected to radioactivity measurement in a-crystallin were being phosphorylated, since this protein is a liquid scintillation counter to obtain the elution profile of the composed of monomers that have apparent molecular radiolabeled material. Fractions to be further analyzed were weights of 22,000 and 20,000 as estimated from mobility on collected, and the protein was precipitated by trichloroacetic NaDodSO4/PAGE (16). Since a-crystallin is present in sig- acid and washed with ethanol and ether as described above. nificant concentration in the lens, has a molecular weight of NaDodSO4/PAGE was carried out according to refs. 8 and approximately 700,000 or more (16), and is much larger than 9, as described previously (3). Mr markers were obtained the other major lens proteins, it can be obtained by HPLC gel from Bio-Rad. Isoelectric focusing (IEF) was carried out filtration procedures (17). Thus, after incubation of calf according to ref. 3. Flat-bed IEF gels containing 2.4% LOCSF preparations with [y-32P]ATP and cAMP, the reac- Ampholine pH 5.0-8.0, 0.6% Ampholine pH 3.5-10.0, and 6 tion mixture was directly fractionated on a TSK G 4000 SW M urea were used. Ampholines were obtained from LKB. column. With this column, high molecular weight proteins Samples containing 50,g ofprotein were applied in a solution such as a-crystallin would be expected to be eluted in the void containing 6 M urea and 1% 2-mercaptoethanol. The pH volume. As shown in Fig. 2 a distinct, well-separated com- measurements in the gels were performed immediately fol- ponent was detected in the void volume peak eluting at lowing the IEF, using a surface pH electrode. Two-dimen- approximately 21 min. The profile of the radioactivity ap- sional PAGE was carried out as previously described (6). pears to follow the profile of the UV absorption. The very To identify the phosphorylated amino acids, [32P]phos- large peak eluting at 37 min is composed of low molecular phorylated LOCSF preparations were subjected to NaDod- weight constituents such as nucleotides, theophylline, and S04/PAGE. The regions of the gel containing phosphoryl- inorganic phosphate. In the absence of theophylline and ated polypeptides located by radioautography were excised cAMP, the absorption peak is essentially eliminated. To and extracted by the method of Beemon and Hunter (10). The characterize the material in the eluted peaks, they were phosphorylated polypeptides extracted from the gel were subjected to hydrolyzed in 6 M HCl at 110°C for 2 hr. The hydrolysates NaDodSO4/PAGE and radioautography (Fig. were dried and dissolved in a marker mixture containing 3). The Coomassie blue-stained gel profile of the material O-phosphotyrosine, O-phosphothreonine, and O-phospho- from peak 1 was characteristic of a-crystallin, with two major serine at 1 mg/ml each. The phosphorylated amino acids components with Mr of22,000 and 20,000 characteristic of the were separated bidimensionally on cellulose thin-layer plates 0.1 mm thick by electrophoresis followed by ascendent CB RA chromatography as described by Hunter and Sefton (11). The marker phosphorylated amino acids were detected by nin- hydrin staining (12). The 32P-labeled phosphorylated amino acids were detected by radioautography of the stained plate. Radioautography of the gels as well as the thin-layer plates was performed with Kodak X-Omat AR film and a DuPont Cronex Lightning Plus intensifying screen at -80°C. a-Crystallin was prepared from calf lens outer cortex as previously described (13). All the solutions used in the procedure were phosphate free. 31P NMR spectra were obtained at 121.5 MHz with a Bruker WM 300 NMR spectrometer operating in the Fourier- transform mode; 3000 scans were accumulated. Samples containing 5 mM a-crystallin (based on a monomer Mr of 20,000) and 20% deuterated water for lock-field stabilization 22.0- 3 were analyzed. The chemical shifts were determined at 25°C 20.0 - and pH 7.4, using an 85% H3PO4 solution as external standard. The amount of phosphate in the samples was estimated by using a 1.25 mM ATP solution as standard. Phosphate measurements in preparations of a-crystallin cAMP Control were carried out according to ref. 14. FIG. 1. NaDodSO4/PAGE analysis of a calf LOCSF phosphoryl- Protein measurements were performed by the method of ated by incubation with [32P]ATP in the presence (cAMP) and Bradford (15). Exhaustive proteolysis was done with pepsin absence (control) of cAMP. CB, Coomassie blue-stained 15% gel; and trypsin. RA, radioautograph. The numbers on the left side indicate M,x lo-3. Downloaded by guest on October 2, 2021 4714 Cell Biology: Spector et al. Proc. Natl. Acad. Sci. USA 82 (1985)

