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Distribution of non-diffusible calcium and sodium in normal and cataractous human lenses
Exp. Eye Rrs. (1977) 25, 183-193
Distribution of Non-diffusible Calcium and Sodium in Normal and Cataractous Human Lenses G. DUNCAN* XqjYeld
AND RUTH
VAN HEE-NIXBEN
Laboratory of Ophthalmology, Waltorb Streetp Oxford,
lhiwrsity
of Oxford.
En~gland
Clear post-mortem (normal) and cataractous lenses (with high sodium and calcium levels) were used in this study. When homogenates of lenses of both types were dialysed against an isosmotic Tris buffer, the diffusion of calcium was found to be much slower than that of sodium. About 30yo of the initial calcium remained after 15 hr whereas less than 5y; of the sodium was still associated with the homogenate. It was concluded that almost all of the sodium ions are in a relatively free and mobile state in both types of lenses, whereas some of the calcium ions appear to be bound to certain species within the lens matrix. Water-soluble and water-insoluble fractions were dialysed separately and, in the normal lens fractions, most of the non-diffusible calcium was found in the soluble fraction. In contrast, the insoluble component contained most of the bound calcium in the cataractous lens fractions after dialysis. In both types of lens the nuclear fract’ions were found t,o contain the highest proportion of bound calcium. Sodium and calcium analyses were also carried out on the soluble proteins separated by column chromatography (Bio-gel A 5 m). The a-crystallin and high molecular-weight fraction contained most of the protein-bound calcium in normal and cataractous lenses. Alt,hough a higher proportion of high molecular weight fract,ion was found in the cat,aractons lenses, t’here was little difference between the calcium density (moles calcium bound per optical density unit at 280 nm) of the high molecular weight and that of the ic-rrystallin fractions; however, the calcium densities of these fractions were greater than the corresponding densities of the fractions from normal lenses. h’ey 1cor~1.s: lens; non-diffusible calcium; sodium; cataract; cc-crystallin; high molecularweight fraction.
1. Introduction There have been many determinations of the changes in ion levels associat,edwith cataract (seeBellows, 1975for a review of the earlier literature) and the most recently reported data indicate that severely disrupted ion distributions are a feature of cortical rather than nuclear cataracts in man (Naraini and Mangili, 1973; Duncan and Bushell. 1975). In some lensesthe sodium concentration exceeds that of the aqueoushumour 11131 over 50 mmol/kg water while the calcium excess is of the order of 15 mmol/kg water. These high values could arise as a result of an internal negative Donnan potential of the order of a few millivolts for sodium and about 30 mV for calcium. Duncan and Bushel1(1976) have indirect evidence to show that the elevatetl sodium concentration could arise from Donnan forces alone, whereas the very high calcium concentration must require the action of additional binding or complexing forces. Ilnncan and van Heyningen (1976) have recently found t’hat both normal and highcalcium cataractous lensesbind significant amounts of 45C:awhen incubated in isosmot,ic sodium-free solutions containing 1 mM-CaCl,, but only the cataractous lenses are capable of complexing calcium in the presence of EGTA. They conclu~led that there mere differences in the calcium complexing sites in thr t,wo types of lens, Ijut did not determine the location of the sites. Jedziniak, Nicoli, Yates and Benedek (1976) have recently determined the calcium associated with different protein fractions of cataractous lenses.After separating thr * present I?
address:
School
of Biological
Sciences,
University 183
of East Anglia.
Norwich,
England.
181
(I.
I)U?u’(‘.-\S
ASI)
I!. \‘AS
HfCYSIIi(:ES
soluble proteins on an agarose column they found that the first l)e;tk (high t~~(~l(!(*~~lit~t~ weight fraction) contained three times as much calcium as the second. lower t11ol(‘~111i~l, weight peak. The calcium densities of the water-insoluble fractions were of t ht. ,S;ilIII1’ order as those of the soluble? low molecular weight fractions. The cataract)orls I~LIIS(Y were classified according to the colour scheme of Pirie (1968) and there \vit~ IIII significant differences in the calcium densities of the various groups. &As sr~vcral lenses have to be pooled for each experiment. the scheme chosen for classification i< of t’he utmost importance in this type of investigation. Van Heyningen (197&L) Ita< shown that the sodium data from lenses classified according to the colour scheme arta quite variable and Duncan and Bushel1 (1975) 1lave also demonst)ratetl a relationship between sodium and calcium content. As a starting point in an investigation of calcium different protein fractions of the lens, we have chosen to investigate lenses that. have a known high calcium content (Group c1 according to the scheme of Duncan antI Bushell, 1975) and we have used clear post mortem lenses as controls. In this study we have determined the relative amounts of non-diffusible calcium associated with the water-soluble and water-insoluble proteins of lens cortex ant1 nucleus. In both normal (post-mortem) and high calcium cataractous lenses we. have found that most of the calcium binding sites are located in the nucleus. In the normal lens non-diffusible calcium is largely associated with the soluhle prot’eins whereas in the cataractous lens (where the total calcium concentration is much higher) most of the bound calcium is found in the insoluble fraction. 2. Materials
and Methods
Materials
Human lensesreferred to as “normal” were clear lensesobtained within 48 hr postmortem. Human cataractous lenseswere those removed in the Oxford Eye Hospital and placed in a weighed polythene pot which was stored frozen until required. Those w&h colourednuclei (Group II and III, Pirie, 1968)were primarily used,but a brief comparative study wasalsomadewith lensesin Groups I to III. Ion
analyses
When required, the lenseswere thawed at room temperature and a one-third segment (approximately) was dissectedout and weighed. The segmentwas then homogenizedin 10 ml ice-cold 4% trichloroacetic acid (TCA) and the homogenatecentrifuged at 5000x g for 15 min. The sedimentwas dried overnight at 60°Cand the difference betweenthe wet weight of the segmentand the dry weight of the precipitate gave the weight of water in the segment. The supernatant was analysed for sodium and calcium by conventional flame-photometric techniques(Duncan and Bushell, 1975).Only cataractous lenseswith :I sodiumvalue of greater than 80 mmol/kg water (group C cataracts by the sodium-ba,sed classificationschemeof Duncan and Bushell, 1975) were analysed in detail in this study as all lensesin this category had a calcium concentration of greater than 10 mmol/kg water. D@LS~OPZ
studies
The detection limit for calcium in the atomic absorption machine used in this study wasof the order of 1 pmol and in order to obtain a sufficient calcium concentration in the various samplesit was necessaryto pool several lenses(usually four) for one experiment. The buffer used for the diffusion studies (100mM-Tris adjusted to pH 7.2 with 6 M-HCl)
