- Email: [email protected]
The subunit structure of chick β-crystallins
Exp. Eye Res. (1974)
18, 485-494
The Subunit Structure of Chick fbCrystallins D. E. hstitute
of An&al
X. TRUMAN
ANII
R. M.
Genetics, Bdinburgl~,
(Received 10 Dectmbw
CLAYTOK
EH9
3JN> Scothd
1973, London)
Chick /3-cryst~allins are a class of proteins containing molecules with a range of size and electrophoretic mobility; there are approximately 15 molecular species, six of which are major constituents of the class. They can be fractionated electrophoretically or by isoelectric focusing. The /3-crystallin molecules can be dissociated into subunits and each aggregate contains a number of different types of subunit, differing immunologically, in electrophoretic mobility and in isoelectric point. The same subunit may be present in several different aggregates, the properties of which are a function of the const,ituent subunits.
1. Introduction The class of chick lens crystallins of lowest. molecular weight is variously known as the long-line crystallins, on account of the appearance of the precipitin arc that they form in immunoelectrophoresis (Zwaan, 1963); y-crystallins, because of their low molecular weight and becausesomeof their numbers are of low electrophoretic mobility (Maisel and Langman, 1961a, 1961b; Zwaan, van Doorenmaalen and Hoenders: 1963); and /3-crystallins becausethey are said to have antigenic similarities to bovine /3-crystallins (van Dam, Schalekamp, Schalekamp-Kuyken and ten Gate, 1963; Zwaan and Ikeda. 1968). We have chosen to use the term P-crystallin. They clearly form a class of some complexity and various estimates of the numbers of different proteins and of their molecular weight are to be found in the literature. Two major classesdiffering in molecular size have beeu demonstrated by Hoenders (1965) and by Truman (1968). Zwaan (1968) found that of nine fi-crystallins, five had a molecular weight of 55 000, two were of 60 000, one was 50 000 and one was 40 000. Using gel filtration to estimate molecular weight, Truman, Brown and Rao (1971) estimated a range of molecular weight up to 59 000 but with someP-crystallins with a molecular weight as low as 16 000. Estimates of the number of crystallins in this class have depended on the analytical technique used, Zwaan (1968) finding nine components. Clayton (1969) 12, Truman (1968) 15 and Bours (1971) 19. Clayton and Truman (1967) argued that the /%crystallins must be a family of heteropolymers, each molecule containing a number of subunits, and the sameconclusion was also supported by the results of a seriesof Ossermantests comparing lens proteins with those of other tissues (Clayton, Campbell and Truman, 1968). Zwaan (1968) also suggestedeither a subunit structure or a disparity of size among the immunologically similar proteins of this class of crystallins. Rana and Maisel (1969, 1970) have given clear evidence of a heteropolymer subunit structure, while estimates of sedimentation coefficients (Clayton and Truman, 1967) and of molecular weights (Truman et al., 1971) in the presenceand absenceof urea have also confirmed this view. In dissociative conditions 1I components were detected in polyacrylamide gel electrophoresis of /3-crystallins, and when individual intact crystallins were isolated and dissociated several of these components were obtained from each one of them (Clayton. 1969), the patterns obtained being consistent with the view that t,he /%crystallins are a family of hetero1)olymers. The data presented in this paper. derived from a number of different, 4x5 E
486
11. E.
S. 1’RCiMAS
AND
R.
111. C’LAYTOS
techniques, give further evidence of the heterogeneous nature their relationship to each other as a series of heteropolymers number of subunits.
of these crystallins antI composed of a limited
2. Materials and Methods Lenses The lenses were dissected from the heads of commercially slaughtered birds supplied by D. B. Marshall (Newbridge) Ltd., Midlothian. They were prepared as described earlier (Truman, 1968), except that the capsules were retained after removing extra-lenticular tissue, since removal of the capsule inevitably also removed some epithelial cells from the lens. A preparation of material rich in cells derived from the lens cortex was sometimes made by taking off the capsules and then shaking both lenses and capsules gently in buffer (10 mM-phosphate 10 in&r-2-mercaptoethanol, pH 7.2) until most of the cortical material appeared to have separated from the lens nuclei. The suspension of cortical cells was removed by decantation, homogenized and centrifuged as described earlier for the whole lenses (Truman, 1968; Clayton, 1969). Isolation
of /3-crystallins
The details of the chromatographic techniques used were described by Truman (1968). Soluble proteins prepared either from whole lenses or from cortical fractions were fractionated by gel filtration on a column of polyacrylamide beads (Bio-Gel P-300) to produce a fraction rich in /?-crystallins. This fraction was further purified by repeated gel filtration, concentrating the proteins between runs by precipitation with (NH&SO, to 600/, saturation. Three runs through the gel titration column were usually sufficient to produce material which when tested by immunoelectrophoresis showed only the long precipitin arc characteristic of the /3-crystallins, with no trace of M- or &crystallin. No differences were found in the properties of the /%crystallins prepared from the cortical material as compared with preparations from the whole lens, but it was easier to obtain pure material from the cortical preparations. A partial fractionation of the @crystallins on a basis of molecular size was achieved by gel filtration on the polyacrylamide beads Bio-Gel P-150. These gel filtration media are manufactured by Bio-Rad Laboratories, Richmond, Calif., U.S.A. and were purchased from V. A. Howe and Co. Ltd., London. Isoelectric focusing The apparatus used was based on that described by Vesterberg, Wadstrom, Vesterberg, Svensson and Malmgren (1967) and was manufactured by LKB-Produkter AB, StockholmBromma, Sweden. Columns of both 110 and 440 ml capacity were used and separations were carried out over a variety of ranges of pH as described in the text. The method used was essentially that described in the manufacturer’s instructions, except that 10 mM2-mercaptoethanol was added to all solutions, and in some cases 7 M-Urea was used as the solvent instead of water. After completion of the separation the contents of the column were passed through a Uvicord II ultraviolet absorbtiometer (LKB-Produkter, StockholmBromma, Sweden), set at 280 nm. The pH of the fractions collected was measured at the same temperature as the isoelectric focusing run, approximately 17°C. Fractions were either tested by immunoelectrophoresis or polyacrylamide gel electrophoresis without prior concentration, or several fractions from a single peak were concentrated by precipitation with (NH&SO, to 60% saturation followed by centrifugation and dialysis. Polyacrylanaide gel electrophoresis For tests in the absence of urea the method used was that described earlier (Truman, 1968), and when tests were made in the presence of 6 M-urea the method was the modifi-
j?-CRYSTALLIN
STRUCTURE
4x5
cation described earlier (Clayton, 1969; Truman, Brown and Campbell (1972). Densitometric scanning of the gels stained with Amido black was in the Joyce-Loebel densitometer, using a red filter. 3. Results The low molecular weight crystallins of the chick lens were prepared by gel filtration and tested by immunoelectrophoresis and by disc electrophoresis on polyacrylamide gels. Both these methods failed to detect proteins which were known from previous experiments to represent the LX- and S-crystallins (Truman, 1968). On the polyacrylamide gels the @crystallin preparations showed some 15 bands after staining with Amid0 black, and of these bands about six were particularly intense and presumably represented major constituents in this class of protein (Figs 1 and 2). The relative intensities of the stained bands varied somewhat between preparations, possibly due to the rejection of varying amounts of the proteins which were less retarded on gel filtration and which were thus removed along with &crystallin.
