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Ribonuclease activity and polysome profile in human senile cataract
Exptl Eye Res. (1968) 7, 546-550
]Ribonuclease A c t i v i t y a n d P o l y s o m e P r o f i l e in H u m a n S e n i l e C a t a r a c t * 5'I. MA:ro.~E, G. MAm~I~'~ A~D F. CAR'rA
University Eye Clinic, University of Parma, Italy (Received 20 May 1968. Lo~don) zk m e a s u r a b l e r i b o n u c l e a s e a c t i v i t y a p p e a r s in h u m a n l e n s duriDg t h e d e v e l o p m e n t o f senile c,~taract; t h e e n z y m a t i c a c t i v i t y , a b s e n t in t h e n o r m a l lens, s e e m s t o b e r o u g h l y p r o ]portiona[ to t.he i n v o l v e m e n t o f t h e l e n s c o r t e x b y t h e o p a c i t i e s a.nd is d e t e c t a b l e e v e n in r e l a t i v e l y e a r l y ph,~ses o f t h e disea.se. T o g e t h e r w i t h She i n c r e a s e d r i b o n u c l e a s e a c t i v i t y a d e c r e a s e o f h e a v y p o l y r i b o s o m e a g g r e g a t e s s e e m a t o b e p r e s e n t in c o r t i c a l senile cats.r,~ct. T h e possible i n t e r p r e t a t i o n s of these d a t a are briefly discussed.
1. Introduction Senile opacificat.ion of h u m a n lens is l
P O L Y S O h I E PrCO:FILE I N H U M A N S E N I L E CA'l:AI~ACT
547
2. M a t e r i a l s and M e t h o d s The assays were c a r r i e d o u t on t h e following tissues: a d u l t bovine, horse a n d dog lenses, rat liver, t r a n s p a r e n t h u m a n lenses o b t a i n e d after e n u c l e a t i o n of t h e eye for ocular t u m o u r s in i n d i v i d u a l s a g e d 50-75 years, senile c a t a r a c t s o b t a i n e d after i n t r a c a p s u l a r ex~traction. As in previous i n v e s t i g a t i o n s (Maraini et el., 1967), d e p e n d i n g on t h e in vivo b i o m i c r o scopicat aspect, c a t a r a c t o u s lmmes were d i v i d e d i n t o different groups a c c o r d i n g to t h e location a n d t h e e x t e n s i o n of t h e opacities:
Groui) 1: n o r m a l lenses Grout) 2: initial cortical opacities in v o lv in g n o t m o r e t h a n a b o u t o n e - h a l f of t h e c o r t e x Group 3: s u b t o t a l cortical c a t a r a c t s leav in g a b o u t o n e - q u a r t e r of cortical fibres free f r o m opacities Groulp 4: c o m p l e t e c o r t i c a l cataract.s Grouip 5: pos t e r i or c a p s u l a r c a t a r a c t (the r e m a i n d e r of t h e lens b ein g p r a c t i c a l l y free f r o m opacities) Group 6: n u c l e a r c a t a r a c t (with clear co rtex ) Lenses were i m m e d i a t e l y frozen a t ~ 1 9 6 ° ( J a n d s t o r e d at this t e m p e r a t u r e u n t i l used.