NON-DIFFUSIBLE
LEES
C’ALCIUX
1x5
contained less than lo? mol/litre calcium and sodium and so as far as the present, studies are concerned, was referred to as calcium and sodium-free. (a) Di$ksio?a a,>~two third sectors. The remaining two third sectors from four normal autl from four cataractous lenses were pooled. Each group was placed in a dialysis SW with 4 ml Tris--HCl buffer and dialysed against 20 ml volumes of the same buffet at 4‘C’. The tliffusates were changed after 1 hr (DJ and 3 hr (D,) ;mtl the experiment terminated after 15 hr (DJ. At the end of the dialysis period. the material in the bags \ras centrifuged at 15 OOO>:g to obtain the supernatant (S,). The sediment was ground up in 4 ml 4’$/, TCA solutions, and centrifuged at NOOi
The content at, I 1 hr was given by the above sum minus D, a,nd similarly, the amount,s itt t 3 11r ant1 f 15 hr were obtained by further subtraction of L), and then L),,. I<) t,llis method a diffusion curve giving the ion content of the lens as a function of time could br drawn (see Fig. 1). The ion contents at the variolls times are expressed as a percentage of their initial value. As both the dry weight and wet weight of the lens material was known. the (l;LtiL could also be expressed in terms of mmol/kg lens water and mmol/kg dry weight. (h) D$M’o~~ ilr ZWS hol)zoyenates. The two third sectors front four lenses were homogenized in 4 ml Tris -HCl buffer and t#he homogenate dialysetl and analysed as tlesc~rihect in section (a). (c) Diffwfo-1~ 1~ cater-soluble and water-imolnble fractions. Two third sectors from foul, ienses were homogenized in 4 ml Tris-HCJ buffer end the homogenates centrifuged ;tt. I.3 000 ;< g for 15 n&l at 10°C to obtain the water-soluble (supernatant) and water-insoluble (sediment) fractions. The supernatant and the sediment (resuspeuded in 4 ml buff’er) wer(’ clialysetl separately against Tris buffer and the ion content at different times and iLlso t,trta tinal dry weightas were determined as in sect,ion (a). ((1) I)ifl~siorr it/ brl~s ~~&us MN? Iells cortex. In some experiments the nuclear (inriel 100 Ilrp) ant1 cortical regioris from four lelises w\-er61 p(~ole(l all(I ctiwlysed separately it’: tlescrit)rtl in sectiou (a).
Frcrctiouution
of
sohble
proteins
Two-third sectors from four to six normal lenses or from four cataractous lenses were homogenized in 4 ml Tris buffer and the water-soluble preparation obtained as described J,ove. This was loaded on to a Bio-Gel A5 m column (0.62 x0.03 m) previously washed ~(1 equilibrated wit#h 100 mM-Tris-HCl buffer (pH 7.3). Th e fl 01s rate was approximately 25 ml hr.1 and the fractions were collected at 15 min intervals. The optical densit,\((I.D. 280 nm) of the odd-numbered fractions was determined and sodium and calcium ;bllalyses carried out on the remaining fractions after they had been acidified with TCA to iL final concentrat,ion of 4%. Because the sodium and calcium concentrations in the fraction tubes were low, it was important to obtain a low and steady “background” from the column. Thorough washing with calcium-free buffer was essential. Experiments where the mean calcium concentration of the first 15 fractions (before the proteins eluted) was greater than 5x10~-6mol litre-.* (i.e. :&bout 3,0~10-5mmol/fraction were abandoned and so were those where the conc*rntration from sample to sample in the initial period varied widely. In the experiments which satisfied our criteria, the mean calcium concentration of the initial 15 samples was F*
3. Results (a)
Conpwisorb
of normal
lenses
and
hiylr
ccclciztn~
cutnrircts
When two-third portions of normal and cataractous lenses (both st’ored f’roxru and then thawed) were dialysed against lG0 mM-Tris--HCl buEer (Fig. I). calcium was found to diffuse more slowly from cataractous than from t,he normal lenses.‘1’11~ rate of loss of calcium was much slower than the concomitant, lossof sodium and thtl slow efflux of calcium was still observed when the lenseswere homogenized l~fore dialysis (closed symbols in Fig. 1). These experiments indicate that a large fraction of the calcium in both normal and cataractous lensesis bouncl to speciest,hat art’ fixed within the lens matrix. The sodium data, on the other hand. suggest,t,hah this iota is mainly in the free ionized state in both normal and cataractous lenses.
-
Dialysis
Pm. 1. Net movements of tous human lenses dialysed Cataractous segments: A, genates. The lines through figure with the diffusion of and Bushell, 1976).
__i
time
(hr)
calcium and sodium from 2/3 sectors and homogenates of normal and cataracagainst sodium-free buffer. Normal segments: 0, calcium: 0, sodium. calcium; @, sodium. The corresponding filled symbols refer to lens homothe sodium points also fitted the data at t = 15 hr. Compare data in this calcium (slow) and sodium (fast) in normal bovine lenses (Fig. 8 in Dunrat~
When the soluble and insoluble fractions are dialysed separately, a clear difference between normal and cataractous lensesemerges(Fig. 2). At the end of the dialysis period the bulk of the non-diffusible calcium in the normal lensesis located in the soluble fraction, whereasby far the greatest proportion of calcium in the cataractous lensesis to be found in the water-insoluble component. As the total calcium content and the relative amounts of soluble and insoluble material are different in the normal and cataractous lens [Fig. 2(a)] it is important in this caseto express the data on a dry weight basis. Table I showsthat in the normal lens, there is little difference in the calcium content of the two fractions after 15 hr dialysis, when the data are expressed
NON-DIFFUSIRLE
LENS
C’ALC’ICTM
1%
100 -Cakium 90
LMS
-
ao-
Total type wet weight Normal 579.9 Cataract 481 . I
2
(0)
fractionation Dry weights soluble insol. 954 53.7 27*3 106.4
q
Calcium content 2.5 17.2
Solvble
fraction
Insoluble
L .-; z: D : E .z z 2 c .t_ z
fraction
I lOO--
(b)
go-
Sodium
fractionation
Lens type
80-
Normal Cataract
70 -
Sodium
content
56 mM /kg water 120 91 11 11
5
GO; 6O-E
:=
E”
cl
50 -
Soluble Insoluble
40-
Dialysis
fraction fraction
time (hr)
FIG. 2. (a) Calcium content of soluble and insoluble fractions from normal and cataractous lenses dialysed separately against sodium-free buffer. The dry weights (TCA precipitates) are given in mg and the concentrations in mM/kg lens water. The first and second columns at each dialysis sampling time show the data from normal and cataractous lenses respectively. (b) Sodium content of soluble and insoluble fractions from normal and cataractous lenses.
on a dry weight, basis.The insoluble fraction from the cataractous lenses,after dialysis, however, contains considerably more calcium on a dry weight basis than the soluble component. The data for the relative amounts of sodium in the two fractions remaining in the sac during dialysis [Pig. 2(b)] indicate that about 2% of the initial sodium is still associatedwith the soluble fractions of both lensesafter dialysis, whereasthe insoluble fractions conta,in insignificant amounts (0.1%). These data prove that the large amounts of calcium found in the different lens fractions after 15 hr dialyses were not tjhe result) of an inefficient dialysing procedure.