I?IQ. 1. Electrophoretic pattern obtained from chick fi-crystallins separated on polyacrylamide the absence of urea. The origin of the electrophoresis is at the top of the diagram and the anode the bottom.
gel in towards
The preparations of /3-crystallin could be further fractionated by gel filtration on polyacrylamide P-150. In this way two major fractions could be distinguished, one containing five components detectable on polyacrylamide electrophoresis, all of which had relatively high mobility towards the anode, while the other fraction contained 10 components, with a somewhat lower electrophoretic mobility. Electrophoretic patterns obtained with such preparations are illustrated in Fig. 3. When a preparation of /3-crystallins is fractionated by isoelectric focusing over a pH range of 3-8, the heterogeneity of these proteins is seen again. Figure 4 represents
488
LJ.
K.
s.
‘ml-J1
AS
A\ s II
I:.
Il.
(‘r,.-iY’I’os
the absorptiometer trace of a typical isoelectric separation. Fonr major com1)onei~t.x occur, with about six minor components. giving moderate agreement with the results of polyacrylamide gel electrophoresis. The peaks in Fig. 4 are identified t)?- their isoelectric point in that run, but as the temperature regulation of the isoelect,ric focusing column was not precise these values cannot be regarded as definitive valnt!s of the isoelectric points but rather as convenient idenbification of the fractions.
FIQ. 2. Electrophoresis of chick /3-crystallins is to the right and the anode to the left.
FIG. 3. Electrophoresis weight fraction. Lower the left.
on polyacrylamide
gel in the absence of urea. The origin
of chick ,%crystallins fractionated by gel filtration. Upper gel: higher molecular gel: lower molecular weight fraction. The origin is to the right and the anode to
FIG. 4. Ultraviolet absorptiometer trace showing separation focusing over the range pH 3-8 in the absence of urea. The pH peaks is indicated.
of chick @xystallins by isoelectric of the fractions containing the main
When the fractions prepared by isoelectric focusing were tested by electrophoresis on polyacrylamide gels most were found to consist of one major fraction. Figure 5 represents the densitometer traces obtained from the gels. In somecasesit can be seen that there was some cross-contamination between fractions: for example, fraction 6.41 also contains some of the material present in fraction 6.70. Nevertheless the separation obtained is generally good. Fraction 7.61 is somewhat anomalous: the minor component shows a relationship between isoelectric point and electrophoretic mobility similar to the other fractions, but the major component of this fraction has
,9-CRYSTALLIK
1x9
STKUCTlIKE
an abnormally high mobility towards the anode, which presumably indicates relatively low molecular weight. The broadness of this peak, suggesting relatively rapid diffusion of the molecules, also indicates low molecular weight. It may be that the material forming the large peak is derived from the breakdown of that in the smaller peak. The production of material of high electrophoretic mobility from some slower /3crystallins on re-running on polyacrylamide gels has been observed previously (Clayton, 1969). 7.61 A
A
6.70 -
A
64 -
A
5.95 -
A
5.82 3.71-
A A
+o Pm. 5. Densitometer t.races of electrophoret.ic separations cm polyacrylamide fractions obtained by isoelectric focusing of chick /3-crystallins. Each fraction focusing columns is designated by its apparent p1. The origin of the electrophoretic right of the diagram snd the anode to the left.
gels without m-tit of from the isoelectric separation is to the
Effect of dissociatiny media on /3-crystalhas Studies of the gel filtration behaviour of chick /3-crystallins in the presence and absence of concentrated urea solutions have indicated that reagents which break hydrogen bonds may reduce the molecular weight of the ,!3-crystallins from about 60 000 to about 16 500 (Truman et al., 1971). Preliminary studies with the ultracentrifuge also confirm this effect of urea on these proteins (Clayton and Truman. 1967; Clayton, 1970). A preparation of total chick P-crystallins examined in the a,nalytical ultracentrifuge in a buffer of 10 nix-phosphate, IO m&I-%mercaptoethanol at pH 7.2 .at a8protein concentration of 8 mg/ml showed a broad peak in schlieren optics with a sedimentation coefficient, sz,,.+ of 6~44% When similar material was dialysed against a similar buffer to which urea had been added to a concentration of 8 M, t)hen the sedimentation velocity was reduced to an s,~,~ of 1*%38.
FIG. 6. Electrophoresis anode to the lvft.
of j%crystallins
in the prescncc
of 6 n~-urca.
The origin
is t,o the right
:uxl C~C
When a preparation of the total @rystallin proteins was dissociated in urea solution and then electrophoresed on polyacrylamide gels in the presenceof 6 M-urea, 11 components were generally seen (Clayton, 1969) (Fig. 6). When electrophoresis in
490
I,.
F:. s.
TKI-MAN
>iSD
1:,. !vl. (‘LAYTON
urea was carried out on starch gel at an acidic pH, eight components were detected (Truman et al., 1971). These results indicate that a variety of polypeptide cheins are involved in the aggregate molecules in the /3-crystallin class. After separation of the /3-crystallins by isoelectric focusing, electrophoresis on polyacrylamide gels in the presence of urea indicated that each fraction contained more than one component after dissociation. The densitometer traces obtained from both undissociated and dissociated samplesof someof the /3-crystallin fractions are set out in Fig. 7. All of these
L I!!-L6.70
6.41
4.78
L-
5
t FIQ. 7. Comparison of of urea of three fractions meter traces is of runs in and anode to the left of
0
0
+
the results of electrophoresis on polyacrylamide gel in the presence and absence obtained by isoelectric focusing of chick b-crystallins. The upper set of densitothe absence of urea, the lower set of runs in 6 M-urea. The origin was to the right each trace. 3.71
4.44
4.78
5.82
5.95
-
-
6.41
6-70
Fro. 8. Diagram showing the major subunits of various fractions of /I-crystallins. Each column represents an electrophoresis on polyacrylamide gel in 6 M-urea, with the origin at the top and the anode at the bottom. The fractions are debignated by their apparent p1 when prepared by isoelectric focusing.
p-CRYSTALLIN
STRUCTURE
491
fractions yield more than one component when tested in dissociating conditions. Each fraction has components similar in electrophoretic mobility to some of the components in other fractions, but the average mobility of the subunits of any one fraction corresponds to the mobility of the aggregate from which they are derived. From the analysis of each fraction in undissociating conditions, we can make some estimate of the likely contamination of each fraction and so can make allowance for the possibility of subunits resulting from contaminating material. It is then possible to estimate the minimum number of types of subunit which must be present in each fraction of the /Scrystallins prepared by isoelectric focusing. Figure 8 is a diagram representing the composition of some of the p-crystallin fractions. If it is assumed that components of the same mobility derived from different fractions are identical, then the total number of different subunits derived by dissociating the /3-crystallins is 12. This is in good agreement with results obtained by dissociating unfractionated preparations of chick /3-crystallins. Ixoekctric focusing in dissociating media Isoelectric focusing carried out with 8 M-urea present in all solutions, over the pH range 5-8, using a preparation of the total /3-crystallins of the chick, gave rise to the elution pattern shown in Fig. 9. About 11 peaks can be distinguished though the quantity of material in some fractions is relatively small. There is a good general agreementbetween this elution pattern obtained by isoelectric focusing and the pattern obtained from polyacrylamide gel electrophoresis in the presenceof urea, the electrophoretic mobilities apparently being a simple function of isoelectric points.
