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Topography of a mitotic inhibitor in the rabbit lens
Exptl Eye Res. (1967) 6, 2 1 3 - 2 1 8
T o p o g r a p h y o f a M i t o t i c I n h i b i t o r in the R a b b i t L e n s ~']ARY J . ¥OADEN a.nct DOI',I¢IT FROOMBERG
Deparlment of Physiolo.c/ical O]~g'iras, Institute of Ophthalmolwy , Ju(ld Street, Lomlon, I I:. C.1, I~b~gland (Received 7 January 1967) A water.soluble mitotic inhibitor, present in rabbit lenses, has b e m t s h o w n to be more con('entrat.ed in the cortex than the mlelcus. A considerable increase in the coneertbratimt of the factor, as the lens ages. has beelt loealized to the len.s eorlAex. Gel-filtr~bion studies ha ~-e shown I h a t t.he rela.t,ive concentration of y-crvst~lllin--the protei~l fl't~ction contai~ting the inhibitor--deerenses with age; and f u r t h e r subfra.ctionation by starch-gel elcctrophoresis has i~Micated that. this dccr(,a.~e is aecompa,~ied by the loss of specific protein fl.aetions.
1. Introduction T h e c o n t i n u a l m i t o t i c a ct, i v i t y w h i c h o c c u r s i n t h e e p i t h e l i u m o f t h e m a m m a l i a n l e n s is n o w w e l l r e c o g n i z e d a n d is r e l a t i v e l y s i m l f l e to e v a l u a t e (see f o r e x a m p l e , . i n m v a s a n a n d ] l a r d i n g , 1965). L i t . t l e is k n o w n , h o w e v e r , o f tire f a c t o r s w h i c h c o n t r o l t h i s p r o c e s s in r i v e . I n a n at, t e m p t t o a s c e r t a . i n t h e m e c h a n i s m w i t h i n t h e t i s s u e t h a t a l l o w s a cell e i t h e r to e n t e r m i t o s i s o r to r e m a i n in t h e r e s t i n g p h a s e , a s e r i e s o f e x p e r i m e n t s h a v e b e e n s t a r t e d in w h i c h y o u n g 6 - 8 ~ : e e k - o l d r a b b i t l e n s e s a r e m a i n t a i n e d in v i t r o for p e r i o d s r a n g i n g f r o m 1 t o 6 h r ( F r o o m b e r g a n d V o a d e n , 1966). T h e m i t o t i c a c t - i v i t y o f t h e t w o l e n s e s f r o m o n e d o n o r r a b b i t a g r e e ~-ery c l o s e l y t h r o u g h o u t t h e i n c u b a t i o n p e r i o d 0 t v e r a g e v a r i a . t i o n a f t e r 5 h r 7"8:t:0"9~/o, 30 e s t i m a t i o n s ) a n d t.his m a d e it p o s s i b l e to e w d u a . t e t h e e f f e c t o f v a r i o u s a d d i t i o n s t o t h e i n c u b a t i n g m e d i u m , 1)v u s i n g o n e l e n s as a c o n t r o l . 13y t h i s m e a n s it w a s e s t a b l i s h e d t.hat a q u e o u s e x t r a c t s o f r a b b i t l e n s e s i n h i b i t t,h e m i t o t i c a c t i v i t y o f t h e y o u n g l e n s e s c u l t u r e d i n v i t r o . I n h i b i t o r y a c t i v i t y w a s found in e x t r a c t s t a k e n f r o m all a r e a s o f t,he y o u n g l e n s a n d w a s s h o w n to b e a s s o c i a t e d with the y-crystallin protein fraction. Preliminary studies indicated that old lenses cont, a i n e d m u c h m o r e o f *l:e i n l f i b i t o r y s u b s t a n c e p e r u n i t w e t w e i g h t t l , a n d i d t h e y o u n g o n e s . T h e pre.sen'c c o m m t m i e a t i o n is c o n c e r n e d w i t h a. c l o s e r i d e n t i f i c a t i o n o f t.he loci o f i n h i b i t o r y a c t i v i t y , t h e r e m t l t s h a v i n g b e e n r e p o r t e d b r i e f l y e l s e w h e r e ( V o a d e n a n d . F r o o m b e r g , 1967). "
.
5'lethods N e w Z e a l a n d w h i t e r a b b i t s were u s e d for these, e x p e r i m e n t , % t h e i n c u b a t e d l e n s e s b e i n g t a k e n f r o m 6 - S - w e e k old a n i m a l s . T h e c o n d i t i o n s for i n c u b a t i o n a n d g e n e r a l m a n i p u l a t i o n o f t h e lenses were d e s c r i b e d p r e v i o u s l y ( I P r o o m b e r g a n d V o a d m I , 1966), o n e lens f r o m e a c h r a b b i t a l w a y s s e r v i n g in a c o n t r o l i n c u b a t i o n . M i t o t i c aetAvity w a s d e t e r m i n e d b y c o u n t i n g m i t o t i c figures in w h o l e p r e p a r a t i o n s of t.he lens e p i t h e l i u m , s t a i n e d b y t h e ti'eulgen t . e e h n i q u e (:Howard, 1952). E x t r a c t s c o n t a i n i n g t h e s o l u b l e lens p r o t e i n s w a s p r e p a r e d a t a c o n c e n t r a t i o n of I 0 0 m g w e t wt~ o f w h o l e l e n s / m l by h o m o g e n i z i n g f r e s h l y excised l e n s e s ira g l a s s - d i s t i l l e d w a t e r in t.he cold. All e x t r a c t s w e r e c e n t r i f u g e d for 10 rain a t 2500 r e v / m i n , tire t e m p e r a t u r e b e i n g m a i n t a i n e d a t 4°C. 213
i'~[ARY J.