Ribonuclease activity assay B r o d y ' s m e t h o d (1957) was e m p l o y e d to d e t e r m i n e R N a s e a c t i v i t y . A f t e r b ein g caref u l l y dried with filter p a p e r , e a c h lens was w e i g h e d a n d h o m o g e n i z e d (30/o w/v) in ice-cold Tris buffer ( p H 7-5; 0.05 ~0; 0-5 m l of t h e h o m o g e n a t e w e r e a d d e d to 0-5 m l of R N A * s o l u t i o n (3 mg/ ml in Tris buffer p H 7-5) a n d i n c u b a t e d a t 37°C for 45". F o r e a c h assay apprc)priate zero-time c on t r ol t u b e s were p r e p a r e d b y i n c u b a t i n g u n d e r t h e s a m e c o n d i t i o n s s u b s t r a t e R N A w i t h o u t lens h o m o g e n a t e . T h e r e a c t i o n was s t o p p e d b y a d d i n g 2 m l of ice-cold 5~/o perchloric acid (PCA) to t h e t e s t t u b e s a n d 2 m l of 5~/o P C A plus 0-5 ml of lens h o m o g e n a t e to the control tubes. A f t e r 5' at 0°C th e t u b e s w e r e c e n t r i f u g e d a t 3000 r e v / m i n a n d t h e increase in op t i c a l dellsity a t 260 mt~ of t h e s u p e r n a t ~ n t s of t h e e x p e r i m e n t a l t u b e s o v e r those of t he z e r o - t i m e t u b e s was t a k e n as t h e R N a s e a c t i v i t y . This last one was e x p r e s s e d as the increase of acid-soluble, u.v. a b s o r b i n g R N A d e g r a d a t i o n p r o d u c t s / m g tissue proteins. Crystalline R N a s e (Boehringer) was ~sed as s t a n d a r d e n z y m e p r e p a r a t i o n . The error of t h e m e t h o d , as e.x-pressed b y t h e v a r i a t i o n coefficient of 5 e n z y m a t i c assays on the s a m e r a t liver h o m o g e n a t e , was ± 4"3135~/o. P r o t e i n c o n t e n t of t h e h o m o g e n a t e was d e t e r m i n e d a c c o r d i n g to L o w r y , R o s e b r o u g h , F a r r a n d R a n d a l l (1951).
Sucrose gradient analysis H o m o g e n i z a t i o n was c a r r i e d o u t in ice-cold M e d i u m A (0-25)I KC1, 0-005 ~ ~gC12, 0-25 ~I sucrose, 0.05 ~ Tris a t p H 7-6) (2 ml/g of tissue) w i t h five strokes ill a t i g h t l y fitting Teflon-glass h o m o g e n i z e r . The p o s t - m i t o c o n d r i a l s u p e r n a t a n t f r a c t i o n was p r e p a r e d b y centrifuging a t 18,000 g for 20 rain. D e o x y c h o l a t e was a d d e d to a final c o n c e n t r a t i o n of 1-2°/o and samples of 1-5 m l were l a y e r e d over 25 ml of a linear g r a d i e n t o f 15 to 35~/o sucrose. C e n t r i f u g a t i o n was p e r f o r m e d in a Spinco L 2 p r e p a r a t i v e u l t r a c e n t r i f u g e u s i n g the SW 25"1 r o t o r a t 24-,000 r e v / m i n for 2 h. Dm-ing all t h e isolation p r o c e d u r e , f r o m homogellization to t h e final c e n t r i f u g a t i o n , a t e m p e r a t u r e c o n d i t i o n of 0 ° C was n ~ i n t a i n e d . •'kfter c e n t r i f u g a t i o n t h e b o t t o m of t h e plastic t u b e was p m l c t u r e d w i t h a h y p o d e r m i c needle a n d fractions of 7 droplets were collected; e a c h f r a c t i o n was a n a l y s e d for a b s o r p t i o n at 260 mt~ in a U n i c a m SiP 500 s p e c t r o p h o t o m e t e r . * Sigma I~NA from yEaSt. Type XI. Purified per Crestfield et a.1. (J. Biol. CherrY. 1955} 216, 185,
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3. Results
Ribonucleasc activity The results are summarized in Table I. No measurable R N a s e a c t i v i t y could be detected in a n y of the n o r m a l lenses so far studied; appreciable enzyme activities were on t h e c o n t r a r y present in c a m r a c t o u s lens homogcnates. R N a s c a c t i v i t y was particularly high in cortical cataract: it, progressively increased from t h e less to t h e more a d w m c c d ibrms and seemed to be r o u g h l y correlated w i t h t h e ext, ension of the lens opacities. TABLE I
Ribm~uclease activity in normal and cataractous h u m a n lenses
31can s.D.