BIG.
focusing
9. Ultraviolet absorptiometer in the presence of 7 M-urea
trace showing separation of subunits over the pH range 5-8. The apparent
of @rystallins by isoelectric p1 of each peak is indicated.
4. Discussion The class of chick lens structural proteins generally known as the long-line or pcrystallins clearly contains a number of different molecular species. The results presented here show that 10 proteins can be separated by isoelectric focusing. Zwaan (1968) has used two-dimensional polyacrylamide gel electrophoresis to determine the molecular weights of nine chick /Scrystallins and Hoenders (1965) used sedimentation behaviour and electrophoretic properties in an extensive study which indicates the
492
n.
E.
s.
TIt!uJIAN
ANI)
IL.
N.
(‘I,AV’I’UN
occurrence of at least five /3-crystallins. In t#he face of such evidence, what arc’ thtb grounds for considering these proteins as a single class? In the development of the ItJns the P-crystallins do not appear simultaneously, the number of P-crystallin co~uponents increasing from the embryonic stage 15, while changes in the proport,ions of /% crystallin components continue after hatching (Rabaey, 1962: 1966; Zmaan. 1963 : van Doorenmaalen, 1964; Zwaan and Ikeda, ~1965: 1968; Ikeda and Zwaan. 1967: Genis-Galvez, Maisel and de Castro, 1968; Genis-Galvcz, de C’astro and Battancr. 1968; C!layton, 1970; Truman et al., 1972). The ontogeny of the subunits of the ,& crystallins also indicates that these proteins do not form a single functional unit, (Clayton and Burns, in preparation). Though the chick P-crystallins have in cotmnou the fact that they are smaller molecules than either M- or %crystallin, they are nevertheless heterogeneous in size (Hoenders, 1965; Truman, 1968; Zwaan, 1968; Truman et al., 1972). The justification for considering these proteins t,ogether must remain the immunological similarity that they shop, especially in immunoelectrophoresis where they appear as conjoint arcs with spurs. This is also particularly clearly seenwhen one of our antisera against Xe~zopusZaevislens is used to react wit,11 chick lens (Clayton and Truman, 1967, 1974). The P-crystallins are then alone in reacting and a continuous precipitin line is formed showing that at least one antigenic determinant is shared by these proteins. Zwaan (1968), in considering his electrophoretic data on chick crystallins, suggested that either the P-crystallins consisted of similar but not identical polypeptide chains, or that they were composedof similar subunits. The evidence put forward here that each P-crystallin molecule contains a number of different subunits, that different crystallins consist of different combinations of subunit,3, with the samesuburlit being shared by several crystallins, and that the electrophoretic behaviour of the aggregates is a function of that of their constituents, is a further confirmation of the data put forward earlier (Clayton and Truman, 1967: 1968; C’layton et al., 1968: Clayton, 1969: Rana and Maisel, 1969, 1970). Much of the evidence for the presence of different types of subunit in aggregate molecules is derived from electrophoretic studies made in the presence of urea ancl the possibility of the formation of artefacts must be borne in mind, in particular the possibility of carbamoylation of proteins by alkaline urea solutions (Stark, Stein and Moore, 1960). However, we find that the major subunits, at least: can be resolved by starch gel electrophoresisin urea at an acidic pH (Truman et al., 1971) and so it scenls probable that only minor components, at the most, could be artefacts. The results of the electrophoresis described here are essentially qualitative and the number of bands seen does not give any indication the proportions of the different subunits within the aggregate. Our previous work (Truman et al., 1971) has indicated that the P-crystallins can exist in a size range from monomersto tetramcrs. Since no rigorously separated /3-crystallin component appears to contain more than three different subunits the results of the subunit analysis are consistent with the molecnlar weight estimations. The exceptional complexity of the chick @rystallins as a class is in agreement with analyses of other species.For example, 11 components were found among the P-crystallins in various speciesof monkey by Ma&e1 and Goodman (1964) using twodimensional electrophoresis and Holt and Kinoshita (1968) found 11 component’sin the rat. Bovine P-crystalline are also known to be complex : Bjijrk (1964) and Spector and Katz (1966) found four fractions and evidence of considerable heterogeneity was found by Testa, Armand and Balazs (1965) van Dam (1966) and Armand, Balazs
j&CRYSTALLIN
STRUCTURE
493
and Testa (1970). A recent study by Zigler and Sidbury (1973) has revealed two groups of /3-crystallins, one containing eight proteins and the other containing at least 10. Evidence that bovine p-crystallins can be dissociated into smaller subunits was found by Bloemendal, Bont, Jongkind and Wisse (1962). Mammalian /I-crystallins appear to contain six to eight different major subunits, and up to 11 have been found when high resolution methods have been used (Spector and Katz, 1965, 1966 ; Shapiro, 1968 ; Day and Clayton, 1972 ; Zigler and Sidbury, 1973). Further references and discussion on the subunit composition of different species will be found in the review by Clayton (1974). Mammalian and bird p-crystallins may have a similar type of subunit composition. Studies by electrophoresis in the presence of sodium dodecyl sulphate (SDS) invariably show that the /3-crystallin subunits have a range of sizes, but electrophoretic studies in dissociating conditions in the absence of SDS indicate that each size class may comprise subunits further distinguished from each other by their charge (Shapiro, 1968; Zigler and Sidbury, 1973). Immunological analysis indicates that these ,&crystallin components are immunologically related (Testa et al., 1965; Rana and Maisel, 1970; Clayton and Truman. 1974). This implies that the p-crystallin polypeptides have been derived by a process of gene duplication followed by divergence (Clayton, 1974). ACKNOWLEDGMENTS
We would like to express our thanks to D. B. Marshall (Newbridge) Ltd. for their continued co-operation in the supply of material for our experiments. We are grateful to Mrs A. G. Brown, Mr A. G. Gillies, Mrs A. Hannah and Mrs H. J. Mackenzie for thei skilled technical assistance. Our work has been supported by grants from the Cancer Research Campaign and the Medical Research Council. REFERENCES Armand, G., Balazs, E. A. and Testa, M. (1970). Separation and partial characterization of two proteins from fraction B of calf lens. Exp. Eye Res. 10, 143. Bjijrk, I. (1964). Fractionation of/?-crystallin from calf lens by gel filtration. Exp. Eye Res. 3,248. Bloemendal, H., Bont, W. S., Jongkind, J. F. and Wisse, J. H. (1962). Splitting and recombination of a-crystallin. Exp. Eye Res. 1, 300. Bours. J. (1971). Isoelectric focusing of lens crystallins in thin-layer polyacrylamide gels. J. Chromatogr. 60,225. Clayton, R. M. (1969). Properties of chick crystallins in terms of their subunit composition. iL:xp. Eye Res. 8, 326. Clayton, R. M. (1970). Problems of differentiation in the vertebrate lens. Curr. Topics De&op. Biol. 5, 115. Clayton, R. M. (1974). Comparative aspects of lens proteins. In The Eye (Ed. Davson, H.). Vol. 5. Academic Press, New York and London. (In press.) Clayton, R. M., Campbell, J. C. and Truman, D. E. S. (1968). A re-examination of the organ specificity of lens antigens. Exp. Eye Res. 7, 11. Clayton, R. M. arid Truman, D. E. S. (1967). Molecular struct’ure and antigenicity of lens proteins. Nature (London) 214, 1201. Clayton, R. M. and Truman, D. E. S. (1968). The structure and cross-reactivity of chick lens proteins. J. Physiol. (London) 198, 72P. Clayton, R. M. and Truman, D. E. S. (1974). The antigenic structure of chickp-crystallin subunits. Exp. Eye Res. 18, 495. van Dam, A. F. (1966). Purification and composition studies ofp crystallin. Exp. Eye Res. 5,255. van Dam, A. F., Schalekamp, M. A., Schalekamp-Kuyken, M. and ten Cate, G. (1963). Immunoelectrophoretio studies on adult and embryonic ocular lenses. Vlth Internat. Embryol. Confwence Helsinki. F
494
D.