214
VOA])EN
AND
1)O]¢,RI.:I' leROO*IBI,IIC, G
P r e p a r a t i o n s of y-erystnllin were m a d e fi'om the fi-eeze-dricd solubh+ p r o t e i n s of wholc lens a q u e o u s e x t r a c t s . 50 mg of fi'eezc-dried lens prot,+.in were applie(l to a c o l u m n (if s e p h a d e x 0 . 7 5 (2 c m × , t 0 era) w h i c h had l',reviously beett prel)ared l)y w a s h i n g wit, It 0.05 .~t Na01 a n d t h e n with 0.05 ~r T r i s / H C l butt\:r p H 7-2 (Bj6rk, |9(il). The c o l u m n was e l u t e d with this buffer, 2 ml vol. being collected at, a rate of 30 m l / h r . P r o t e i n was d e t e c t e d by m e a s u r i n g the opi.i('aI d e n s i t y at, 280 ml+ on a Unimm~ S t 500 spect, r o p h o t o m e t e r . T h e ft'act.ious COllt++lJllillg the y-crystallin were pooled, d i a l y s e d o v e r n i g h t +~t. 4°0 in ,t litres of dist, illed water, freeze-dried -tn(l stored at, - - 2 0 ° 0 . S t a r c h gel e l e c t r o p h o r e s i s ( B D I I hydrolyse(I s t a r c h ) was p e r f o r m e d h o r i z o n t a l l y using the T r i s - d i s e o n t i n u o u s buffer s y s t e m (Poulik, 1957). It, was r u n for 2 hr at .t°C with a s t a r t i n g c u r r e n t of 2 mA/cm strip. Starch blocks of 20 c m x 10 em ;-: ~ c m were p r e p a r e d at t.he r e c o m m e n d e d coneentrat.ion for e a c h b a t c h of s t a r c h - - a p p r o x i n u t t e l y 1(/g starch pet" 100 ml T r i s / e i t r i c acid buffer p H 8.0 (18-3g Tris a n d 2-I g cit.rie acid in 2 litres of dist.illcd water). 10 mg protein in 0"05 ml distilled w a t e r were applie[I free or with W h a t m n n 17 c h r o m a t o g r a p h y p a p e r as s u p p o r t i n g m e d i u m , to 1½-2 cm wide t r o u g h s - - c o m p a r a b l e resuit.s w e r e o b t a i n e d in both eases, e l e c t r o - d e c a n t a t i o n bcit~g critical; in the. former (see Smithies, 1959 for discussion). 13orate buffer p H 8.(3 (5 g s o d i u m h y d r o x i d e and 37 g. Boric acid dissolved in 2 litres distilled w a t e r ) was p r e s e n t in the e l e c t r o d e trays. Protein was d e t e c t e d by s t a i n i n g w i t h a m i d o black 10B (Smithies, 1959). 3. R e s u l t s and D i s c u s s i o n
The i~H+ibitoru propco'Hcs of l.ens ea:tracts T h e o b s e r v a t i o n t.haC old l e n s e x t r a c t s c o n t a i n e d c o n s i d e r n b l y m o r e i n h i b i t o r y s u b s t a n c e p e r unit, ",vet, w e i g h t t i t a n d i d y o u n g o n e s h a s n o w b e e n e x t e n d e d , a n d t h e i n h i b i t o r y propert.ies of t h e c o r t e x a n d n u c l e u s of y o u n g a n d old h;nses s e p a r a t e l y
o--O C 0 r t e x s t o - - o Nucleu
100
.--oCortex }3-Sv~eeks o - - e Nucleus
90 -------------BO
6-Sweeks
-
Q .__.____
Q
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•
O
O-
o< g -Q
5O 40
6
2O
_i-2~o ....
I0
0
I
I
I-0
2-0
_
_
~
3-0 Mq lens (.wet w t )
.
I.
<,-0
.
.I 5"0
| 6"0
.Fie. 1. I n h i b i t i o n o f m i t o s i s b y e x t r a c t s of y o u n g a n d ok[ r a b b i t lenses. I l l t w o a d d i t i o n a l c x p c r i 1 m c n t s t h e e q u i v a l e n t o f 6 m g web w e i g h t ot" 3 " 5 - y e a r l e n s n u c l e i g a v e 90 a n d 1,- - O,o s t i m u l a t i o n s of m i t o t i c activity. T h e vorbieal l i n e s r e p r e s e n t s t a n d a r d e r r o r s o f t h e m o a n .