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
0(*) 0(~') 0(.~) 0(1:) 0(:~) o($)
0"362 0 0 0-550 0
0"264 0"443 0"089 0'639 0
0"460 0'094. 0.25l 0"864 0'634 o 0 0-840 0.324 0.572 0.135 0.432 0.441 0-688
0-378 0"127 0 0"370 0 o
0 0 0"221 0"110 0 o 0-061 0 0.046 0
0 0
0"182 -4=0-256
0-287 =~__0.258
0"409 ~0-288
0"145 :k0"181
0-043 :]: 0.070
(*) H o r s e ; (~) bovine; C[:) h u m a n . RNa.se a c t i v i t y is e x p r e s s e d as ~he increase in t h e u.v. a b s o r p t i o n a t 260 mt~/mg t i s s u e p r o t e i n s r e c o r d e d d u r i n g the i n c u b a t i o n t i m e o f 45".
A measurable e n z y m a t i c a c t i v i t y was less reguh~rly recorded in posterior a n d nuclear cataracts. The analysis of v a r i a n c e between t h e different groups of c a t a r a c t o u s lenses is lfighly sigrfifical~t (aY = 4-153; F 0-01 = 3-91); t h e D u n c a n - K r a m e r test shows a significant difference between complete cortical c a t a r a c t s a n d nuclear a n d posterior opacities. No significant difference m a y on the c o n t r a r y be d e m o n s t r a t e d between t h e different groups of cortical cataracts.
Sucrose gradient analysis I n contrast to w h a t o b t a i n e d with t h e same t e c h n i q u e w i t h r a t liver (Fig. 1), satisfactory polysome profiles were difficult to o b t a i n in a d u l t lens due to poor resolution in t h e oligosome region, This was p r o b a b l y a t least in p a r t l i n k e d to
POLYSOME
PR, O F I L E
IN
I - [ U M A N SE~N~ILE C A T A R A C T
649
considerable contamination with stn'ucturM i)rotehm which became apparent after the 30th fraction and was reflected b y an inv(,rsion of the 26c~/280 m/~ extinctio~x ratio. A sucrose gradient analysis of polyribosomes from horse lens is illustrated in Fig. 2; similar results were obtained from adult bovine and dog lenses.
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0"4
0-4
0-2
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30
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I
40
0
50
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.Fro. 1. P o l y s o m e profile f r o m I)r)st-mitochondrial s u p e r n a t ~ n t f r a c t i o n f r o m r a t liver. L i n e a r sucrose g r a d i e n t 10-35~/o.
I
IO Bottom
I
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I
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40
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50 Top
~ m . 2. P o l y s o m e profile f r o m p o s t - m i t o e h o n drial s u p e r n a t a n $ f r a c t i o n f r o m horse lens. L i n e a r sucrose gradielxt 1 0 - 3 5 % .
Due to the obvious difficulty in obtaining an adequate n u m b e r of h u m a n transparent lenses, polysome profile studies have been limited to three groups of h u m a n cata.ractous lenses: early corticM cataracts (group 2), complete cortical cataracts (gn'oup 4) a nd posterior capsular cataracts (group 5). The results, graphically illustrated in :Fig. 3, show a lowering of the profile in the he a vy polysome region in total cortical, cataracts, compared with early cortical a nd posterior capsular cataracts.
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:FIe,. 3. (a) I ) o l y s o m e profiles f r o m e a r l y ( ) a n d c o m p l e t e (. . . . . ) c o r t i c a l c a t a r a c t . (b) P o l y s o m e profile f r o m p o s t e r i o r c a p s u l a r c a t a r a c t . L i n e a r s u c r o s e g r a d i e n t 15-35°/o.