E.
S. TRUMAN
AND
R.
N.
CLAYTOK
Day,
T. H. and Clayton, R. M. (1972). Multiple changes in lens proteins composition associated with Catrr. gene in the mouse. Genet. Res. Camb. 19, 241. van Doorenmaalen, W. J. (1964). Immunohistological studies of proteins during the development of chick lens. In Methodes Nouvelles en Embryologic (Ed. Wolff, Et.). P. 151. Hermann, Paris. Genis-GBlvez, J. M., de Castro. J. M. and Battaner, E. (1968). Lens soluble proteins: Correlation with the cytological differentiation in the young and adult organ in the chick. xatu.rr
(Londm)217,652. Getis-G&lvez, J. M., Maisel, H. and de Castro, J. (1968). Changes in chick lens prot,eins with ageing. Exp. Eye Res. 7, 593. Hoenders, H. J. (1965). Uber die Proteinzusammensetzung der Augenlinse des Huhns. Thesis, Amsterdam. Holt, W. S. and Kinoshita, J. H. (1968). Starch gel electrophoresis of the soluble lens proteins from normal and galactosemic animals. Invest. Ophthdmol. 7, 169. Ikeda, A. and Zwaan, J. (1967). The changing cellular localization of a-crystallin in the lens of the chick embryo studied by immunofluorescence. Develop. Biol. 15,348. Maisel, H. and Goodman, M. (1965). Comparative electrophoretic study of vertebrate lens proteins. Amer. J. Ophthulmol. 59, 697. Maisel, H. and Langman, J. (1961a). An immuno-embryological study on t,he chick lens. J. Embryol. Exp. Morphol. 9, 191. Maisel, H. and Langman, J. (1961b). Lens proteins in various tissues of the chick eye and in the lens of animals throughout the vertebrate series. Anat. Rec. 14, 183. Rabaey, M. (1962). Electrophoretic and immunoelectrophoretic studies on the soluble proteins in the developing lens of birds. Exp. Eye Res. 1, 310. Rabaey, M. (1968). Immunology in relation to the proteins of the normal lens. In Biochemistry of the E’ye. XX Int. Congr. Ophthalmol. (Ed. Dardenne, M. U. and Nordmann, J.). P. 301. Karger, Basel. Rana, M. W. and Maisel, H. (1969). Subunit composition of chick lens a-crystallin. Exp. Eye Res. 8, 216. Rana, M. W. and Maisel, H. (1970). Th e subunit structure of chick lens beta-crystallins. Ophthal. Res. 1, 156. Shapiro, A. L. (1968). Subunit comparison of rabbit lens beta-crystallin. Invest. Ophthalmol. 7,554. Spector, A. and Katz, E. (1965). The deaggregation of bovine lens a-crystallins. J. Biol. Chem. 240, 1979. Spector, A. and Katz, E. (1966). Studies upon beta-crystallin. Docum. Ophthalmol. 20,44. Stark, G. R., Stein, W. H. and Moore, S. (1960). Reactions of the cyanate present in aqueous urea with amino acids and proteins. J. Biol. Chem. 235,3177. Testa, M., Armand, G. and Balazs, E. S. (1965). Separation of the soluble protein bovine lens in polyacrylamide gels. Exp. Eye Res. 4, 327. Truman, D. E. S. (1968). Gel filtration of lens proteins. Exp. Eye Res. 7, 358. Truman, D. E. S., Brown, A. G. and Campbell, J. C. (1972). The relationship between the ontogeny of antigens and the polypeptide chains of the crystallins during chick lens development. Exp. Eye Res. 13,58. Truman, D. E. S., Brown, A. G. and Rao, K. V. (1971). Estimates of the molecular weights of chick /% and S-crystallins and their subunits by gel filtration. Exp. Eye Res. 12,304. Vesterberg, O., Wadstrijm, T., Vesterberg, K. Svensson, H. and Malmgren, B. (1967). Studies on extracellular proteins from StaphyZococus aureus. I. Separation and characterization of enzymes and toxins by isoelectric focusing. Biochim. Biophys. Acta. 133,435. Zigler, J. S. and Sidbury, J. B. (1973). Structure of calf lens /?-crystallins. Exp. Eye Res. 16, 207. Zwaan, J. (1963). Immunochemical analysis of the eye lens during development. Thesis, Amsterdam. Zwaan, J. (1968). Electrophoretic studies on the heterogeneity of the chicken lens crystallins. Exp. Eye Res. 7, 461. Zwaan, J., van Doorenmaalen, W. J. and Hoenders, H. J. (1963). Antigenen van de Kippen-ooglens. 4 de Fed. Vergad. &led. Biol. Veren., Nijmegen. Zwaan, J. and Ikeda, A. (1965). An immunochemical study of the beta-crystallins of the chicken lens. Ontogenic and phylogenetic aspects. In The Structure of the Eye. 2nd. Symposium (Ed. Rohen, J. W.). Sohattaner-Verlag, Stuttgart. Zwaan, J. and Ikeda, A. (1968). Macromolecular events during differentiation of the chick lens. Exp. Eye Res. 7,301.