TOPOGI~APH.Y
OF
A MITO'.IJ[C
INIIIBITOR
IN
TIlE
I~ABBIT
LENS
215
invcsl, igated. The results arc sho~m in Fig. 1 and Table I. The rise in c o n c e n t r a t i o n in ohl lenses has been confirmed but it is notewort, h y tha~ this occurs only in tim lens cortex. The erragic results obtained with nuclear extracts (which m a y be.assessed from t,he large standard errors of the mean shown for ~he y o u n g lens mmleus) m a y possibly be a t t r i b u t e d to a non-specific i n h i b i t i o n being produced by a i b m n i n o i d which was nee rcmovcd b y the procedures eml)loyed. This has n o t been investigated TA I~LE I
Effect of .varyin 9 concentrations of rabbit lens homogenate on the mitotic activity of rabbit lenses incubated in vit.ro R e ( l u c ~ i o n o f t o t a l mitotAc (:o,mt (°/o) Conccntratitm of l e a s homogena~c, ( r a g w e t weight,) o f lca~
6 - 8 wcol~ lens cortex
3"5 y c n r lens cortex
7 9 - : 0"57(3) (:~4:~ 1.0(4) 39~_ 1.7(4)
0.5
17(1)
3-5 y e a r lens nucleus (see f o o t h a t e F i g . 1)
390)
47:!~ 12-0(4)
6-0 5.0 4-0 3"0 2"0 1.0 0.25 0-1
6-8 week lens nucleus
82(2) 332.9"0(3)
23(I) l~):~s-s(4)
st(1) sol2)
~3(s)
79C2) 69A:3-4(3) 54~_2-I(4)
T h e n u m b e r o f e s t i m a t i o n s is s h o w n i,x p a r e n t h e s e s . l ~ c s u l t s a r e e x p r e s s e d a s means -z.S.l~;.~l,
furt,her as our interest was focused on the increased i~lhibitory a c t i v i t y t h a t occurs with aging; and this was localized to the lens cortex. Of three old lens nuclei examined, the extrae, t.s f,'om two of them gave no inhibition, whereas the third b e h a v e d like tlm.t from a y o u n g lens. This l a t t e r result is sito~nl in Fig. 1.
The fractio~ation of soluble lens protein on Sephadex, G.75 I t was esta.blishcd previously (iV,:oombcrg aud \:oaden, 1966) t h a t the i n h i b i t i o n of mitosis in r a b b i t lenses cultured in v i t r o was a specific p r o p e r t y of the y-crystallin protein f r a c t i o n - - a s obtained by gel-filtration on Sephadex G.75 (Bj6rk, 1961). Using this supporting m e d i u m the filtration p a t t e r n s obtained ~dth ageing cortex and mmleus have now been i n v e s t i g a t e d and are shown in Figs 2 and 3. Tim m a x i m u m n u m b e r of peaks obtained corresponds t o / t h e f o u r reported b y J3jSrk (1961) for calf lens proteins. I-[owever, a.s can be seen, the second peak in the cortex f r a c t i o n a t i o n shows a progressive increase with age and, in addition, is v i r t u a l l y absent from all the nuclear extracts. The significance of this is not known, b u t w h e n t h e four peaks of the 3.5-year cortex were s e p a r a t e l y dialysed and freeze-dried, i n h i b i t i o n remained associated with the bhird p e a k ~ t h e y-erystallin fl'action. The changes in the relative c o n c e n t r a t i o n of this fraction t h a t occur with age are of p a r t i c u l a r interest as the t r e n d is towards a decrease: this change is opposite to ghat of the inhibition, which
MARY
216
J.
VOAI;EN
AND
I)ORR1T
FIIOOMBEI~G
i n c r e a s e s w i t h age, a n d i m p l i e s b l l a t i n h i b i t i o n is a c h a r a c t e r i s t i c , n o t of t h e ~,c r y s t a l l i n p r o t e i n s as a whole, b u t is a s s o c i a t e d w i t h one c o m p o n e n t of t,hem. I~ s h o u l d be n o t e d t h a t t h e apparent, d e c r e a s e in ~,-crystallin t h a t occurs in t h e ageing lens m m l c u s is only r e l a t i v e (ct'. T a b l e [l). W h e n a b s o h l t e values were calcuhLtcd, it, w a s f o u n d that, t.here was a t,wofold increase, t he weight, of soluble m a t e r i a l in tlle lens 7-0
6-0
5"0
4-0 E o
I
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q
fxJ
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3-0
2-0
I-0 #
¢"
0
! I0
[ 20
I 30
,L 40
I 50
Effluent
I " - - " - g" 60 70 volume
~-~780
w 90
I00
(ml)
FJo. 2. l Z r a c t i , m a t i o n o f t h e s o l u b l e p r o t e i n front r a b b i t l e n s c o r t e x on S e l ) h a t l c x C:75 ( 5 0 ¢ n g t're~'ze(h'ied (.xt.raet a p p l i e d t(, c o l u m n ) . ( . ) 6 - 8 wc~:ks; ( - - - ) S m o n t h s ; (. . . . . . ) 1..'5 y e a r s ; ( . . . . . . ) 3-5 y e a r s .
Age of rabbit
]legion (,f lens
Wet wt of tissue (rag)
Dry wt of insoluble matter (rag)
6-7 6-7 3-5 3"5
Cortex Nucleus Cortex Nucleus
184 85 305 345
2-7 0.7 9-7 31-2
wks wks yrs yrs
D r y wt of soluble matter (rag)
49-9 22-9 77-0 116-5
o,,,o ~Vater
Relative amounts o f ycryst~allin*
71 71 72 57
I'00 0.68 l-O0 1-38
* The amounts of y-crystallin present have been estimated by weighing the areas under the rclevanb c u r v e s ( F i g s 2 a n d 3). T h o q u a n t i t y o f y - c r y s t a l l i n p r e s e n t in 50 m g o f s ( , l u b l e m a t t e r f r o m t h e 6 - 7 week l e n s c o r t e x h a s b e e n t a k e n as u n i t y a n d t h e r e m a i n i n g v a l u e s c a ] c u l a t e d r e l a t i v e t o t h i s .