The sodium data from the same lenses [Fig. 3(h)] indicate onw mow t hat t II in species is readily exchangeable and the little sodium that remains after tlialvfis i< located mainly in the soluble fractions.
1004-n
Calcium
fractionation
Sodium
(nucleus
fractlonotion
(nucleus Sodium
Normal cataract
64 m&kg water 110 1’ 1’ 8’
Soluble
cortex
Soluble
nucleus
Oiolysis
3. (a) Calcium content of nuclear and cortical separately. See legend to Fig. 2 and text for further fractions from human lenses analysed separately. FIG.
and
Lens type
0
(0)
and cortex)
cortex)
content
q n
Insoluble
cortex
insoluble
nucleus
time (hrl
fractions from human lenses dialysed and analyscd details. (h) Sodium content of nuclear and cortical
NON-DIFFUSIBLE
LESS
CALC’lIThI
I X!l
It is obviously important to fractionate t,he insoluble component. still further to determine the nature of calcium binding aites in the cataractous lens. However, most. of the procedures we have tried so far, including enzymat,ic digestion. urea solubilization and lipid extraction have all introduced intolerably high calcium contamination levels and our data from these procedures are not yet reliable. An indirect method of’ attack on this problem may now he possible as Duncan and van Heyningen (1976) have recently shown that over 50yb of t’he complexed calciun~ in cataractous lenses can be replaced by radioa.ctive calcium after incubating for 15 hr in a sodium fret: In&~r. This preloading method may provide a means of obtaining a detailed eqtimattb of the calcium in various sub-fractions of the insoiuble component, where a nalciutll calcium exchange occurs. ‘VVe did. however, find that it was possible to det~ermiue the calcium antl aotliurll ihsSOc:i:~t~ed with i-he different protein components iti the solul)le fractions of lct~s IIO~IO--
(0)
+B+y-i
(0)
3
2-
I
2.25
A
-1 - IfI 7 j-0’15; 0 I ;
.g 0” = .o ‘i o
I
0.8
I
j t
(b) 0.7
-
0.6
-
0.5
-
0.4
-
- 3.0
i 8 s
- 2.25
-
;
I.5
- 0.75
0
20
40
60
60
100 Fmction
120 140 number
FIG. 4. (a) Calcium density of proteins isolated from normal was washed and equilibrated with calcium-free 0.1 x-Tris-HCI collected. HM, high molecular weight fraction; cx, ar-crystallin; weight of lenses, 1184 mg. (b) Calcium denqity of cataractous 435
mg.
160
160
I 206
human lenses. The column (Bio- Gel Mm) buffer, pH 7-3 and 4 ml fractions wcrc 8, fi-crystallin: y, y-crystallin. Total wrt lens proteins. Total wet weight of lensrs.
cr-crystallin
[Fig. 5(a),( 1J)]. (a)
-6
0.4 -
Fraction 5. (a) Sodium legend to Fig. 4 and wet weight of lenses, FIG.
number
Total associated with normal lens proteins. text for further details. (b) Sodium associated 425 mg.
wet with
weight of cataractous
lemen, 1184 lens proteins.
mg. See Total
As many laboratories use nuclear colour alone as a basis for classifying (Brie. 1968). rather than colour combined with sodium content as we have done, we carried out a brief comparative study of the distribut,ion of indiffusible calciunl in I he tlifferent groups. The calcium associated with tbe soluble and insoluble fractions were determined after 15 hr dialysis as described in Xet’hotl. Y:.sect’ion C’. ant1 the intl icate (Tahlr 3) that as the soluble/insoluble weight ratio tlecreases, t,hr: proportion of the total c;~kium associated with the insohtble fraction increases. \Vr: (lit1 JW~. howcwr. carry out a comprehensive st,udy as both the sodiunI anti calciu~t~ conwntr:tt ions varietl gr&ly within each class [see also van Hrpningrn. 1972(l))]. Cat~2UiWl
Clitt;l.
4. Discussion The calcium and sodium concentrations of the post-mortem lenses are higher t hall those normally, found in mammalian lenses and are indeed higher than those found ill certain cataracts (Group A4according to the scheme of Duncan and Busl~ell. 197.5). .\ net influx of both sodium (van Hey&ngen, 1972) and calcium has probal~ly occurre(I cluring the rela,tively long period (up to 48 hr) before the lenses were availalJt~ to IIS. However, we have some confidence that these lenses can be treated as normal UNtrols. firstly because the sodium and calcium diffusion characteristics are similar to those found in fresh bovine lenses (Duncan and Hushell. 1976) and secondly I)ecaltscb their calcium binding properties are also similar to those of fresh bovine lenses (I)unoan and van He.yningen, 1976). The slow rate of loss of calcium from the cataractous Itln~ (Fig. 1) is especially interesting as it implies that the large amounts of calcium t,tiat enter these lenses do not remain in a free form. This confirms the suggestion (Duncan and Rushell, 1!375) that the high concentration of calcium in t)heir group C cataracts wa,s not brought about simply by the lens Donnan potential. They suggostet1 that calcium complexing sites were present and it follows from t)his t.hat the rat,t! of loss of calcium from lens homogenates should be slower t.han the concomitant loss 01’ sodium. Fig. I shows this to be the case. When the soluble and insoluble fractions are dialysed separately (Fig. 2) clear clitkrwxs in the calcium distribution between normal and cataractous lenses flmergc’. In the post-mortem lens almost SOY0 of the total calcium is in the soluble fraction ii,n(1 after tlialysis over 20% of the total calcium still adheres t’o this fraction. Only al)out 10?{I of the total is located in the insoluble fraction at the end of t,he experinwnt. Howver, in cataractous lenses, where the dry weight, of the insoluble fractiotl is considerably greater, 30:/, of the total calcium is now locat,ed in this fraction at thcb end of dialysis with only about 2% in the soluble fraction. The calculat,ions in Table 1 show that the calcium content of both soluble and insoluble fractions. expresstld on it. dry weight basis. increases as a result of cataract format,ion hut the relative incwaw in the insoluble fraction is the greater. ‘l’hr~ major proportion of calcium remaining after dialysis in ljoth nornln,l an(l aataractous lenses is in the nucleus [Fig. 3(a)] ant I. when the data are expresstl( 1 on ib clrv weight basis (Table II), the insoluble fraction in the nucleus of cat’aractous lermsf~s b&Is by far the greatest amount of calcium . A41though the proportion of calciunl bound by the soluble fraction in the nucleus of the cataraatons lens is relatively small. t hc soluble proteins have more calcium adhering to theIt at tbc: enct of t,lre (li;l.lvsi< pc~riocl than their counterparts in the normal lens.