18, 485-494
The Subunit Structure of Chick fbCrystallins D. E. hstitute
of An&al
X. TRUMAN
ANII
R. M.
Genetics, Bdinburgl~,
(Received 10 Dectmbw
CLAYTOK
EH9
3JN> Scothd
1973, London)
Chick /3-cryst~allins are a class of proteins containing molecules with a range of size and electrophoretic mobility; there are approximately 15 molecular species, six of which are major constituents of the class. They can be fractionated electrophoretically or by isoelectric focusing. The /3-crystallin molecules can be dissociated into subunits and each aggregate contains a number of different types of subunit, differing immunologically, in electrophoretic mobility and in isoelectric point. The same subunit may be present in several different aggregates, the properties of which are a function of the const,ituent subunits.
1. Introduction The class of chick lens crystallins of lowest. molecular weight is variously known as the long-line crystallins, on account of the appearance of the precipitin arc that they form in immunoelectrophoresis (Zwaan, 1963); y-crystallins, because of their low molecular weight and becausesomeof their numbers are of low electrophoretic mobility (Maisel and Langman, 1961a, 1961b; Zwaan, van Doorenmaalen and Hoenders: 1963); and /3-crystallins becausethey are said to have antigenic similarities to bovine /3-crystallins (van Dam, Schalekamp, Schalekamp-Kuyken and ten Gate, 1963; Zwaan and Ikeda. 1968). We have chosen to use the term P-crystallin. They clearly form a class of some complexity and various estimates of the numbers of different proteins and of their molecular weight are to be found in the literature. Two major classesdiffering in molecular size have beeu demonstrated by Hoenders (1965) and by Truman (1968). Zwaan (1968) found that of nine fi-crystallins, five had a molecular weight of 55 000, two were of 60 000, one was 50 000 and one was 40 000. Using gel filtration to estimate molecular weight, Truman, Brown and Rao (1971) estimated a range of molecular weight up to 59 000 but with someP-crystallins with a molecular weight as low as 16 000. Estimates of the number of crystallins in this class have depended on the analytical technique used, Zwaan (1968) finding nine components. Clayton (1969) 12, Truman (1968) 15 and Bours (1971) 19. Clayton and Truman (1967) argued that the /%crystallins must be a family of heteropolymers, each molecule containing a number of subunits, and the sameconclusion was also supported by the results of a seriesof Ossermantests comparing lens proteins with those of other tissues (Clayton, Campbell and Truman, 1968). Zwaan (1968) also suggestedeither a subunit structure or a disparity of size among the immunologically similar proteins of this class of crystallins. Rana and Maisel (1969, 1970) have given clear evidence of a heteropolymer subunit structure, while estimates of sedimentation coefficients (Clayton and Truman, 1967) and of molecular weights (Truman et al., 1971) in the presenceand absenceof urea have also confirmed this view. In dissociative conditions 1I components were detected in polyacrylamide gel electrophoresis of /3-crystallins, and when individual intact crystallins were isolated and dissociated several of these components were obtained from each one of them (Clayton. 1969), the patterns obtained being consistent with the view that t,he /%crystallins are a family of hetero1)olymers. The data presented in this paper. derived from a number of different, 4x5 E
486
11. E.
S. 1’RCiMAS
AND
R.
111. C’LAYTOS
techniques, give further evidence of the heterogeneous nature their relationship to each other as a series of heteropolymers number of subunits.
of these crystallins antI composed of a limited
2. Materials and Methods Lenses The lenses were dissected from the heads of commercially slaughtered birds supplied by D. B. Marshall (Newbridge) Ltd., Midlothian. They were prepared as described earlier (Truman, 1968), except that the capsules were retained after removing extra-lenticular tissue, since removal of the capsule inevitably also removed some epithelial cells from the lens. A preparation of material rich in cells derived from the lens cortex was sometimes made by taking off the capsules and then shaking both lenses and capsules gently in buffer (10 mM-phosphate 10 in&r-2-mercaptoethanol, pH 7.2) until most of the cortical material appeared to have separated from the lens nuclei. The suspension of cortical cells was removed by decantation, homogenized and centrifuged as described earlier for the whole lenses (Truman, 1968; Clayton, 1969). Isolation
of /3-crystallins
The details of the chromatographic techniques used were described by Truman (1968). Soluble proteins prepared either from whole lenses or from cortical fractions were fractionated by gel filtration on a column of polyacrylamide beads (Bio-Gel P-300) to produce a fraction rich in /?-crystallins. This fraction was further purified by repeated gel filtration, concentrating the proteins between runs by precipitation with (NH&SO, to 600/, saturation. Three runs through the gel titration column were usually sufficient to produce material which when tested by immunoelectrophoresis showed only the long precipitin arc characteristic of the /3-crystallins, with no trace of M- or &crystallin. No differences were found in the properties of the /%crystallins prepared from the cortical material as compared with preparations from the whole lens, but it was easier to obtain pure material from the cortical preparations. A partial fractionation of the @crystallins on a basis of molecular size was achieved by gel filtration on the polyacrylamide beads Bio-Gel P-150. These gel filtration media are manufactured by Bio-Rad Laboratories, Richmond, Calif., U.S.A. and were purchased from V. A. Howe and Co. Ltd., London. Isoelectric focusing The apparatus used was based on that described by Vesterberg, Wadstrom, Vesterberg, Svensson and Malmgren (1967) and was manufactured by LKB-Produkter AB, StockholmBromma, Sweden. Columns of both 110 and 440 ml capacity were used and separations were carried out over a variety of ranges of pH as described in the text. The method used was essentially that described in the manufacturer’s instructions, except that 10 mM2-mercaptoethanol was added to all solutions, and in some cases 7 M-Urea was used as the solvent instead of water. After completion of the separation the contents of the column were passed through a Uvicord II ultraviolet absorbtiometer (LKB-Produkter, StockholmBromma, Sweden), set at 280 nm. The pH of the fractions collected was measured at the same temperature as the isoelectric focusing run, approximately 17°C. Fractions were either tested by immunoelectrophoresis or polyacrylamide gel electrophoresis without prior concentration, or several fractions from a single peak were concentrated by precipitation with (NH&SO, to 60% saturation followed by centrifugation and dialysis. Polyacrylanaide gel electrophoresis For tests in the absence of urea the method used was that described earlier (Truman, 1968), and when tests were made in the presence of 6 M-urea the method was the modifi-
j?-CRYSTALLIN
STRUCTURE
4x5
cation described earlier (Clayton, 1969; Truman, Brown and Campbell (1972). Densitometric scanning of the gels stained with Amido black was in the Joyce-Loebel densitometer, using a red filter. 3. Results The low molecular weight crystallins of the chick lens were prepared by gel filtration and tested by immunoelectrophoresis and by disc electrophoresis on polyacrylamide gels. Both these methods failed to detect proteins which were known from previous experiments to represent the LX- and S-crystallins (Truman, 1968). On the polyacrylamide gels the @crystallin preparations showed some 15 bands after staining with Amid0 black, and of these bands about six were particularly intense and presumably represented major constituents in this class of protein (Figs 1 and 2). The relative intensities of the stained bands varied somewhat between preparations, possibly due to the rejection of varying amounts of the proteins which were less retarded on gel filtration and which were thus removed along with &crystallin.