4. D i s c u s s i o n
The absence of a nleasurable :RNase a c t i v i t y in normal lens was already known b y the work of Z igman et al. (1963) and of Swanson, Je te r and R a l e y (1967); our present data confirm the results of these authors. However, no data were a v a i l a b l e , to our knowledge, on RNase a c t i v i t y changes in senile cataract; our results demonstrate t h a t
550
hi. ~1[AIONE, G. B I A R A I N I A N D I,'. C A R T A
tile a p p e a r a n c e of a n appreciable e n z y m e aetivi{.y accompanies the d e v e l o p m e n t of senile lens opacifieation. This increased R N a s e a c t i v i t y is p a r t i c u l a r l y evident in cortical cataracts, a n d seems to be r o u g h l y correlated to the e x t e n t of cortical opacification; the increase of t h e e n z y m e a c t i v i t y is far less constant and m u c h lower in nuclear a n d capsular cataracts, a n d this m i g h t be il~terpreted, in the positive cases, as reflecting a minor cortical i n v o l v e m e n t so f r e q u e n t l y coexisting in these lenses. We t h i n k t h a t two m a i n interpretations of this increased R N a s e a c t i v i t y m a y be advanced: (a) a n increased enzylq~e synthesis, (b) a decreased synthesis of a n R N a s e inhibitor. Considering t h a t the lens total protein content is specifically decreased in cortical cataract, p r o b a b l y reflecbing a lowered proteh~ synthesis, and t h a t the increase in R N a s e a c t i v i t y seems to be proportional to the i n v o l v e m e n t of the lens cortex, we would be a t present more inclined to support the second of these possibilities, i.e. the decreased synthesis of a n R N a s e inhibitor. Together w i t h the changes in R N a s e a c t i v i t y our d a t a point out the probable decrease of h e a v y polyribosomal aggregates in cortical cataract; if the relative a m o u n t s of the different species of ribosome aggregates on the g r a d i e n t reflects accurately the proportions really present in the living cell, this finding m i g h t give a possible explanation of the decreased protein synthesis in cataractous h u m a n lens. Different possibilities m u s t be considered in discussing this change of the polysome profile ill cortical cataract: (1) it m i g h t reflect a n increased rate of lenticular R N A degradation occurring in vivo, possibly linked to the increased R N a s e a c t i v i t y (2) it migtlt be the consequence of a change in the kinds of messenger R N A e v e n t u a l l y occurring durb~g the d e v e l o p m e n t of the cataract. I n this respect, it m a y be recalled, however, t h a t the peptide m a p s obtained from h u m a n cataractous ~-crystallin a n d a l b u m i n o i d do not a p p r e c i a b l y differ from those obtained from n o r m a l bovine ~-crystallin and a l b u m i n o i d (Chi-Ching Mok a n d 1,Valey, 1967). (3) it m i g h t be the result of ICNase action during the isolation procedure a n d therefore not reflect a condition existing in vivo in the lens. We do not consider it possible, on the basis of t h e presently available data, to support one or the other of these possibilities. F u r t h e r studies are clearly needed o~., this problem, together w i t h i m p r o v e d technicM procedure allowing better resolution in t h e oligosome region. I~EFERENCES Brody, S. (1957) JBiochim. Biophys. iIcta 2J4, 502. Chi-Ching ~iok and Waley, S. G. (1967). 1~iochem. J. ][04, 128. I)ardenne, U. and I)rechsler, G. (1961). Arch. 029hthalmoL 164, 156. Devi, A. (1962). XI~.Oonc. Ophthal. vol. 2, p. 1149. Friedburg, D. (1966). Arch. Ophthalmol. 170, 365. :H[oekwin, O. and Berghoff, 1~. (1966). Klin. J~[onatsbl. Augenheilk. 149, 87. Lowry, O. ~., Rosebrough, ~N. J., Farr, A. L. and Randall, 1%. J. (1951). J. Biol. Chem. 193, 265.] Maeh, H. (1963). l(lin, i~lonatsbl. Augenheilk. 143, 689. Biaione, hi., ~a;raini, G. ~nd Carla, F. (1967). Ann. Ottalmol. Clin. Oculist. 93, I125. ~raini, G., Sant0ri, Bi. and Carta, F. (1967). JF,xptl Eye Res. ~, 126. Rabaey, ~I. (1959). Over de eiwitsaraenstelling der ooglens. Thesis, Ghent, ]3e]gium. Swanson, A. A., J e t e r , J. and R~ley, K . ' W . (i967). Exptl Eye ires. ~, 351. Zigman, S., Burton, M., Font~ine, J. ~nd Lerman, S. (1963). Invest. Ophthalmol. 2, 621.