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~ ' D " {gr~m'~,ga ~ ,,;.~ "
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Pl..,TI-: l. 8t,tu'eh-gel el~'ctv~,phovesis o f v~lhbit, lens v,,rtex y-cry.-1.allin. Age t~f rabbit.s: A. 8 - 7 weeks; 13.8 m,nt,]ts; ~, 2.F) ye,~lr.~; D. 3.5 y e a r s .
. ,
-, -
'r()POGI~APiIY
OF
A I~[I'I'OTI(~
[NHIB1TOI¢.
IN
T.I-[E
I¢~ABBIT
LENS
217
l~ucleus increasing ~tDl)roximately fivef0ld fl'om 6-7 wceles to 3-5 years. Similarly, corot)arid)It calcuh~tions for the c o r t e x show t h a t the total ~v-crystallin present in this arc;~ remains relabively c o n s t a n t between these a g c s ~ t h e sohtble material in the cortex i~creasi~zg 1.5-fold in weight.
8.0 7.0
i
, "ll
6.0 :1
5.0 =k
E
~
0
oo ¢%1
t
;
4-o~-
!
i
i: I
I
t
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1
i
i: !
3-0
!
i
t
i
!
i
I
'
i t
I i
'.;,-,,
I -0 l-
/ ~ -~,
...-
•
i
o
I0
20
50
40 Effluent
50 volume
60
70
80
90
I00
(ml)
I:~c.. 3. F r a c t i o ~ m t i t , n o f t h e s o l u b l e p r o t e i l ~ f r o m r a b b i t l e n s n u c l e u s o n S e p h a d e x C,.75 (50 m g f r e e z e d r i e d c.xtrur.t a p p J i c d t t> c(>lumt 0. { - ~ ) 6 - S w e e k s : {. . . . ) 8 months; (. . . . . ) 1-5 3"ears; ( . . . . . ) 3-5 y e a r s .
Values obtained for the weights of the cortex and nucleus of single lenses at. these ages, and for the relative a m o u n t s of soluble al~d insoluble protein present, are also shown in Table II. T h e y agree with the va)ues reported by other workers (see van I-Ieyningen, ] 962).
The fractionatio.n of rabbit le~s ~,-erystallin by starch-gel electrop~oresis In an a t t e m p t to extend the observation on the changes t h a t occur in the Vcrystallin protein derived ~'rom the lens cortcx, wc have s.ub-fractionate'd it f u r t h e r b y starch-gel electrophoresis. The results obtained are s~]own in ~]ate 1 ; they confirm pre~dous reports of the heterogene.ity of this fraction (for example, Bj6rk, 1964; Spector, 1965; P a p a c o n s t a n t i n o u , 1965). *~Vhether the s e p a r a t e fractions detected represent in~iividual proteins cannot be determined at present but BjSrk (1964), wor}ci)~g with calf lens ~,-crystaliin, detected a t least six fractions and was able to
218
MARY J. VOAI)EN AND .DOI1R1T I,~ROOMBE]~C,
t)rovidc evidence for the homogcueit.y of four of them. Tim presm~t, results show a marked variation in the clectrophoretie pa.ttcrn wit,b age, the nmximmn number of fractions (8) being found in the young adult, y-cryst,allin. Tlmre is ~shen a striking loss of the more posit, ively charged and/or larger molecular wc,ight~ prot.eins as the lens continues to age. The significaJmc of t.his for lhc amint,enance of the homeostatic balancc of tit(: ageing lens is ats present unknown, b u t the results are of pa.rlicular interest, i~ the present, context, as t.hey would sugge.st, t,h.at~ inhibition is associated wit.h some or all ot" the remainiug protein fl'nctions. The elution and testil~g of the individual protein fl'actions is being at,templed, but at. present, no definite coneltlsions have beel~ reached. A C1¢, N 0 W L E D C .~I E N'l'8
We exteml our thanks, as al\v:/)-s, to Dr. F,. 4. Weale for his patience nnd his eont.imiii~g interest in t [",s work. The technical assistance of Miss Barbara ,_cott£ nnd Mr. P. ({oklwater is gr~tefully acknowledged. Financial SUplx~rt, was received from the Medical Research Council. R E FE. I{V , N C" 1~,~:-'
Ilj6rk. ]. (t961). Exl)tl Ere ICes. l, 145. .Bj/Srk. 1. (1964). Exptl Ere lies. 3, 254. ]"roomberg, 1). and Voaden. M. (1966). E.rptl Eye ]~es. 5, 1. van ]-Icyninge.n. ]l. (1962). ]rt :lVtc Erie, vol, l. Ed. H. Davstm. Academic Press, New York. I[oward, A. {]952). Stain 2'ccMwl. 27, 313. I~apaconstantinou, J. (1965). Biochim. Biot,]~ffs...Ida ..trust. 107, S1. Poulik. M. D. (1957). :Nature 180, 1477. Smithics, O. (1959). ]n Advances in Prolein Cllemistry. Eds. (',. B. ,.\~ginsen, 3I. l,. ,a,nsm~, K. :Bailey and J. T. EdsalL -kcadcmic Pres,% New York. Spcctor, A. (1965). 1rived. 01~hthalmol. 4, 5"79. Sri,fiva~a~/, B. ]3. and Harding, (2. V. (1965). In, rcsl. Opht],ahnoi. 4, 452, Voaden, ~I. J. and ],~roomberg, D. (1967). In Syml,osium o~. thc Biochemistry of the Eye, August, (1966), ]~d. J. Nordmmm; S. Kargcr A.G. (Basel, Ncw York), .In preparation.