Xormal Senile cataract
2.6 4.4
2.3 168
The data were obtained from the soluble and insoluble separately for 15 hr (Fig. 2). See text for further details.
Calcium Soluble cortex ~~~~
Lens type _.__..._ ~
content Soluble nucleus
fractions
of lenses that
had been
dialys(,tl
(m&r/kg dry weight) Insoluble Tnsolublo cortex nWA?lX
Normal Senile cataract
Soluble and insoluble fractions were obtained from computations
from both cortex and nucleus were dialysed separately and the valurs on the data presented in Fig. 3(a) (after 15 hr dialysis).
TABLE
Lens type
III
o/o Ca in soluble fraction
y& Ca in insoluble fraction
Soluble/ insoluble ratio
20.2 10.0 8.2 6.1
13.8 13.5 30.1 35.3
1.3 0.83 0.39 0.20
Totai Na WO% water)
Totai Ca (KlM/iq water)
.Normal Group I Group II Group III
54 68 106 184
1.X 4.2 9.8 12.5
The data represent the mean of two separate experiments (four lenses from each group) cium associated with the soluble and insolubk fractions was obtained after I.5 hr dialysis in the text (see data at t = 15 hr in Fig. 2 for comparison).
and the calas described
There has been much interest over the past few years in the association between calcium and the two soluble protein fractions, high molecular weight and cc-crystallin (Jedziniak, Kinoshita, Yates, Hacker and Benedek, 1973; Spector, Adams and Krul, 1974; Jedziniak, Kinoshita, Yates and Benedek, 1975). In the present experiments, most of the calcium in the soluble fraction of the human lens, that does not appear
SON-DIFFUSIBLE
LENS
CALCIUM
193
to bo in free solution, is associated with the high molecular weight fraction and r,-crystallin [I?& 4(a),(b)]. The high molecular u-eight fraction accounts for a larger proportion of t’he total soluble protein of the cataractous lens [Fig. 4(b)] than of the normal lens [Fig. 4(a)], but this fraction does not appear to have a greater affinity for calcium than cr-crystallin. Expressed on an optical density basis, read at 280 nm, the calcium O.D. ratio (nmol Ca/O.D. unit, calculated from the areas under the peaks) are 90 and 111 for the high molecular weight fraction and x-crystallin respectively in t’he normal lens and 166 and 190 for cataractous lens [Fig. 4 (a),(b):. This is in contrast to t!he findings of Jedziniak, Nicoli, Yates and Benedek (1976) who find clear tliff’erences between t)he high and low molecular weight fractions in cataractous lensea. The! did not, however. find significant differences between the calcium densities of thcl Amble and insoluble fractions that we find, but as they did not determine the calcium in t,he dialysis medium and so cannot express the data on a percentage ha&. it is impossible to carry out a direct comparison. We conclude that the higher moleculatr weight and cr-crystallin fractions bind much more calcium than the ot)her solublt: proteins and that more calcium is bound in the cataractous state. ACKNOWLEDGMESTS
We thank Dr Keith J. Dilley and Dr John J. Harding for many stimulating discussions and Miss Joy Rosserfor excellent technical assistance.G. D. thanks Mr Ant,hony J. Bran for the hospit,ality and facilities of the laboratory and also the Royal National Instit.ute for the Blind, and Researchinto Child Blindness(RICB) for providing financial support. We are also indebted to Rlr Mervyn Gascoyneof the Inorganic Chemistry laboratory of the I7niversity of Oxford for his assist’ance with the atomic absorption measurements. REFEREXCES Bellows, J. G. (1!)75). Cataract ad Abnormalities of the Lens. Prune and Stratton, New York. Duncan, G. and Bushell, A. R. (1975). Ion analyses of human cataractons lenses. Exp. Eye Rex. 20, 223-30. Duncan, G. and Bushell, A. R. (1976). The bovine lens as an ion-exchanger: a comparison with ion IcLvels in human cat,aractous lenses. Exp. &ye Res. 23,341-5X Duncan, G. and van Heyningen, R. (1976). Differences in t.he calcium binding ca.pacit,y of normal and cataractous lenses. DOG. Ophthnl. Proceedings Series 3’0. 9, 15-18. van Heyningen, R. (1972a). The human lens I. A comparison of cataracts ext,racted in Oxford (England) and Shikarpur (W. Pakistan). h’xp. Eye Res. 13, 13647. van Heyningen, R. (1972b). The human lens III. Some observations on the post-mortem lens. Exp. Eye Res. 13, 155-60. Jedziniak, J. A., Kinoshita, J. H., Yates, E. M., Hacker, L. 0. and Benedek, G. B. (1973). On the presence and mechanism of formation of heavy molecular weight aggregates in human normal and cataractous lenses. Exp. Eye Res. 15, 185-92. Jedziniak, J. A., Kinoshita, J. H., Yates, E. M. and Benedek, (:. B. (1975). The concentration and localization of heavy molecular weight aggregates in aging normal and cataractoIls human lenses. Exp. &e Res. 20,367-g. Jedziniak, J. A.. Sicoli, D. F., Yates, E. M. and Benedek, 0. B. (1976). On the calcium concent,ration of catarantous and normal human lenses and protein fractions of cataract,ous lenses. Exp. Eye Rex. 23, 325-32. &s, H. A., Hoenders, H. J. and Wollensak, J. (1976). Protein changes in the human lens during development of senile nuclear cataract. Biochim. Biophys. Acta 434,3243. Maraini, G. and Mangili, R. (1973). Differences in proteins and in the water balance of the lens in nuclear and cortical types of senile cataract. In CIBA Symposium : The Human Lens i,n Relntion to Catamcf. Elsevier. Amsterdam. Pirie, A. (1968). Color and solubility of the proteins of the lens. Inuest. Ophthulmol. 7, 634-42. Spector, A., Adams, D. and Krul, K. (1974). Calcium and high molecular weight protein aggregates in bovine and human lens. Invest. Ophthalmol. 13, 982-W.