]Ribonuclease A c t i v i t y a n d P o l y s o m e P r o f i l e in H u m a n S e n i l e C a t a r a c t * 5'I. MA:ro.~E, G. MAm~I~'~ A~D F. CAR'rA
University Eye Clinic, University of Parma, Italy (Received 20 May 1968. Lo~don) zk m e a s u r a b l e r i b o n u c l e a s e a c t i v i t y a p p e a r s in h u m a n l e n s duriDg t h e d e v e l o p m e n t o f senile c,~taract; t h e e n z y m a t i c a c t i v i t y , a b s e n t in t h e n o r m a l lens, s e e m s t o b e r o u g h l y p r o ]portiona[ to t.he i n v o l v e m e n t o f t h e l e n s c o r t e x b y t h e o p a c i t i e s a.nd is d e t e c t a b l e e v e n in r e l a t i v e l y e a r l y ph,~ses o f t h e disea.se. T o g e t h e r w i t h She i n c r e a s e d r i b o n u c l e a s e a c t i v i t y a d e c r e a s e o f h e a v y p o l y r i b o s o m e a g g r e g a t e s s e e m a t o b e p r e s e n t in c o r t i c a l senile cats.r,~ct. T h e possible i n t e r p r e t a t i o n s of these d a t a are briefly discussed.
1. Introduction Senile opacificat.ion of h u m a n lens is l
P O L Y S O h I E PrCO:FILE I N H U M A N S E N I L E CA'l:AI~ACT
547
2. M a t e r i a l s and M e t h o d s The assays were c a r r i e d o u t on t h e following tissues: a d u l t bovine, horse a n d dog lenses, rat liver, t r a n s p a r e n t h u m a n lenses o b t a i n e d after e n u c l e a t i o n of t h e eye for ocular t u m o u r s in i n d i v i d u a l s a g e d 50-75 years, senile c a t a r a c t s o b t a i n e d after i n t r a c a p s u l a r ex~traction. As in previous i n v e s t i g a t i o n s (Maraini et el., 1967), d e p e n d i n g on t h e in vivo b i o m i c r o scopicat aspect, c a t a r a c t o u s lmmes were d i v i d e d i n t o different groups a c c o r d i n g to t h e location a n d t h e e x t e n s i o n of t h e opacities:
Groui) 1: n o r m a l lenses Grout) 2: initial cortical opacities in v o lv in g n o t m o r e t h a n a b o u t o n e - h a l f of t h e c o r t e x Group 3: s u b t o t a l cortical c a t a r a c t s leav in g a b o u t o n e - q u a r t e r of cortical fibres free f r o m opacities Groulp 4: c o m p l e t e c o r t i c a l cataract.s Grouip 5: pos t e r i or c a p s u l a r c a t a r a c t (the r e m a i n d e r of t h e lens b ein g p r a c t i c a l l y free f r o m opacities) Group 6: n u c l e a r c a t a r a c t (with clear co rtex ) Lenses were i m m e d i a t e l y frozen a t ~ 1 9 6 ° ( J a n d s t o r e d at this t e m p e r a t u r e u n t i l used.