T o p o g r a p h y o f a M i t o t i c I n h i b i t o r in the R a b b i t L e n s ~']ARY J . ¥OADEN a.nct DOI',I¢IT FROOMBERG
Deparlment of Physiolo.c/ical O]~g'iras, Institute of Ophthalmolwy , Ju(ld Street, Lomlon, I I:. C.1, I~b~gland (Received 7 January 1967) A water.soluble mitotic inhibitor, present in rabbit lenses, has b e m t s h o w n to be more con('entrat.ed in the cortex than the mlelcus. A considerable increase in the coneertbratimt of the factor, as the lens ages. has beelt loealized to the len.s eorlAex. Gel-filtr~bion studies ha ~-e shown I h a t t.he rela.t,ive concentration of y-crvst~lllin--the protei~l fl't~ction contai~ting the inhibitor--deerenses with age; and f u r t h e r subfra.ctionation by starch-gel elcctrophoresis has i~Micated that. this dccr(,a.~e is aecompa,~ied by the loss of specific protein fl.aetions.
1. Introduction T h e c o n t i n u a l m i t o t i c a ct, i v i t y w h i c h o c c u r s i n t h e e p i t h e l i u m o f t h e m a m m a l i a n l e n s is n o w w e l l r e c o g n i z e d a n d is r e l a t i v e l y s i m l f l e to e v a l u a t e (see f o r e x a m p l e , . i n m v a s a n a n d ] l a r d i n g , 1965). L i t . t l e is k n o w n , h o w e v e r , o f tire f a c t o r s w h i c h c o n t r o l t h i s p r o c e s s in r i v e . I n a n at, t e m p t t o a s c e r t a . i n t h e m e c h a n i s m w i t h i n t h e t i s s u e t h a t a l l o w s a cell e i t h e r to e n t e r m i t o s i s o r to r e m a i n in t h e r e s t i n g p h a s e , a s e r i e s o f e x p e r i m e n t s h a v e b e e n s t a r t e d in w h i c h y o u n g 6 - 8 ~ : e e k - o l d r a b b i t l e n s e s a r e m a i n t a i n e d in v i t r o for p e r i o d s r a n g i n g f r o m 1 t o 6 h r ( F r o o m b e r g a n d V o a d e n , 1966). T h e m i t o t i c a c t - i v i t y o f t h e t w o l e n s e s f r o m o n e d o n o r r a b b i t a g r e e ~-ery c l o s e l y t h r o u g h o u t t h e i n c u b a t i o n p e r i o d 0 t v e r a g e v a r i a . t i o n a f t e r 5 h r 7"8:t:0"9~/o, 30 e s t i m a t i o n s ) a n d t.his m a d e it p o s s i b l e to e w d u a . t e t h e e f f e c t o f v a r i o u s a d d i t i o n s t o t h e i n c u b a t i n g m e d i u m , 1)v u s i n g o n e l e n s as a c o n t r o l . 13y t h i s m e a n s it w a s e s t a b l i s h e d t.hat a q u e o u s e x t r a c t s o f r a b b i t l e n s e s i n h i b i t t,h e m i t o t i c a c t i v i t y o f t h e y o u n g l e n s e s c u l t u r e d i n v i t r o . I n h i b i t o r y a c t i v i t y w a s found in e x t r a c t s t a k e n f r o m all a r e a s o f t,he y o u n g l e n s a n d w a s s h o w n to b e a s s o c i a t e d with the y-crystallin protein fraction. Preliminary studies indicated that old lenses cont, a i n e d m u c h m o r e o f *l:e i n l f i b i t o r y s u b s t a n c e p e r u n i t w e t w e i g h t t l , a n d i d t h e y o u n g o n e s . T h e pre.sen'c c o m m t m i e a t i o n is c o n c e r n e d w i t h a. c l o s e r i d e n t i f i c a t i o n o f t.he loci o f i n h i b i t o r y a c t i v i t y , t h e r e m t l t s h a v i n g b e e n r e p o r t e d b r i e f l y e l s e w h e r e ( V o a d e n a n d . F r o o m b e r g , 1967). "
.
5'lethods N e w Z e a l a n d w h i t e r a b b i t s were u s e d for these, e x p e r i m e n t , % t h e i n c u b a t e d l e n s e s b e i n g t a k e n f r o m 6 - S - w e e k old a n i m a l s . T h e c o n d i t i o n s for i n c u b a t i o n a n d g e n e r a l m a n i p u l a t i o n o f t h e lenses were d e s c r i b e d p r e v i o u s l y ( I P r o o m b e r g a n d V o a d m I , 1966), o n e lens f r o m e a c h r a b b i t a l w a y s s e r v i n g in a c o n t r o l i n c u b a t i o n . M i t o t i c aetAvity w a s d e t e r m i n e d b y c o u n t i n g m i t o t i c figures in w h o l e p r e p a r a t i o n s of t.he lens e p i t h e l i u m , s t a i n e d b y t h e ti'eulgen t . e e h n i q u e (:Howard, 1952). E x t r a c t s c o n t a i n i n g t h e s o l u b l e lens p r o t e i n s w a s p r e p a r e d a t a c o n c e n t r a t i o n of I 0 0 m g w e t wt~ o f w h o l e l e n s / m l by h o m o g e n i z i n g f r e s h l y excised l e n s e s ira g l a s s - d i s t i l l e d w a t e r in t.he cold. All e x t r a c t s w e r e c e n t r i f u g e d for 10 rain a t 2500 r e v / m i n , tire t e m p e r a t u r e b e i n g m a i n t a i n e d a t 4°C. 213
i'~[ARY J.