Distribution of Non-diffusible Calcium and Sodium in Normal and Cataractous Human Lenses G. DUNCAN* XqjYeld
AND RUTH
VAN HEE-NIXBEN
Laboratory of Ophthalmology, Waltorb Streetp Oxford,
lhiwrsity
of Oxford.
En~gland
Clear post-mortem (normal) and cataractous lenses (with high sodium and calcium levels) were used in this study. When homogenates of lenses of both types were dialysed against an isosmotic Tris buffer, the diffusion of calcium was found to be much slower than that of sodium. About 30yo of the initial calcium remained after 15 hr whereas less than 5y; of the sodium was still associated with the homogenate. It was concluded that almost all of the sodium ions are in a relatively free and mobile state in both types of lenses, whereas some of the calcium ions appear to be bound to certain species within the lens matrix. Water-soluble and water-insoluble fractions were dialysed separately and, in the normal lens fractions, most of the non-diffusible calcium was found in the soluble fraction. In contrast, the insoluble component contained most of the bound calcium in the cataractous lens fractions after dialysis. In both types of lens the nuclear fract’ions were found t,o contain the highest proportion of bound calcium. Sodium and calcium analyses were also carried out on the soluble proteins separated by column chromatography (Bio-gel A 5 m). The a-crystallin and high molecular-weight fraction contained most of the protein-bound calcium in normal and cataractous lenses. Alt,hough a higher proportion of high molecular weight fract,ion was found in the cat,aractons lenses, t’here was little difference between the calcium density (moles calcium bound per optical density unit at 280 nm) of the high molecular weight and that of the ic-rrystallin fractions; however, the calcium densities of these fractions were greater than the corresponding densities of the fractions from normal lenses. h’ey 1cor~1.s: lens; non-diffusible calcium; sodium; cataract; cc-crystallin; high molecularweight fraction.
1. Introduction There have been many determinations of the changes in ion levels associat,edwith cataract (seeBellows, 1975for a review of the earlier literature) and the most recently reported data indicate that severely disrupted ion distributions are a feature of cortical rather than nuclear cataracts in man (Naraini and Mangili, 1973; Duncan and Bushell. 1975). In some lensesthe sodium concentration exceeds that of the aqueoushumour 11131 over 50 mmol/kg water while the calcium excess is of the order of 15 mmol/kg water. These high values could arise as a result of an internal negative Donnan potential of the order of a few millivolts for sodium and about 30 mV for calcium. Duncan and Bushel1(1976) have indirect evidence to show that the elevatetl sodium concentration could arise from Donnan forces alone, whereas the very high calcium concentration must require the action of additional binding or complexing forces. Ilnncan and van Heyningen (1976) have recently found t’hat both normal and highcalcium cataractous lensesbind significant amounts of 45C:awhen incubated in isosmot,ic sodium-free solutions containing 1 mM-CaCl,, but only the cataractous lenses are capable of complexing calcium in the presence of EGTA. They conclu~led that there mere differences in the calcium complexing sites in thr t,wo types of lens, Ijut did not determine the location of the sites. Jedziniak, Nicoli, Yates and Benedek (1976) have recently determined the calcium associated with different protein fractions of cataractous lenses.After separating thr * present I?
address:
School
of Biological
Sciences,
University 183
of East Anglia.
Norwich,
England.
181
(I.
I)U?u’(‘.-\S
ASI)
I!. \‘AS
HfCYSIIi(:ES
soluble proteins on an agarose column they found that the first l)e;tk (high t~~(~l(!(*~~lit~t~ weight fraction) contained three times as much calcium as the second. lower t11ol(‘~111i~l, weight peak. The calcium densities of the water-insoluble fractions were of t ht. ,S;ilIII1’ order as those of the soluble? low molecular weight fractions. The cataract)orls I~LIIS(Y were classified according to the colour scheme of Pirie (1968) and there \vit~ IIII significant differences in the calcium densities of the various groups. &As sr~vcral lenses have to be pooled for each experiment. the scheme chosen for classification i< of t’he utmost importance in this type of investigation. Van Heyningen (197&L) Ita< shown that the sodium data from lenses classified according to the colour scheme arta quite variable and Duncan and Bushel1 (1975) 1lave also demonst)ratetl a relationship between sodium and calcium content. As a starting point in an investigation of calcium different protein fractions of the lens, we have chosen to investigate lenses that. have a known high calcium content (Group c1 according to the scheme of Duncan antI Bushell, 1975) and we have used clear post mortem lenses as controls. In this study we have determined the relative amounts of non-diffusible calcium associated with the water-soluble and water-insoluble proteins of lens cortex ant1 nucleus. In both normal (post-mortem) and high calcium cataractous lenses we. have found that most of the calcium binding sites are located in the nucleus. In the normal lens non-diffusible calcium is largely associated with the soluhle prot’eins whereas in the cataractous lens (where the total calcium concentration is much higher) most of the bound calcium is found in the insoluble fraction. 2. Materials
and Methods
Materials
Human lensesreferred to as “normal” were clear lensesobtained within 48 hr postmortem. Human cataractous lenseswere those removed in the Oxford Eye Hospital and placed in a weighed polythene pot which was stored frozen until required. Those w&h colourednuclei (Group II and III, Pirie, 1968)were primarily used,but a brief comparative study wasalsomadewith lensesin Groups I to III. Ion
analyses
When required, the lenseswere thawed at room temperature and a one-third segment (approximately) was dissectedout and weighed. The segmentwas then homogenizedin 10 ml ice-cold 4% trichloroacetic acid (TCA) and the homogenatecentrifuged at 5000x g for 15 min. The sedimentwas dried overnight at 60°Cand the difference betweenthe wet weight of the segmentand the dry weight of the precipitate gave the weight of water in the segment. The supernatant was analysed for sodium and calcium by conventional flame-photometric techniques(Duncan and Bushell, 1975).Only cataractous lenseswith :I sodiumvalue of greater than 80 mmol/kg water (group C cataracts by the sodium-ba,sed classificationschemeof Duncan and Bushell, 1975) were analysed in detail in this study as all lensesin this category had a calcium concentration of greater than 10 mmol/kg water. D@LS~OPZ
studies
The detection limit for calcium in the atomic absorption machine used in this study wasof the order of 1 pmol and in order to obtain a sufficient calcium concentration in the various samplesit was necessaryto pool several lenses(usually four) for one experiment. The buffer used for the diffusion studies (100mM-Tris adjusted to pH 7.2 with 6 M-HCl)
NON-DIFFUSIBLE
LEES
C’ALCIUX
1x5