Ribonuclease activity assay B r o d y ' s m e t h o d (1957) was e m p l o y e d to d e t e r m i n e R N a s e a c t i v i t y . A f t e r b ein g caref u l l y dried with filter p a p e r , e a c h lens was w e i g h e d a n d h o m o g e n i z e d (30/o w/v) in ice-cold Tris buffer ( p H 7-5; 0.05 ~0; 0-5 m l of t h e h o m o g e n a t e w e r e a d d e d to 0-5 m l of R N A * s o l u t i o n (3 mg/ ml in Tris buffer p H 7-5) a n d i n c u b a t e d a t 37°C for 45". F o r e a c h assay apprc)priate zero-time c on t r ol t u b e s were p r e p a r e d b y i n c u b a t i n g u n d e r t h e s a m e c o n d i t i o n s s u b s t r a t e R N A w i t h o u t lens h o m o g e n a t e . T h e r e a c t i o n was s t o p p e d b y a d d i n g 2 m l of ice-cold 5~/o perchloric acid (PCA) to t h e t e s t t u b e s a n d 2 m l of 5~/o P C A plus 0-5 ml of lens h o m o g e n a t e to the control tubes. A f t e r 5' at 0°C th e t u b e s w e r e c e n t r i f u g e d a t 3000 r e v / m i n a n d t h e increase in op t i c a l dellsity a t 260 mt~ of t h e s u p e r n a t ~ n t s of t h e e x p e r i m e n t a l t u b e s o v e r those of t he z e r o - t i m e t u b e s was t a k e n as t h e R N a s e a c t i v i t y . This last one was e x p r e s s e d as the increase of acid-soluble, u.v. a b s o r b i n g R N A d e g r a d a t i o n p r o d u c t s / m g tissue proteins. Crystalline R N a s e (Boehringer) was ~sed as s t a n d a r d e n z y m e p r e p a r a t i o n . The error of t h e m e t h o d , as e.x-pressed b y t h e v a r i a t i o n coefficient of 5 e n z y m a t i c assays on the s a m e r a t liver h o m o g e n a t e , was ± 4"3135~/o. P r o t e i n c o n t e n t of t h e h o m o g e n a t e was d e t e r m i n e d a c c o r d i n g to L o w r y , R o s e b r o u g h , F a r r a n d R a n d a l l (1951).
Sucrose gradient analysis H o m o g e n i z a t i o n was c a r r i e d o u t in ice-cold M e d i u m A (0-25)I KC1, 0-005 ~ ~gC12, 0-25 ~I sucrose, 0.05 ~ Tris a t p H 7-6) (2 ml/g of tissue) w i t h five strokes ill a t i g h t l y fitting Teflon-glass h o m o g e n i z e r . The p o s t - m i t o c o n d r i a l s u p e r n a t a n t f r a c t i o n was p r e p a r e d b y centrifuging a t 18,000 g for 20 rain. D e o x y c h o l a t e was a d d e d to a final c o n c e n t r a t i o n of 1-2°/o and samples of 1-5 m l were l a y e r e d over 25 ml of a linear g r a d i e n t o f 15 to 35~/o sucrose. C e n t r i f u g a t i o n was p e r f o r m e d in a Spinco L 2 p r e p a r a t i v e u l t r a c e n t r i f u g e u s i n g the SW 25"1 r o t o r a t 24-,000 r e v / m i n for 2 h. Dm-ing all t h e isolation p r o c e d u r e , f r o m homogellization to t h e final c e n t r i f u g a t i o n , a t e m p e r a t u r e c o n d i t i o n of 0 ° C was n ~ i n t a i n e d . •'kfter c e n t r i f u g a t i o n t h e b o t t o m of t h e plastic t u b e was p m l c t u r e d w i t h a h y p o d e r m i c needle a n d fractions of 7 droplets were collected; e a c h f r a c t i o n was a n a l y s e d for a b s o r p t i o n at 260 mt~ in a U n i c a m SiP 500 s p e c t r o p h o t o m e t e r . * Sigma I~NA from yEaSt. Type XI. Purified per Crestfield et a.1. (J. Biol. CherrY. 1955} 216, 185,
548
5I. 3 [ A I O N E ,
G. M A I C A I N . [
AND
F. CAR TA
3. Results
Ribonucleasc activity The results are summarized in Table I. No measurable R N a s e a c t i v i t y could be detected in a n y of the n o r m a l lenses so far studied; appreciable enzyme activities were on t h e c o n t r a r y present in c a m r a c t o u s lens homogcnates. R N a s c a c t i v i t y was particularly high in cortical cataract: it, progressively increased from t h e less to t h e more a d w m c c d ibrms and seemed to be r o u g h l y correlated w i t h t h e ext, ension of the lens opacities. TABLE I
Ribm~uclease activity in normal and cataractous h u m a n lenses
31can s.D.