I?IQ. 1. Electrophoretic pattern obtained from chick fi-crystallins separated on polyacrylamide the absence of urea. The origin of the electrophoresis is at the top of the diagram and the anode the bottom.
gel in towards
The preparations of /3-crystallin could be further fractionated by gel filtration on polyacrylamide P-150. In this way two major fractions could be distinguished, one containing five components detectable on polyacrylamide electrophoresis, all of which had relatively high mobility towards the anode, while the other fraction contained 10 components, with a somewhat lower electrophoretic mobility. Electrophoretic patterns obtained with such preparations are illustrated in Fig. 3. When a preparation of /3-crystallins is fractionated by isoelectric focusing over a pH range of 3-8, the heterogeneity of these proteins is seen again. Figure 4 represents
488
LJ.
K.
s.
‘ml-J1
AS
A\ s II
I:.
Il.
(‘r,.-iY’I’os
the absorptiometer trace of a typical isoelectric separation. Fonr major com1)onei~t.x occur, with about six minor components. giving moderate agreement with the results of polyacrylamide gel electrophoresis. The peaks in Fig. 4 are identified t)?- their isoelectric point in that run, but as the temperature regulation of the isoelect,ric focusing column was not precise these values cannot be regarded as definitive valnt!s of the isoelectric points but rather as convenient idenbification of the fractions.
FIQ. 2. Electrophoresis of chick /3-crystallins is to the right and the anode to the left.
FIG. 3. Electrophoresis weight fraction. Lower the left.
on polyacrylamide
gel in the absence of urea. The origin
of chick ,%crystallins fractionated by gel filtration. Upper gel: higher molecular gel: lower molecular weight fraction. The origin is to the right and the anode to
FIG. 4. Ultraviolet absorptiometer trace showing separation focusing over the range pH 3-8 in the absence of urea. The pH peaks is indicated.
of chick @xystallins by isoelectric of the fractions containing the main
When the fractions prepared by isoelectric focusing were tested by electrophoresis on polyacrylamide gels most were found to consist of one major fraction. Figure 5 represents the densitometer traces obtained from the gels. In somecasesit can be seen that there was some cross-contamination between fractions: for example, fraction 6.41 also contains some of the material present in fraction 6.70. Nevertheless the separation obtained is generally good. Fraction 7.61 is somewhat anomalous: the minor component shows a relationship between isoelectric point and electrophoretic mobility similar to the other fractions, but the major component of this fraction has
,9-CRYSTALLIK
1x9
STKUCTlIKE
an abnormally high mobility towards the anode, which presumably indicates relatively low molecular weight. The broadness of this peak, suggesting relatively rapid diffusion of the molecules, also indicates low molecular weight. It may be that the material forming the large peak is derived from the breakdown of that in the smaller peak. The production of material of high electrophoretic mobility from some slower /3crystallins on re-running on polyacrylamide gels has been observed previously (Clayton, 1969). 7.61 A
A
6.70 -
A
64 -
A
5.95 -
A
5.82 3.71-
A A
+o Pm. 5. Densitometer t.races of electrophoret.ic separations cm polyacrylamide fractions obtained by isoelectric focusing of chick /3-crystallins. Each fraction focusing columns is designated by its apparent p1. The origin of the electrophoretic right of the diagram snd the anode to the left.
gels without m-tit of from the isoelectric separation is to the
Effect of dissociatiny media on /3-crystalhas Studies of the gel filtration behaviour of chick /3-crystallins in the presence and absence of concentrated urea solutions have indicated that reagents which break hydrogen bonds may reduce the molecular weight of the ,!3-crystallins from about 60 000 to about 16 500 (Truman et al., 1971). Preliminary studies with the ultracentrifuge also confirm this effect of urea on these proteins (Clayton and Truman. 1967; Clayton, 1970). A preparation of total chick P-crystallins examined in the a,nalytical ultracentrifuge in a buffer of 10 nix-phosphate, IO m&I-%mercaptoethanol at pH 7.2 .at a8protein concentration of 8 mg/ml showed a broad peak in schlieren optics with a sedimentation coefficient, sz,,.+ of 6~44% When similar material was dialysed against a similar buffer to which urea had been added to a concentration of 8 M, t)hen the sedimentation velocity was reduced to an s,~,~ of 1*%38.
FIG. 6. Electrophoresis anode to the lvft.
of j%crystallins
in the prescncc
of 6 n~-urca.
The origin
is t,o the right
:uxl C~C
When a preparation of the total @rystallin proteins was dissociated in urea solution and then electrophoresed on polyacrylamide gels in the presenceof 6 M-urea, 11 components were generally seen (Clayton, 1969) (Fig. 6). When electrophoresis in
490
I,.
F:. s.
TKI-MAN
>iSD
1:,. !vl. (‘LAYTON
urea was carried out on starch gel at an acidic pH, eight components were detected (Truman et al., 1971). These results indicate that a variety of polypeptide cheins are involved in the aggregate molecules in the /3-crystallin class. After separation of the /3-crystallins by isoelectric focusing, electrophoresis on polyacrylamide gels in the presence of urea indicated that each fraction contained more than one component after dissociation. The densitometer traces obtained from both undissociated and dissociated samplesof someof the /3-crystallin fractions are set out in Fig. 7. All of these
L I!!-L6.70
6.41
4.78
L-
5
t FIQ. 7. Comparison of of urea of three fractions meter traces is of runs in and anode to the left of
0
0
+
the results of electrophoresis on polyacrylamide gel in the presence and absence obtained by isoelectric focusing of chick b-crystallins. The upper set of densitothe absence of urea, the lower set of runs in 6 M-urea. The origin was to the right each trace. 3.71
4.44
4.78
5.82
5.95
-
-
6.41
6-70
Fro. 8. Diagram showing the major subunits of various fractions of /I-crystallins. Each column represents an electrophoresis on polyacrylamide gel in 6 M-urea, with the origin at the top and the anode at the bottom. The fractions are debignated by their apparent p1 when prepared by isoelectric focusing.
p-CRYSTALLIN
STRUCTURE
491
fractions yield more than one component when tested in dissociating conditions. Each fraction has components similar in electrophoretic mobility to some of the components in other fractions, but the average mobility of the subunits of any one fraction corresponds to the mobility of the aggregate from which they are derived. From the analysis of each fraction in undissociating conditions, we can make some estimate of the likely contamination of each fraction and so can make allowance for the possibility of subunits resulting from contaminating material. It is then possible to estimate the minimum number of types of subunit which must be present in each fraction of the /Scrystallins prepared by isoelectric focusing. Figure 8 is a diagram representing the composition of some of the p-crystallin fractions. If it is assumed that components of the same mobility derived from different fractions are identical, then the total number of different subunits derived by dissociating the /3-crystallins is 12. This is in good agreement with results obtained by dissociating unfractionated preparations of chick /3-crystallins. Ixoekctric focusing in dissociating media Isoelectric focusing carried out with 8 M-urea present in all solutions, over the pH range 5-8, using a preparation of the total /3-crystallins of the chick, gave rise to the elution pattern shown in Fig. 9. About 11 peaks can be distinguished though the quantity of material in some fractions is relatively small. There is a good general agreementbetween this elution pattern obtained by isoelectric focusing and the pattern obtained from polyacrylamide gel electrophoresis in the presenceof urea, the electrophoretic mobilities apparently being a simple function of isoelectric points.