214
VOA])EN
AND
1)O]¢,RI.:I' leROO*IBI,IIC, G
P r e p a r a t i o n s of y-erystnllin were m a d e fi'om the fi-eeze-dricd solubh+ p r o t e i n s of wholc lens a q u e o u s e x t r a c t s . 50 mg of fi'eezc-dried lens prot,+.in were applie(l to a c o l u m n (if s e p h a d e x 0 . 7 5 (2 c m × , t 0 era) w h i c h had l',reviously beett prel)ared l)y w a s h i n g wit, It 0.05 .~t Na01 a n d t h e n with 0.05 ~r T r i s / H C l butt\:r p H 7-2 (Bj6rk, |9(il). The c o l u m n was e l u t e d with this buffer, 2 ml vol. being collected at, a rate of 30 m l / h r . P r o t e i n was d e t e c t e d by m e a s u r i n g the opi.i('aI d e n s i t y at, 280 ml+ on a Unimm~ S t 500 spect, r o p h o t o m e t e r . T h e ft'act.ious COllt++lJllillg the y-crystallin were pooled, d i a l y s e d o v e r n i g h t +~t. 4°0 in ,t litres of dist, illed water, freeze-dried -tn(l stored at, - - 2 0 ° 0 . S t a r c h gel e l e c t r o p h o r e s i s ( B D I I hydrolyse(I s t a r c h ) was p e r f o r m e d h o r i z o n t a l l y using the T r i s - d i s e o n t i n u o u s buffer s y s t e m (Poulik, 1957). It, was r u n for 2 hr at .t°C with a s t a r t i n g c u r r e n t of 2 mA/cm strip. Starch blocks of 20 c m x 10 em ;-: ~ c m were p r e p a r e d at t.he r e c o m m e n d e d coneentrat.ion for e a c h b a t c h of s t a r c h - - a p p r o x i n u t t e l y 1(/g starch pet" 100 ml T r i s / e i t r i c acid buffer p H 8.0 (18-3g Tris a n d 2-I g cit.rie acid in 2 litres of dist.illcd water). 10 mg protein in 0"05 ml distilled w a t e r were applie[I free or with W h a t m n n 17 c h r o m a t o g r a p h y p a p e r as s u p p o r t i n g m e d i u m , to 1½-2 cm wide t r o u g h s - - c o m p a r a b l e resuit.s w e r e o b t a i n e d in both eases, e l e c t r o - d e c a n t a t i o n bcit~g critical; in the. former (see Smithies, 1959 for discussion). 13orate buffer p H 8.(3 (5 g s o d i u m h y d r o x i d e and 37 g. Boric acid dissolved in 2 litres distilled w a t e r ) was p r e s e n t in the e l e c t r o d e trays. Protein was d e t e c t e d by s t a i n i n g w i t h a m i d o black 10B (Smithies, 1959). 3. R e s u l t s and D i s c u s s i o n
The i~H+ibitoru propco'Hcs of l.ens ea:tracts T h e o b s e r v a t i o n t.haC old l e n s e x t r a c t s c o n t a i n e d c o n s i d e r n b l y m o r e i n h i b i t o r y s u b s t a n c e p e r unit, ",vet, w e i g h t t i t a n d i d y o u n g o n e s h a s n o w b e e n e x t e n d e d , a n d t h e i n h i b i t o r y propert.ies of t h e c o r t e x a n d n u c l e u s of y o u n g a n d old h;nses s e p a r a t e l y
o--O C 0 r t e x s t o - - o Nucleu
100
.--oCortex }3-Sv~eeks o - - e Nucleus
90 -------------BO
6-Sweeks
-
Q .__.____
Q
. . . .
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O
O-
o< g -Q
5O 40
6
2O
_i-2~o ....
I0
0
I
I
I-0
2-0
_
_
~
3-0 Mq lens (.wet w t )
.
I.
<,-0
.
.I 5"0
| 6"0
.Fie. 1. I n h i b i t i o n o f m i t o s i s b y e x t r a c t s of y o u n g a n d ok[ r a b b i t lenses. I l l t w o a d d i t i o n a l c x p c r i 1 m c n t s t h e e q u i v a l e n t o f 6 m g web w e i g h t ot" 3 " 5 - y e a r l e n s n u c l e i g a v e 90 a n d 1,- - O,o s t i m u l a t i o n s of m i t o t i c activity. T h e vorbieal l i n e s r e p r e s e n t s t a n d a r d e r r o r s o f t h e m o a n .