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
0(*) 0(~') 0(.~) 0(1:) 0(:~) o($)
0"362 0 0 0-550 0
0"264 0"443 0"089 0'639 0
0"460 0'094. 0.25l 0"864 0'634 o 0 0-840 0.324 0.572 0.135 0.432 0.441 0-688
0-378 0"127 0 0"370 0 o
0 0 0"221 0"110 0 o 0-061 0 0.046 0
0 0
0"182 -4=0-256
0-287 =~__0.258
0"409 ~0-288
0"145 :k0"181
0-043 :]: 0.070
(*) H o r s e ; (~) bovine; C[:) h u m a n . RNa.se a c t i v i t y is e x p r e s s e d as ~he increase in t h e u.v. a b s o r p t i o n a t 260 mt~/mg t i s s u e p r o t e i n s r e c o r d e d d u r i n g the i n c u b a t i o n t i m e o f 45".
A measurable e n z y m a t i c a c t i v i t y was less reguh~rly recorded in posterior a n d nuclear cataracts. The analysis of v a r i a n c e between t h e different groups of c a t a r a c t o u s lenses is lfighly sigrfifical~t (aY = 4-153; F 0-01 = 3-91); t h e D u n c a n - K r a m e r test shows a significant difference between complete cortical c a t a r a c t s a n d nuclear a n d posterior opacities. No significant difference m a y on the c o n t r a r y be d e m o n s t r a t e d between t h e different groups of cortical cataracts.
Sucrose gradient analysis I n contrast to w h a t o b t a i n e d with t h e same t e c h n i q u e w i t h r a t liver (Fig. 1), satisfactory polysome profiles were difficult to o b t a i n in a d u l t lens due to poor resolution in t h e oligosome region, This was p r o b a b l y a t least in p a r t l i n k e d to
POLYSOME
PR, O F I L E
IN
I - [ U M A N SE~N~ILE C A T A R A C T
649
considerable contamination with stn'ucturM i)rotehm which became apparent after the 30th fraction and was reflected b y an inv(,rsion of the 26c~/280 m/~ extinctio~x ratio. A sucrose gradient analysis of polyribosomes from horse lens is illustrated in Fig. 2; similar results were obtained from adult bovine and dog lenses.
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0.8
0"4
0-4
0-2
o
_
I
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30
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I
40
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50
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.Fro. 1. P o l y s o m e profile f r o m I)r)st-mitochondrial s u p e r n a t ~ n t f r a c t i o n f r o m r a t liver. L i n e a r sucrose g r a d i e n t 10-35~/o.
I
IO Bottom
I
20
I
30
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40
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50 Top
~ m . 2. P o l y s o m e profile f r o m p o s t - m i t o e h o n drial s u p e r n a t a n $ f r a c t i o n f r o m horse lens. L i n e a r sucrose gradielxt 1 0 - 3 5 % .
Due to the obvious difficulty in obtaining an adequate n u m b e r of h u m a n transparent lenses, polysome profile studies have been limited to three groups of h u m a n cata.ractous lenses: early corticM cataracts (group 2), complete cortical cataracts (gn'oup 4) a nd posterior capsular cataracts (group 5). The results, graphically illustrated in :Fig. 3, show a lowering of the profile in the he a vy polysome region in total cortical, cataracts, compared with early cortical a nd posterior capsular cataracts.