contained less than lo? mol/litre calcium and sodium and so as far as the present, studies are concerned, was referred to as calcium and sodium-free. (a) Di$ksio?a a,>~two third sectors. The remaining two third sectors from four normal autl from four cataractous lenses were pooled. Each group was placed in a dialysis SW with 4 ml Tris--HCl buffer and dialysed against 20 ml volumes of the same buffet at 4‘C’. The tliffusates were changed after 1 hr (DJ and 3 hr (D,) ;mtl the experiment terminated after 15 hr (DJ. At the end of the dialysis period. the material in the bags \ras centrifuged at 15 OOO>:g to obtain the supernatant (S,). The sediment was ground up in 4 ml 4’$/, TCA solutions, and centrifuged at NOOi
The content at, I 1 hr was given by the above sum minus D, a,nd similarly, the amount,s itt t 3 11r ant1 f 15 hr were obtained by further subtraction of L), and then L),,. I<) t,llis method a diffusion curve giving the ion content of the lens as a function of time could br drawn (see Fig. 1). The ion contents at the variolls times are expressed as a percentage of their initial value. As both the dry weight and wet weight of the lens material was known. the (l;LtiL could also be expressed in terms of mmol/kg lens water and mmol/kg dry weight. (h) D$M’o~~ ilr ZWS hol)zoyenates. The two third sectors front four lenses were homogenized in 4 ml Tris -HCl buffer and t#he homogenate dialysetl and analysed as tlesc~rihect in section (a). (c) Diffwfo-1~ 1~ cater-soluble and water-imolnble fractions. Two third sectors from foul, ienses were homogenized in 4 ml Tris-HCJ buffer end the homogenates centrifuged ;tt. I.3 000 ;< g for 15 n&l at 10°C to obtain the water-soluble (supernatant) and water-insoluble (sediment) fractions. The supernatant and the sediment (resuspeuded in 4 ml buff’er) wer(’ clialysetl separately against Tris buffer and the ion content at different times and iLlso t,trta tinal dry weightas were determined as in sect,ion (a). ((1) I)ifl~siorr it/ brl~s ~~&us MN? Iells cortex. In some experiments the nuclear (inriel 100 Ilrp) ant1 cortical regioris from four lelises w\-er61 p(~ole(l all(I ctiwlysed separately it’: tlescrit)rtl in sectiou (a).
Frcrctiouution
of
sohble
proteins
Two-third sectors from four to six normal lenses or from four cataractous lenses were homogenized in 4 ml Tris buffer and the water-soluble preparation obtained as described J,ove. This was loaded on to a Bio-Gel A5 m column (0.62 x0.03 m) previously washed ~(1 equilibrated wit#h 100 mM-Tris-HCl buffer (pH 7.3). Th e fl 01s rate was approximately 25 ml hr.1 and the fractions were collected at 15 min intervals. The optical densit,\((I.D. 280 nm) of the odd-numbered fractions was determined and sodium and calcium ;bllalyses carried out on the remaining fractions after they had been acidified with TCA to iL final concentrat,ion of 4%. Because the sodium and calcium concentrations in the fraction tubes were low, it was important to obtain a low and steady “background” from the column. Thorough washing with calcium-free buffer was essential. Experiments where the mean calcium concentration of the first 15 fractions (before the proteins eluted) was greater than 5x10~-6mol litre-.* (i.e. :&bout 3,0~10-5mmol/fraction were abandoned and so were those where the conc*rntration from sample to sample in the initial period varied widely. In the experiments which satisfied our criteria, the mean calcium concentration of the initial 15 samples was F*
3. Results (a)
Conpwisorb
of normal
lenses
and
hiylr
ccclciztn~
cutnrircts
When two-third portions of normal and cataractous lenses (both st’ored f’roxru and then thawed) were dialysed against lG0 mM-Tris--HCl buEer (Fig. I). calcium was found to diffuse more slowly from cataractous than from t,he normal lenses.‘1’11~ rate of loss of calcium was much slower than the concomitant, lossof sodium and thtl slow efflux of calcium was still observed when the lenseswere homogenized l~fore dialysis (closed symbols in Fig. 1). These experiments indicate that a large fraction of the calcium in both normal and cataractous lensesis bouncl to speciest,hat art’ fixed within the lens matrix. The sodium data, on the other hand. suggest,t,hah this iota is mainly in the free ionized state in both normal and cataractous lenses.
-
Dialysis
Pm. 1. Net movements of tous human lenses dialysed Cataractous segments: A, genates. The lines through figure with the diffusion of and Bushell, 1976).
__i
time
(hr)
calcium and sodium from 2/3 sectors and homogenates of normal and cataracagainst sodium-free buffer. Normal segments: 0, calcium: 0, sodium. calcium; @, sodium. The corresponding filled symbols refer to lens homothe sodium points also fitted the data at t = 15 hr. Compare data in this calcium (slow) and sodium (fast) in normal bovine lenses (Fig. 8 in Dunrat~
When the soluble and insoluble fractions are dialysed separately, a clear difference between normal and cataractous lensesemerges(Fig. 2). At the end of the dialysis period the bulk of the non-diffusible calcium in the normal lensesis located in the soluble fraction, whereasby far the greatest proportion of calcium in the cataractous lensesis to be found in the water-insoluble component. As the total calcium content and the relative amounts of soluble and insoluble material are different in the normal and cataractous lens [Fig. 2(a)] it is important in this caseto express the data on a dry weight basis. Table I showsthat in the normal lens, there is little difference in the calcium content of the two fractions after 15 hr dialysis, when the data are expressed
NON-DIFFUSIRLE
LENS
C’ALC’ICTM
1%
100 -Cakium 90
LMS
-
ao-
Total type wet weight Normal 579.9 Cataract 481 . I
2
(0)
fractionation Dry weights soluble insol. 954 53.7 27*3 106.4
q
Calcium content 2.5 17.2
Solvble
fraction
Insoluble
L .-; z: D : E .z z 2 c .t_ z
fraction
I lOO--
(b)
go-
Sodium
fractionation
Lens type
80-
Normal Cataract
70 -
Sodium
content
56 mM /kg water 120 91 11 11
5
GO; 6O-E
:=
E”
cl
50 -
Soluble Insoluble
40-
Dialysis
fraction fraction
time (hr)
FIG. 2. (a) Calcium content of soluble and insoluble fractions from normal and cataractous lenses dialysed separately against sodium-free buffer. The dry weights (TCA precipitates) are given in mg and the concentrations in mM/kg lens water. The first and second columns at each dialysis sampling time show the data from normal and cataractous lenses respectively. (b) Sodium content of soluble and insoluble fractions from normal and cataractous lenses.
on a dry weight, basis.The insoluble fraction from the cataractous lenses,after dialysis, however, contains considerably more calcium on a dry weight basis than the soluble component. The data for the relative amounts of sodium in the two fractions remaining in the sac during dialysis [Pig. 2(b)] indicate that about 2% of the initial sodium is still associatedwith the soluble fractions of both lensesafter dialysis, whereasthe insoluble fractions conta,in insignificant amounts (0.1%). These data prove that the large amounts of calcium found in the different lens fractions after 15 hr dialyses were not tjhe result) of an inefficient dialysing procedure.