TOPOGI~APH.Y
OF
A MITO'.IJ[C
INIIIBITOR
IN
TIlE
I~ABBIT
LENS
215
invcsl, igated. The results arc sho~m in Fig. 1 and Table I. The rise in c o n c e n t r a t i o n in ohl lenses has been confirmed but it is notewort, h y tha~ this occurs only in tim lens cortex. The erragic results obtained with nuclear extracts (which m a y be.assessed from t,he large standard errors of the mean shown for ~he y o u n g lens mmleus) m a y possibly be a t t r i b u t e d to a non-specific i n h i b i t i o n being produced by a i b m n i n o i d which was nee rcmovcd b y the procedures eml)loyed. This has n o t been investigated TA I~LE I
Effect of .varyin 9 concentrations of rabbit lens homogenate on the mitotic activity of rabbit lenses incubated in vit.ro R e ( l u c ~ i o n o f t o t a l mitotAc (:o,mt (°/o) Conccntratitm of l e a s homogena~c, ( r a g w e t weight,) o f lca~
6 - 8 wcol~ lens cortex
3"5 y c n r lens cortex
7 9 - : 0"57(3) (:~4:~ 1.0(4) 39~_ 1.7(4)
0.5
17(1)
3-5 y e a r lens nucleus (see f o o t h a t e F i g . 1)
390)
47:!~ 12-0(4)
6-0 5.0 4-0 3"0 2"0 1.0 0.25 0-1
6-8 week lens nucleus
82(2) 332.9"0(3)
23(I) l~):~s-s(4)
st(1) sol2)
~3(s)
79C2) 69A:3-4(3) 54~_2-I(4)
T h e n u m b e r o f e s t i m a t i o n s is s h o w n i,x p a r e n t h e s e s . l ~ c s u l t s a r e e x p r e s s e d a s means -z.S.l~;.~l,
furt,her as our interest was focused on the increased i~lhibitory a c t i v i t y t h a t occurs with aging; and this was localized to the lens cortex. Of three old lens nuclei examined, the extrae, t.s f,'om two of them gave no inhibition, whereas the third b e h a v e d like tlm.t from a y o u n g lens. This l a t t e r result is sito~nl in Fig. 1.
The fractio~ation of soluble lens protein on Sephadex, G.75 I t was esta.blishcd previously (iV,:oombcrg aud \:oaden, 1966) t h a t the i n h i b i t i o n of mitosis in r a b b i t lenses cultured in v i t r o was a specific p r o p e r t y of the y-crystallin protein f r a c t i o n - - a s obtained by gel-filtration on Sephadex G.75 (Bj6rk, 1961). Using this supporting m e d i u m the filtration p a t t e r n s obtained ~dth ageing cortex and mmleus have now been i n v e s t i g a t e d and are shown in Figs 2 and 3. Tim m a x i m u m n u m b e r of peaks obtained corresponds t o / t h e f o u r reported b y J3jSrk (1961) for calf lens proteins. I-[owever, a.s can be seen, the second peak in the cortex f r a c t i o n a t i o n shows a progressive increase with age and, in addition, is v i r t u a l l y absent from all the nuclear extracts. The significance of this is not known, b u t w h e n t h e four peaks of the 3.5-year cortex were s e p a r a t e l y dialysed and freeze-dried, i n h i b i t i o n remained associated with the bhird p e a k ~ t h e y-erystallin fl'action. The changes in the relative c o n c e n t r a t i o n of this fraction t h a t occur with age are of p a r t i c u l a r interest as the t r e n d is towards a decrease: this change is opposite to ghat of the inhibition, which
MARY
216
J.
VOAI;EN
AND
I)ORR1T
FIIOOMBEI~G
i n c r e a s e s w i t h age, a n d i m p l i e s b l l a t i n h i b i t i o n is a c h a r a c t e r i s t i c , n o t of t h e ~,c r y s t a l l i n p r o t e i n s as a whole, b u t is a s s o c i a t e d w i t h one c o m p o n e n t of t,hem. I~ s h o u l d be n o t e d t h a t t h e apparent, d e c r e a s e in ~,-crystallin t h a t occurs in t h e ageing lens m m l c u s is only r e l a t i v e (ct'. T a b l e [l). W h e n a b s o h l t e values were calcuhLtcd, it, w a s f o u n d that, t.here was a t,wofold increase, t he weight, of soluble m a t e r i a l in tlle lens 7-0
6-0
5"0
4-0 E o
I
Ii
aD
q
fxJ
c;
3-0
2-0
I-0 #
¢"
0
! I0
[ 20
I 30
,L 40
I 50
Effluent
I " - - " - g" 60 70 volume
~-~780
w 90
I00
(ml)
FJo. 2. l Z r a c t i , m a t i o n o f t h e s o l u b l e p r o t e i n front r a b b i t l e n s c o r t e x on S e l ) h a t l c x C:75 ( 5 0 ¢ n g t're~'ze(h'ied (.xt.raet a p p l i e d t(, c o l u m n ) . ( . ) 6 - 8 wc~:ks; ( - - - ) S m o n t h s ; (. . . . . . ) 1..'5 y e a r s ; ( . . . . . . ) 3-5 y e a r s .
Age of rabbit
]legion (,f lens
Wet wt of tissue (rag)
Dry wt of insoluble matter (rag)
6-7 6-7 3-5 3"5
Cortex Nucleus Cortex Nucleus
184 85 305 345
2-7 0.7 9-7 31-2
wks wks yrs yrs
D r y wt of soluble matter (rag)
49-9 22-9 77-0 116-5
o,,,o ~Vater
Relative amounts o f ycryst~allin*
71 71 72 57
I'00 0.68 l-O0 1-38
* The amounts of y-crystallin present have been estimated by weighing the areas under the rclevanb c u r v e s ( F i g s 2 a n d 3). T h o q u a n t i t y o f y - c r y s t a l l i n p r e s e n t in 50 m g o f s ( , l u b l e m a t t e r f r o m t h e 6 - 7 week l e n s c o r t e x h a s b e e n t a k e n as u n i t y a n d t h e r e m a i n i n g v a l u e s c a ] c u l a t e d r e l a t i v e t o t h i s .