1.0 0 CB RA I -0.8 0I -0.6 1 x -0.4 c4 5 58- 4 -AI E 5.92- -A2 -0.2 CL -0 6.70- .411-B 15 20 25 30 35 40 45 Elution time, min 7.08- -B2 FIG. 2. Elution profile of [32P]phosphorylated calf LOCSF frac- tionated by gel filtration HPLC in a TSK 4000 SW column. , Absorbance at 280 nm; *-, 32P radioactivity measured in 0.33-ml aliquots. The fractions pooled for further analyses by NaDodSO4/ PAGE are indicated by the numbers 1 through 5. B and A chains, respectively. Almost all of the radioactivity was observed in positions corresponding to the A and B chains. A minor radioactive band ofMr approximately 57,000 was also observed. Examination of the other peaks indicated that no significant level of radioactivity is found in any other FIG. 4. IEF analysis of the void volume fraction (fraction 1; see major lens polypeptide. No radioactivity was found associ- Fig. 2) obtained by gel filtration HPLC. CB, Coomassie blue-stained gel; RA, radioautograph of the same gel. The numbers on the left ated with the ,/- or y-crystallins. The radioactive components indicate the observed pI values obtained by direct pH measurement of Mr 43,000 and 57,000 have previously been shown to be using a surface electrode. When the radioautographs were exposed actin and vimentin, respectively (6). The radioactive bands for shorter periods, the radioactive spots could be precisely super- with Mr 22,000 and 20,000 in peak 2 and possibly peak 3 imposed on the Coomassie blue bands of pH 5.58 and 6.70. apparently represent contamination of a-crystallin compo- nents. While only two a-crystallin polypeptides, A2 and B2, are have isoelectric points of 7.08, 5.92, and 5.58, corresponding directly synthesized, post-translational reactions lead to the very closely to the reported values for B2, A2, and A1 chains, each of the respectively (18). The B1 chain appears as a minor component production of additional species. Since with an isoelectric point of 6.70, also very close to the polypeptides has a characteristic isoelectric point, the peak 1 reported value (18). However, the radioactivity was essen- material obtained by the HPLC gel filtration was analyzed by tially confined to two bands corresponding to the A1 and B1 IEF (Fig. 4). Coomassie blue staining of the gels indicated a chains. It was consistently observed that while much more A1 typical pattern. Three major components representing more than B1 was detected, similar levels of32p incorporation were than 75% ofthe total material (measured by scanning the gels) found in both polypeptides. Such results suggest that the B polypeptide is a better substrate than its A counterpart, CB RA CB RA CB RA CB RA CB RA perhaps due to accessibility to the kinase or specificity requirements. Similar results were obtained from IEF anal- yses over narrower pH ranges. Two-dimensional separations

Me...... (IEF followed by NaDodSO4/PAGE) were also performed ..... :;. ..:. . with the material from peak 1 (see Fig. 2). As shown in Fig. 5, only two major radioactive spots were observed, which can be superimposed on the respective A1 and B1 spots from the Coomassie blue-stained gel. Since the cAMP-dependent

a. D8 - 6.70 70 : 22.0- cv~~~~~~~v9 .. .,vSlT 9nrf - ...... :.. 22.0 22.0- 20.0. ... 20.0- 2 3 4 5 FIG. 3. NaDodSO4/PAGE analysis of the fractions obtained by FIG. 5. Two-dimensional analysis (IEF followed by NaDodSO4/ gel filtration HPLC indicated in Fig. 2. The numbers 1 through 5 PAGE) of the void volume fraction (fraction 1; see Fig. 2) obtained indicate the fraction number. CB, Coomassie blue-stained gel; RA, by gel filtration HPLC. (Upper) Coomassie blue-stained gel. (Lower) radioautograph of the stained gel. The numbers on the left indicate Radioautograph of the same gel. The Mr x 10-3 is indicated on the Mr X 1O-3. left. The observed pI values are indicated above the spots. Downloaded by guest on October 2, 2021 Cell Biology: Spector et al. Proc. Natl. Acad. Sci. USA 82 (1985) 4715 phosphorylation of actin and vimentin in bovine LOCSF reactions (21-23). However, it is difficult to obtain a- preparations