BIG.
focusing
9. Ultraviolet absorptiometer in the presence of 7 M-urea
trace showing separation of subunits over the pH range 5-8. The apparent
of @rystallins by isoelectric p1 of each peak is indicated.
4. Discussion The class of chick lens structural proteins generally known as the long-line or pcrystallins clearly contains a number of different molecular species. The results presented here show that 10 proteins can be separated by isoelectric focusing. Zwaan (1968) has used two-dimensional polyacrylamide gel electrophoresis to determine the molecular weights of nine chick /Scrystallins and Hoenders (1965) used sedimentation behaviour and electrophoretic properties in an extensive study which indicates the
492
n.
E.
s.
TIt!uJIAN
ANI)
IL.
N.
(‘I,AV’I’UN
occurrence of at least five /3-crystallins. In t#he face of such evidence, what arc’ thtb grounds for considering these proteins as a single class? In the development of the ItJns the P-crystallins do not appear simultaneously, the number of P-crystallin co~uponents increasing from the embryonic stage 15, while changes in the proport,ions of /% crystallin components continue after hatching (Rabaey, 1962: 1966; Zmaan. 1963 : van Doorenmaalen, 1964; Zwaan and Ikeda, ~1965: 1968; Ikeda and Zwaan. 1967: Genis-Galvez, Maisel and de Castro, 1968; Genis-Galvcz, de C’astro and Battancr. 1968; C!layton, 1970; Truman et al., 1972). The ontogeny of the subunits of the ,& crystallins also indicates that these proteins do not form a single functional unit, (Clayton and Burns, in preparation). Though the chick P-crystallins have in cotmnou the fact that they are smaller molecules than either M- or %crystallin, they are nevertheless heterogeneous in size (Hoenders, 1965; Truman, 1968; Zwaan, 1968; Truman et al., 1972). The justification for considering these proteins t,ogether must remain the immunological similarity that they shop, especially in immunoelectrophoresis where they appear as conjoint arcs with spurs. This is also particularly clearly seenwhen one of our antisera against Xe~zopusZaevislens is used to react wit,11 chick lens (Clayton and Truman, 1967, 1974). The P-crystallins are then alone in reacting and a continuous precipitin line is formed showing that at least one antigenic determinant is shared by these proteins. Zwaan (1968), in considering his electrophoretic data on chick crystallins, suggested that either the P-crystallins consisted of similar but not identical polypeptide chains, or that they were composedof similar subunits. The evidence put forward here that each P-crystallin molecule contains a number of different subunits, that different crystallins consist of different combinations of subunit,3, with the samesuburlit being shared by several crystallins, and that the electrophoretic behaviour of the aggregates is a function of that of their constituents, is a further confirmation of the data put forward earlier (Clayton and Truman, 1967: 1968; C’layton et al., 1968: Clayton, 1969: Rana and Maisel, 1969, 1970). Much of the evidence for the presence of different types of subunit in aggregate molecules is derived from electrophoretic studies made in the presence of urea ancl the possibility of the formation of artefacts must be borne in mind, in particular the possibility of carbamoylation of proteins by alkaline urea solutions (Stark, Stein and Moore, 1960). However, we find that the major subunits, at least: can be resolved by starch gel electrophoresisin urea at an acidic pH (Truman et al., 1971) and so it scenls probable that only minor components, at the most, could be artefacts. The results of the electrophoresis described here are essentially qualitative and the number of bands seen does not give any indication the proportions of the different subunits within the aggregate. Our previous work (Truman et al., 1971) has indicated that the P-crystallins can exist in a size range from monomersto tetramcrs. Since no rigorously separated /3-crystallin component appears to contain more than three different subunits the results of the subunit analysis are consistent with the molecnlar weight estimations. The exceptional complexity of the chick @rystallins as a class is in agreement with analyses of other species.For example, 11 components were found among the P-crystallins in various speciesof monkey by Ma&e1 and Goodman (1964) using twodimensional electrophoresis and Holt and Kinoshita (1968) found 11 component’sin the rat. Bovine P-crystalline are also known to be complex : Bjijrk (1964) and Spector and Katz (1966) found four fractions and evidence of considerable heterogeneity was found by Testa, Armand and Balazs (1965) van Dam (1966) and Armand, Balazs
j&CRYSTALLIN
STRUCTURE
493
and Testa (1970). A recent study by Zigler and Sidbury (1973) has revealed two groups of /3-crystallins, one containing eight proteins and the other containing at least 10. Evidence that bovine p-crystallins can be dissociated into smaller subunits was found by Bloemendal, Bont, Jongkind and Wisse (1962). Mammalian /I-crystallins appear to contain six to eight different major subunits, and up to 11 have been found when high resolution methods have been used (Spector and Katz, 1965, 1966 ; Shapiro, 1968 ; Day and Clayton, 1972 ; Zigler and Sidbury, 1973). Further references and discussion on the subunit composition of different species will be found in the review by Clayton (1974). Mammalian and bird p-crystallins may have a similar type of subunit composition. Studies by electrophoresis in the presence of sodium dodecyl sulphate (SDS) invariably show that the /3-crystallin subunits have a range of sizes, but electrophoretic studies in dissociating conditions in the absence of SDS indicate that each size class may comprise subunits further distinguished from each other by their charge (Shapiro, 1968; Zigler and Sidbury, 1973). Immunological analysis indicates that these ,&crystallin components are immunologically related (Testa et al., 1965; Rana and Maisel, 1970; Clayton and Truman. 1974). This implies that the p-crystallin polypeptides have been derived by a process of gene duplication followed by divergence (Clayton, 1974). ACKNOWLEDGMENTS
We would like to express our thanks to D. B. Marshall (Newbridge) Ltd. for their continued co-operation in the supply of material for our experiments. We are grateful to Mrs A. G. Brown, Mr A. G. Gillies, Mrs A. Hannah and Mrs H. J. Mackenzie for thei skilled technical assistance. Our work has been supported by grants from the Cancer Research Campaign and the Medical Research Council. REFERENCES Armand, G., Balazs, E. A. and Testa, M. (1970). Separation and partial characterization of two proteins from fraction B of calf lens. Exp. Eye Res. 10, 143. Bjijrk, I. (1964). Fractionation of/?-crystallin from calf lens by gel filtration. Exp. Eye Res. 3,248. Bloemendal, H., Bont, W. S., Jongkind, J. F. and Wisse, J. H. (1962). Splitting and recombination of a-crystallin. Exp. Eye Res. 1, 300. Bours. J. (1971). Isoelectric focusing of lens crystallins in thin-layer polyacrylamide gels. J. Chromatogr. 60,225. Clayton, R. M. (1969). Properties of chick crystallins in terms of their subunit composition. iL:xp. Eye Res. 8, 326. Clayton, R. M. (1970). Problems of differentiation in the vertebrate lens. Curr. Topics De&op. Biol. 5, 115. Clayton, R. M. (1974). Comparative aspects of lens proteins. In The Eye (Ed. Davson, H.). Vol. 5. Academic Press, New York and London. (In press.) Clayton, R. M., Campbell, J. C. and Truman, D. E. S. (1968). A re-examination of the organ specificity of lens antigens. Exp. Eye Res. 7, 11. Clayton, R. M. arid Truman, D. E. S. (1967). Molecular struct’ure and antigenicity of lens proteins. Nature (London) 214, 1201. Clayton, R. M. and Truman, D. E. S. (1968). The structure and cross-reactivity of chick lens proteins. J. Physiol. (London) 198, 72P. Clayton, R. M. and Truman, D. E. S. (1974). The antigenic structure of chickp-crystallin subunits. Exp. Eye Res. 18, 495. van Dam, A. F. (1966). Purification and composition studies ofp crystallin. Exp. Eye Res. 5,255. van Dam, A. F., Schalekamp, M. A., Schalekamp-Kuyken, M. and ten Cate, G. (1963). Immunoelectrophoretio studies on adult and embryonic ocular lenses. Vlth Internat. Embryol. Confwence Helsinki. F
494
D.