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1
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Pl..,TI-: l. 8t,tu'eh-gel el~'ctv~,phovesis o f v~lhbit, lens v,,rtex y-cry.-1.allin. Age t~f rabbit.s: A. 8 - 7 weeks; 13.8 m,nt,]ts; ~, 2.F) ye,~lr.~; D. 3.5 y e a r s .
. ,
-, -
'r()POGI~APiIY
OF
A I~[I'I'OTI(~
[NHIB1TOI¢.
IN
T.I-[E
I¢~ABBIT
LENS
217
l~ucleus increasing ~tDl)roximately fivef0ld fl'om 6-7 wceles to 3-5 years. Similarly, corot)arid)It calcuh~tions for the c o r t e x show t h a t the total ~v-crystallin present in this arc;~ remains relabively c o n s t a n t between these a g c s ~ t h e sohtble material in the cortex i~creasi~zg 1.5-fold in weight.
8.0 7.0
i
, "ll
6.0 :1
5.0 =k
E
~
0
oo ¢%1
t
;
4-o~-
!
i
i: I
I
t
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1
i
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!
i
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i
!
i
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'
i t
I i
'.;,-,,
I -0 l-
/ ~ -~,
...-
•
i
o
I0
20
50
40 Effluent
50 volume
60
70
80
90
I00
(ml)
I:~c.. 3. F r a c t i o ~ m t i t , n o f t h e s o l u b l e p r o t e i l ~ f r o m r a b b i t l e n s n u c l e u s o n S e p h a d e x C,.75 (50 m g f r e e z e d r i e d c.xtrur.t a p p J i c d t t> c(>lumt 0. { - ~ ) 6 - S w e e k s : {. . . . ) 8 months; (. . . . . ) 1-5 3"ears; ( . . . . . ) 3-5 y e a r s .
Values obtained for the weights of the cortex and nucleus of single lenses at. these ages, and for the relative a m o u n t s of soluble al~d insoluble protein present, are also shown in Table II. T h e y agree with the va)ues reported by other workers (see van I-Ieyningen, ] 962).
The fractionatio.n of rabbit le~s ~,-erystallin by starch-gel electrop~oresis In an a t t e m p t to extend the observation on the changes t h a t occur in the Vcrystallin protein derived ~'rom the lens cortcx, wc have s.ub-fractionate'd it f u r t h e r b y starch-gel electrophoresis. The results obtained are s~]own in ~]ate 1 ; they confirm pre~dous reports of the heterogene.ity of this fraction (for example, Bj6rk, 1964; Spector, 1965; P a p a c o n s t a n t i n o u , 1965). *~Vhether the s e p a r a t e fractions detected represent in~iividual proteins cannot be determined at present but BjSrk (1964), wor}ci)~g with calf lens ~,-crystaliin, detected a t least six fractions and was able to
218
MARY J. VOAI)EN AND .DOI1R1T I,~ROOMBE]~C,
t)rovidc evidence for the homogcueit.y of four of them. Tim presm~t, results show a marked variation in the clectrophoretie pa.ttcrn wit,b age, the nmximmn number of fractions (8) being found in the young adult, y-cryst,allin. Tlmre is ~shen a striking loss of the more posit, ively charged and/or larger molecular wc,ight~ prot.eins as the lens continues to age. The significaJmc of t.his for lhc amint,enance of the homeostatic balancc of tit(: ageing lens is ats present unknown, b u t the results are of pa.rlicular interest, i~ the present, context, as t.hey would sugge.st, t,h.at~ inhibition is associated wit.h some or all ot" the remainiug protein fl'nctions. The elution and testil~g of the individual protein fl'actions is being at,templed, but at. present, no definite coneltlsions have beel~ reached. A C1¢, N 0 W L E D C .~I E N'l'8
We exteml our thanks, as al\v:/)-s, to Dr. F,. 4. Weale for his patience nnd his eont.imiii~g interest in t [",s work. The technical assistance of Miss Barbara ,_cott£ nnd Mr. P. ({oklwater is gr~tefully acknowledged. Financial SUplx~rt, was received from the Medical Research Council. R E FE. I{V , N C" 1~,~:-'
Ilj6rk. ]. (t961). Exl)tl Ere ICes. l, 145. .Bj/Srk. 1. (1964). Exptl Ere lies. 3, 254. ]"roomberg, 1). and Voaden. M. (1966). E.rptl Eye ]~es. 5, 1. van ]-Icyninge.n. ]l. (1962). ]rt :lVtc Erie, vol, l. Ed. H. Davstm. Academic Press, New York. I[oward, A. {]952). Stain 2'ccMwl. 27, 313. I~apaconstantinou, J. (1965). Biochim. Biot,]~ffs...Ida ..trust. 107, S1. Poulik. M. D. (1957). :Nature 180, 1477. Smithics, O. (1959). ]n Advances in Prolein Cllemistry. Eds. (',. B. ,.\~ginsen, 3I. l,. ,a,nsm~, K. :Bailey and J. T. EdsalL -kcadcmic Pres,% New York. Spcctor, A. (1965). 1rived. 01~hthalmol. 4, 5"79. Sri,fiva~a~/, B. ]3. and Harding, (2. V. (1965). In, rcsl. Opht],ahnoi. 4, 452, Voaden, ~I. J. and ],~roomberg, D. (1967). In Syml,osium o~. thc Biochemistry of the Eye, August, (1966), ]~d. J. Nordmmm; S. Kargcr A.G. (Basel, Ncw York), .In preparation.