results in the incorporation of phosphate exclu- crystallin even in the newly formed fiber cells that does not sively in serine residues (6), it was of interest to identify the contain Al and B1 chains. Such observations suggest that a phosphorylated amino acids present in the presumptive more rapid, perhaps enzymatically controlled, specific reac- phosphorylated monomers of a-crystallin. Therefore, tion may be involved. It is not possible at the present time to [32P]phosphorylated A and B chains, obtained by preparative definitively conclude that all transformation to Al and B1 is NaDodSO4/PAGE, were subjected to partial acid hydrolysis due to the phosphorylation reaction or that the deamidation and the [32P]phospho amino acids in the hydrolysates were reaction does not occur. It is possible that both reactions investigated by using a two-dimensional analytical separation occur. Indeed, on the basis of our present information, the system on thin-layer chromatography plates. This method direct phosphorylation of Al and B1 cannot be definitely permits the simultaneous identification ofphosphothreonine, excluded. phosphotyrosine, and phosphoserine. [32P]Phosphoserine It is likely, on the basis of the work reported in this was the only 32P-labeled phospho amino acid detected in the communication, that a-crystallin is phosphorylated in what phosphorylated preparations of both A and B chains of appears to be a specific reaction. The labeling is found almost a-crystallin. exclusively in the Al and B1 chains. None of the other major [32P]Orthophosphate incorporation experiments were also lens proteins appear to be phosphorylated, and the phospho- performed with calflenses in organ culture. After incubation, rylation occurs solely on serine residues. The finding that the the outer cortex was separated, homogenized, precipitated process is cAMP dependent suggests it is subject to regula- with trichloroacetic acid, and then subjected to NaDod- tion by extracellular signals. An experiment recently report- S04/PAGE. As with the incorporation in the homogenates, ed by Simonneau et al. (24) is ofinterest in this respect. They major labeled bands were found with mobilities comparable found that incubation of bovine epithelial cells in the pres- to those of the A and B polypeptides of a-crystallin. IEF ence of a retinal extract and labeled amino acid causes indicated that these bands correspond to the A1 and B1 apparent incorporation into the B1 chain ofa-crystallin rather polypeptides. These investigations support the viewpoint than incorporation into the B2 chain found in the absence of that similar phosphorylation reactions occur in intact organ the factor. If this observation is correct it suggests, on the cultures and homogenates. basis of the observations in this communication, that the Since the A1 and B1 chains represent approximately 30%o of retinal extract contains a component that stimulates the the total a-crystallin (16), it was of interest to ascertain if cAMP-dependent phosphorylation of the B2 chain. It should phosphate could be detected in several a-crystallin samples be noted that in epithelial cells, a-crystallin makes up only a prepared independently and in the absence of phosphate. few percent of the total protein and that, unlike the situation Determinations of phosphate indicated approximately 0.2 in the fiber cell, the protein in the B chain predominates (20). mol of phosphate per mol of a-crystallin monomer (assuming No B1 or Al has been previously reported in epithelial cells. a monomer Mr of 20,000). Repeated treatment with 6 M urea It is possible that the marked increase in a-crystallin pro- (1 mg of protein per 7.5 ml) and precipitation with trichloro- duction, the change in the ratio of A to B chains, and the acetic acid caused little change in the results. phosphorylation are all aspects ofthe terminal differentiation The phosphate content of a-crystallin was also examined process. by 31P NMR spectroscopy. A major phosphorus resonance While the function ofthe phosphorylation of a-crystallin is (signal to noise greater than 5:1) was observed at 4.5 ppm, a not understood, its possible control by extracellular factor(s) chemical shift expected for phosphoserine-containing perhaps involved with terminal differentiation suggests that polypeptides (19). No change in this observed chemical shift the