The sodium data from the same lenses [Fig. 3(h)] indicate onw mow t hat t II in species is readily exchangeable and the little sodium that remains after tlialvfis i< located mainly in the soluble fractions.
1004-n
Calcium
fractionation
Sodium
(nucleus
fractlonotion
(nucleus Sodium
Normal cataract
64 m&kg water 110 1’ 1’ 8’
Soluble
cortex
Soluble
nucleus
Oiolysis
3. (a) Calcium content of nuclear and cortical separately. See legend to Fig. 2 and text for further fractions from human lenses analysed separately. FIG.
and
Lens type
0
(0)
and cortex)
cortex)
content
q n
Insoluble
cortex
insoluble
nucleus
time (hrl
fractions from human lenses dialysed and analyscd details. (h) Sodium content of nuclear and cortical
NON-DIFFUSIBLE
LESS
CALC’lIThI
I X!l
It is obviously important to fractionate t,he insoluble component. still further to determine the nature of calcium binding aites in the cataractous lens. However, most. of the procedures we have tried so far, including enzymat,ic digestion. urea solubilization and lipid extraction have all introduced intolerably high calcium contamination levels and our data from these procedures are not yet reliable. An indirect method of’ attack on this problem may now he possible as Duncan and van Heyningen (1976) have recently shown that over 50yb of t’he complexed calciun~ in cataractous lenses can be replaced by radioa.ctive calcium after incubating for 15 hr in a sodium fret: In&~r. This preloading method may provide a means of obtaining a detailed eqtimattb of the calcium in various sub-fractions of the insoiuble component, where a nalciutll calcium exchange occurs. ‘VVe did. however, find that it was possible to det~ermiue the calcium antl aotliurll ihsSOc:i:~t~ed with i-he different protein components iti the solul)le fractions of lct~s IIO~IO--
(0)
+B+y-i
(0)
3
2-
I
2.25
A
-1 - IfI 7 j-0’15; 0 I ;
.g 0” = .o ‘i o
I
0.8
I
j t
(b) 0.7
-
0.6
-
0.5
-
0.4
-
- 3.0
i 8 s
- 2.25
-
;
I.5
- 0.75
0
20
40
60
60
100 Fmction
120 140 number
FIG. 4. (a) Calcium density of proteins isolated from normal was washed and equilibrated with calcium-free 0.1 x-Tris-HCI collected. HM, high molecular weight fraction; cx, ar-crystallin; weight of lenses, 1184 mg. (b) Calcium denqity of cataractous 435
mg.
160
160
I 206
human lenses. The column (Bio- Gel Mm) buffer, pH 7-3 and 4 ml fractions wcrc 8, fi-crystallin: y, y-crystallin. Total wrt lens proteins. Total wet weight of lensrs.
cr-crystallin
[Fig. 5(a),( 1J)]. (a)
-6
0.4 -
Fraction 5. (a) Sodium legend to Fig. 4 and wet weight of lenses, FIG.
number
Total associated with normal lens proteins. text for further details. (b) Sodium associated 425 mg.
wet with
weight of cataractous
lemen, 1184 lens proteins.
mg. See Total
As many laboratories use nuclear colour alone as a basis for classifying (Brie. 1968). rather than colour combined with sodium content as we have done, we carried out a brief comparative study of the distribut,ion of indiffusible calciunl in I he tlifferent groups. The calcium associated with tbe soluble and insoluble fractions were determined after 15 hr dialysis as described in Xet’hotl. Y:.sect’ion C’. ant1 the intl icate (Tahlr 3) that as the soluble/insoluble weight ratio tlecreases, t,hr: proportion of the total c;~kium associated with the insohtble fraction increases. \Vr: (lit1 JW~. howcwr. carry out a comprehensive st,udy as both the sodiunI anti calciu~t~ conwntr:tt ions varietl gr&ly within each class [see also van Hrpningrn. 1972(l))]. Cat~2UiWl
Clitt;l.
4. Discussion The calcium and sodium concentrations of the post-mortem lenses are higher t hall those normally, found in mammalian lenses and are indeed higher than those found ill certain cataracts (Group A4according to the scheme of Duncan and Busl~ell. 197.5). .\ net influx of both sodium (van Hey&ngen, 1972) and calcium has probal~ly occurre(I cluring the rela,tively long period (up to 48 hr) before the lenses were availalJt~ to IIS. However, we have some confidence that these lenses can be treated as normal UNtrols. firstly because the sodium and calcium diffusion characteristics are similar to those found in fresh bovine lenses (Duncan and Hushell. 1976) and secondly I)ecaltscb their calcium binding properties are also similar to those of fresh bovine lenses (I)unoan and van He.yningen, 1976). The slow rate of loss of calcium from the cataractous Itln~ (Fig. 1) is especially interesting as it implies that the large amounts of calcium t,tiat enter these lenses do not remain in a free form. This confirms the suggestion (Duncan and Rushell, 1!375) that the high concentration of calcium in t)heir group C cataracts wa,s not brought about simply by the lens Donnan potential. They suggostet1 that calcium complexing sites were present and it follows from t)his t.hat the rat,t! of loss of calcium from lens homogenates should be slower t.han the concomitant loss 01’ sodium. Fig. I shows this to be the case. When the soluble and insoluble fractions are dialysed separately (Fig. 2) clear clitkrwxs in the calcium distribution between normal and cataractous lenses flmergc’. In the post-mortem lens almost SOY0 of the total calcium is in the soluble fraction ii,n(1 after tlialysis over 20% of the total calcium still adheres t’o this fraction. Only al)out 10?{I of the total is located in the insoluble fraction at the end of t,he experinwnt. Howver, in cataractous lenses, where the dry weight, of the insoluble fractiotl is considerably greater, 30:/, of the total calcium is now locat,ed in this fraction at thcb end of dialysis with only about 2% in the soluble fraction. The calculat,ions in Table 1 show that the calcium content of both soluble and insoluble fractions. expresstld on it. dry weight basis. increases as a result of cataract format,ion hut the relative incwaw in the insoluble fraction is the greater. ‘l’hr~ major proportion of calcium remaining after dialysis in ljoth nornln,l an(l aataractous lenses is in the nucleus [Fig. 3(a)] ant I. when the data are expresstl( 1 on ib clrv weight basis (Table II), the insoluble fraction in the nucleus of cat’aractous lermsf~s b&Is by far the greatest amount of calcium . A41though the proportion of calciunl bound by the soluble fraction in the nucleus of the cataraatons lens is relatively small. t hc soluble proteins have more calcium adhering to theIt at tbc: enct of t,lre (li;l.lvsi< pc~riocl than their counterparts in the normal lens.