E.
S. TRUMAN
AND
R.
N.
CLAYTOK
Day,
T. H. and Clayton, R. M. (1972). Multiple changes in lens proteins composition associated with Catrr. gene in the mouse. Genet. Res. Camb. 19, 241. van Doorenmaalen, W. J. (1964). Immunohistological studies of proteins during the development of chick lens. In Methodes Nouvelles en Embryologic (Ed. Wolff, Et.). P. 151. Hermann, Paris. Genis-GBlvez, J. M., de Castro. J. M. and Battaner, E. (1968). Lens soluble proteins: Correlation with the cytological differentiation in the young and adult organ in the chick. xatu.rr
(Londm)217,652. Getis-G&lvez, J. M., Maisel, H. and de Castro, J. (1968). Changes in chick lens prot,eins with ageing. Exp. Eye Res. 7, 593. Hoenders, H. J. (1965). Uber die Proteinzusammensetzung der Augenlinse des Huhns. Thesis, Amsterdam. Holt, W. S. and Kinoshita, J. H. (1968). Starch gel electrophoresis of the soluble lens proteins from normal and galactosemic animals. Invest. Ophthdmol. 7, 169. Ikeda, A. and Zwaan, J. (1967). The changing cellular localization of a-crystallin in the lens of the chick embryo studied by immunofluorescence. Develop. Biol. 15,348. Maisel, H. and Goodman, M. (1965). Comparative electrophoretic study of vertebrate lens proteins. Amer. J. Ophthulmol. 59, 697. Maisel, H. and Langman, J. (1961a). An immuno-embryological study on t,he chick lens. J. Embryol. Exp. Morphol. 9, 191. Maisel, H. and Langman, J. (1961b). Lens proteins in various tissues of the chick eye and in the lens of animals throughout the vertebrate series. Anat. Rec. 14, 183. Rabaey, M. (1962). Electrophoretic and immunoelectrophoretic studies on the soluble proteins in the developing lens of birds. Exp. Eye Res. 1, 310. Rabaey, M. (1968). Immunology in relation to the proteins of the normal lens. In Biochemistry of the E’ye. XX Int. Congr. Ophthalmol. (Ed. Dardenne, M. U. and Nordmann, J.). P. 301. Karger, Basel. Rana, M. W. and Maisel, H. (1969). Subunit composition of chick lens a-crystallin. Exp. Eye Res. 8, 216. Rana, M. W. and Maisel, H. (1970). Th e subunit structure of chick lens beta-crystallins. Ophthal. Res. 1, 156. Shapiro, A. L. (1968). Subunit comparison of rabbit lens beta-crystallin. Invest. Ophthalmol. 7,554. Spector, A. and Katz, E. (1965). The deaggregation of bovine lens a-crystallins. J. Biol. Chem. 240, 1979. Spector, A. and Katz, E. (1966). Studies upon beta-crystallin. Docum. Ophthalmol. 20,44. Stark, G. R., Stein, W. H. and Moore, S. (1960). Reactions of the cyanate present in aqueous urea with amino acids and proteins. J. Biol. Chem. 235,3177. Testa, M., Armand, G. and Balazs, E. S. (1965). Separation of the soluble protein bovine lens in polyacrylamide gels. Exp. Eye Res. 4, 327. Truman, D. E. S. (1968). Gel filtration of lens proteins. Exp. Eye Res. 7, 358. Truman, D. E. S., Brown, A. G. and Campbell, J. C. (1972). The relationship between the ontogeny of antigens and the polypeptide chains of the crystallins during chick lens development. Exp. Eye Res. 13,58. Truman, D. E. S., Brown, A. G. and Rao, K. V. (1971). Estimates of the molecular weights of chick /% and S-crystallins and their subunits by gel filtration. Exp. Eye Res. 12,304. Vesterberg, O., Wadstrijm, T., Vesterberg, K. Svensson, H. and Malmgren, B. (1967). Studies on extracellular proteins from StaphyZococus aureus. I. Separation and characterization of enzymes and toxins by isoelectric focusing. Biochim. Biophys. Acta. 133,435. Zigler, J. S. and Sidbury, J. B. (1973). Structure of calf lens /?-crystallins. Exp. Eye Res. 16, 207. Zwaan, J. (1963). Immunochemical analysis of the eye lens during development. Thesis, Amsterdam. Zwaan, J. (1968). Electrophoretic studies on the heterogeneity of the chicken lens crystallins. Exp. Eye Res. 7, 461. Zwaan, J., van Doorenmaalen, W. J. and Hoenders, H. J. (1963). Antigenen van de Kippen-ooglens. 4 de Fed. Vergad. &led. Biol. Veren., Nijmegen. Zwaan, J. and Ikeda, A. (1965). An immunochemical study of the beta-crystallins of the chicken lens. Ontogenic and phylogenetic aspects. In The Structure of the Eye. 2nd. Symposium (Ed. Rohen, J. W.). Sohattaner-Verlag, Stuttgart. Zwaan, J. and Ikeda, A. (1968). Macromolecular events during differentiation of the chick lens. Exp. Eye Res. 7,301.