phosphorylated macromolecule is involved with some position was obtained when identical samples were either aspect of the biology of the fiber cell. Experiments reporting exhaustively digested with proteolytic enzymes or treated a relationship of a-crystallin with membrane and matrix with 8 M urea. A phosphoserine sample was found to give a structure (25, 26) would suggest that the phosphorylated chemical shift identical to that previously reported (19). The protein interacts with such structures, contributing to the estimated amount of phosphate associated with the protein overall organization of the fiber cell. was approximately 0.27 mol per mol of a-crystallin (assuming We acknowledge the competent technical assistance of Leslie a monomer Mr of 20,000). These results, in conjunction with Houghton. We are indebted to Great American Veal Company for the chemical analyses, are consistent with the metabolic supplying bovine eyes. This work was supported by grants from the labeling experiments and the identification ofphosphoserine, National Eye Institute. R.C. is a Fulbright Fellow. J.S. was sup- suggesting that a-crystallin contains covalently linked phos- ported by a National Eye Institute Training Program Grant. phate. It is interesting to note that while the level of labeled 1. Krebs, E. G. (1983) Philos. Trans. R. Soc. London Ser. B 302, phosphate incorporated into a-crystallin was low, the anal- 3-11. yses of isolated a-crystallin indicate an abundance of phos- 2. Davson, H. (1980) Physiology of the Eye (Academic, New phate sufficient to account for the abundance of the A1 and York), 4th Ed., p. 116. B1 chains. 3. Sredy, J. & Spector, A. (1984) Exp. Eye Res. 39, 653-664. 4. Johnson, K. R. & Johnson, R. (1982) Fed. Proc. Fed. Am. Soc. Exp. Biol. 41, 755. DISCUSSION 5. Garland, D. & Russell, P. (1985) Proc. Nat!. Acad. Sci. USA The synthesis of a-crystallin can be considered to occur in 82, 653-657. 6. Sredy, J., Roy, D. & Spector, A. (1984) Curr. Eye Res. 3, two stages. First, there is the direct gene-controlled expres- 1423-1431. sion of the A2 and B2 chains and the formation of a 7. Ireland, M. & Maisel, H. (1984) Curr. Eye Res. 3, 961-968. homogeneous macromolecule of Mr approximately 700,000 8. Laemmli, U. K. (1971) Nature (London) 227, 680-685. (16). This is followed by post-translational reactions leading 9. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, to the formation ofthe A1 and B1 chains, changes in aggregate 4406-4412. structure reflected in physical heterogeneity and higher 10. Beemon, K. & Hunter, T. (1978) J. Virol. 28, 251-266. molecular weight and, finally, limited proteolytic degradation 11. Hunter, T. & Sefton, B. M. (1980) Proc. Natl. Acad. Sci. USA and racemization primarily detected in the protein isolated 77, 1311-1315. from older sections of the tissue (20). 12. Dreyer, W. J. & Bynum, E. (1967) Methods Enzymol. 10, Previous work has suggested that the appearance of the Al 32-39. and B1 chains is due to slow nonenzymatic deamidation 13. Li, L.-K. (1978) Exp. Eye Res. 27, 553-566. Downloaded by guest on October 2, 2021 4716 Cell Biology: Spector et al. Proc. Natl. Acad. Sci. USA 82 (1985)

14. Buss, J. E. & Stull, J. T. (1983) Methods Enzymol. 99, 7-14. 21. Bloemendal, H., Berns, A. J. M., van Der Ouderaa, F. & de 15. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. Jong, W. W. (1972) Exp. Eye Res. 14, 80-81. 16. Stauffer, J., Rothschild, C., Wandel, T. & Spector, A. (1974) 22. van Venrooij, W. J., de Jong, W. W., Janssen, A. & Invest. Ophthalmol. 13, 151-153. Bloemendal, H. (1974) Exp. Eye Res. 19, 157-162. 17. Bindels, J. G., DeMan, B. M. & Hoenders, H. J. (1982) J. 23. van Kleef, F. S. M., de Jong, W. W. & Hoenders, H. J. (1975) Chromatogr. 252, 255-267. Nature (London) 258, 264-266. 18. Bindels, J. G. (1982) Dissertation (Univ. of Nijmegen, 24. Simonneau, L., Merve, B., Jacquemin, E. & Courtois, Y. Nijmegen, The Netherlands), p. 60. (1983) Cell Differ. 13, 185-190. 19. Matheis, G. & Whitaker, J. R. (1984) Int. J. Biochem. 16, 25. Ramaekers, F. C. S., Selten-Versteegen, A. E. & Bloemen- 867-873. dal, H. (1980) Blochim. Biophys. Acta 596, 57-63. 20. Bloemendal, H. (1981) Molecular and Cellular Biology of the 26. Bloemendal, H., Hermsen, T., Dunia, I. & Benedetti, E. L. Eye Lens (Wiley, New York), p. 191. (1982) Exp. Eye Res. 35, 61-67. Downloaded by guest on October 2, 2021