Xormal Senile cataract
2.6 4.4
2.3 168
The data were obtained from the soluble and insoluble separately for 15 hr (Fig. 2). See text for further details.
Calcium Soluble cortex ~~~~
Lens type _.__..._ ~
content Soluble nucleus
fractions
of lenses that
had been
dialys(,tl
(m&r/kg dry weight) Insoluble Tnsolublo cortex nWA?lX
Normal Senile cataract
Soluble and insoluble fractions were obtained from computations
from both cortex and nucleus were dialysed separately and the valurs on the data presented in Fig. 3(a) (after 15 hr dialysis).
TABLE
Lens type
III
o/o Ca in soluble fraction
y& Ca in insoluble fraction
Soluble/ insoluble ratio
20.2 10.0 8.2 6.1
13.8 13.5 30.1 35.3
1.3 0.83 0.39 0.20
Totai Na WO% water)
Totai Ca (KlM/iq water)
.Normal Group I Group II Group III
54 68 106 184
1.X 4.2 9.8 12.5
The data represent the mean of two separate experiments (four lenses from each group) cium associated with the soluble and insolubk fractions was obtained after I.5 hr dialysis in the text (see data at t = 15 hr in Fig. 2 for comparison).
and the calas described
There has been much interest over the past few years in the association between calcium and the two soluble protein fractions, high molecular weight and cc-crystallin (Jedziniak, Kinoshita, Yates, Hacker and Benedek, 1973; Spector, Adams and Krul, 1974; Jedziniak, Kinoshita, Yates and Benedek, 1975). In the present experiments, most of the calcium in the soluble fraction of the human lens, that does not appear
SON-DIFFUSIBLE
LENS
CALCIUM
193
to bo in free solution, is associated with the high molecular weight fraction and r,-crystallin [I?& 4(a),(b)]. The high molecular u-eight fraction accounts for a larger proportion of t’he total soluble protein of the cataractous lens [Fig. 4(b)] than of the normal lens [Fig. 4(a)], but this fraction does not appear to have a greater affinity for calcium than cr-crystallin. Expressed on an optical density basis, read at 280 nm, the calcium O.D. ratio (nmol Ca/O.D. unit, calculated from the areas under the peaks) are 90 and 111 for the high molecular weight fraction and x-crystallin respectively in t’he normal lens and 166 and 190 for cataractous lens [Fig. 4 (a),(b):. This is in contrast to t!he findings of Jedziniak, Nicoli, Yates and Benedek (1976) who find clear tliff’erences between t)he high and low molecular weight fractions in cataractous lensea. The! did not, however. find significant differences between the calcium densities of thcl Amble and insoluble fractions that we find, but as they did not determine the calcium in t,he dialysis medium and so cannot express the data on a percentage ha&. it is impossible to carry out a direct comparison. We conclude that the higher moleculatr weight and cr-crystallin fractions bind much more calcium than the ot)her solublt: proteins and that more calcium is bound in the cataractous state. ACKNOWLEDGMESTS
We thank Dr Keith J. Dilley and Dr John J. Harding for many stimulating discussions and Miss Joy Rosserfor excellent technical assistance.G. D. thanks Mr Ant,hony J. Bran for the hospit,ality and facilities of the laboratory and also the Royal National Instit.ute for the Blind, and Researchinto Child Blindness(RICB) for providing financial support. We are also indebted to Rlr Mervyn Gascoyneof the Inorganic Chemistry laboratory of the I7niversity of Oxford for his assist’ance with the atomic absorption measurements. REFEREXCES Bellows, J. G. (1!)75). Cataract ad Abnormalities of the Lens. Prune and Stratton, New York. Duncan, G. and Bushell, A. R. (1975). Ion analyses of human cataractons lenses. Exp. Eye Rex. 20, 223-30. Duncan, G. and Bushell, A. R. (1976). The bovine lens as an ion-exchanger: a comparison with ion IcLvels in human cat,aractous lenses. Exp. &ye Res. 23,341-5X Duncan, G. and van Heyningen, R. (1976). Differences in t.he calcium binding ca.pacit,y of normal and cataractous lenses. DOG. Ophthnl. Proceedings Series 3’0. 9, 15-18. van Heyningen, R. (1972a). The human lens I. A comparison of cataracts ext,racted in Oxford (England) and Shikarpur (W. Pakistan). h’xp. Eye Res. 13, 13647. van Heyningen, R. (1972b). The human lens III. Some observations on the post-mortem lens. Exp. Eye Res. 13, 155-60. Jedziniak, J. A., Kinoshita, J. H., Yates, E. M., Hacker, L. 0. and Benedek, G. B. (1973). On the presence and mechanism of formation of heavy molecular weight aggregates in human normal and cataractous lenses. Exp. Eye Res. 15, 185-92. Jedziniak, J. A., Kinoshita, J. H., Yates, E. M. and Benedek, (:. B. (1975). The concentration and localization of heavy molecular weight aggregates in aging normal and cataractoIls human lenses. Exp. &e Res. 20,367-g. Jedziniak, J. A.. Sicoli, D. F., Yates, E. M. and Benedek, 0. B. (1976). On the calcium concent,ration of catarantous and normal human lenses and protein fractions of cataract,ous lenses. Exp. Eye Rex. 23, 325-32. &s, H. A., Hoenders, H. J. and Wollensak, J. (1976). Protein changes in the human lens during development of senile nuclear cataract. Biochim. Biophys. Acta 434,3243. Maraini, G. and Mangili, R. (1973). Differences in proteins and in the water balance of the lens in nuclear and cortical types of senile cataract. In CIBA Symposium : The Human Lens i,n Relntion to Catamcf. Elsevier. Amsterdam. Pirie, A. (1968). Color and solubility of the proteins of the lens. Inuest. Ophthulmol. 7, 634-42. Spector, A., Adams, D. and Krul, K. (1974). Calcium and high molecular weight protein aggregates in bovine and human lens. Invest. Ophthalmol. 13, 982-W.