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:FIe,. 3. (a) I ) o l y s o m e profiles f r o m e a r l y ( ) a n d c o m p l e t e (. . . . . ) c o r t i c a l c a t a r a c t . (b) P o l y s o m e profile f r o m p o s t e r i o r c a p s u l a r c a t a r a c t . L i n e a r s u c r o s e g r a d i e n t 15-35°/o.
4. D i s c u s s i o n
The absence of a nleasurable :RNase a c t i v i t y in normal lens was already known b y the work of Z igman et al. (1963) and of Swanson, Je te r and R a l e y (1967); our present data confirm the results of these authors. However, no data were a v a i l a b l e , to our knowledge, on RNase a c t i v i t y changes in senile cataract; our results demonstrate t h a t
550
hi. ~1[AIONE, G. B I A R A I N I A N D I,'. C A R T A
tile a p p e a r a n c e of a n appreciable e n z y m e aetivi{.y accompanies the d e v e l o p m e n t of senile lens opacifieation. This increased R N a s e a c t i v i t y is p a r t i c u l a r l y evident in cortical cataracts, a n d seems to be r o u g h l y correlated to the e x t e n t of cortical opacification; the increase of t h e e n z y m e a c t i v i t y is far less constant and m u c h lower in nuclear a n d capsular cataracts, a n d this m i g h t be il~terpreted, in the positive cases, as reflecting a minor cortical i n v o l v e m e n t so f r e q u e n t l y coexisting in these lenses. We t h i n k t h a t two m a i n interpretations of this increased R N a s e a c t i v i t y m a y be advanced: (a) a n increased enzylq~e synthesis, (b) a decreased synthesis of a n R N a s e inhibitor. Considering t h a t the lens total protein content is specifically decreased in cortical cataract, p r o b a b l y reflecbing a lowered proteh~ synthesis, and t h a t the increase in R N a s e a c t i v i t y seems to be proportional to the i n v o l v e m e n t of the lens cortex, we would be a t present more inclined to support the second of these possibilities, i.e. the decreased synthesis of a n R N a s e inhibitor. Together w i t h the changes in R N a s e a c t i v i t y our d a t a point out the probable decrease of h e a v y polyribosomal aggregates in cortical cataract; if the relative a m o u n t s of the different species of ribosome aggregates on the g r a d i e n t reflects accurately the proportions really present in the living cell, this finding m i g h t give a possible explanation of the decreased protein synthesis in cataractous h u m a n lens. Different possibilities m u s t be considered in discussing this change of the polysome profile ill cortical cataract: (1) it m i g h t reflect a n increased rate of lenticular R N A degradation occurring in vivo, possibly linked to the increased R N a s e a c t i v i t y (2) it migtlt be the consequence of a change in the kinds of messenger R N A e v e n t u a l l y occurring durb~g the d e v e l o p m e n t of the cataract. I n this respect, it m a y be recalled, however, t h a t the peptide m a p s obtained from h u m a n cataractous ~-crystallin a n d a l b u m i n o i d do not a p p r e c i a b l y differ from those obtained from n o r m a l bovine ~-crystallin and a l b u m i n o i d (Chi-Ching Mok a n d 1,Valey, 1967). (3) it m i g h t be the result of ICNase action during the isolation procedure a n d therefore not reflect a condition existing in vivo in the lens. We do not consider it possible, on the basis of t h e presently available data, to support one or the other of these possibilities. F u r t h e r studies are clearly needed o~., this problem, together w i t h i m p r o v e d technicM procedure allowing better resolution in t h e oligosome region. I~EFERENCES Brody, S. (1957) JBiochim. Biophys. iIcta 2J4, 502. Chi-Ching ~iok and Waley, S. G. (1967). 1~iochem. J. ][04, 128. I)ardenne, U. and I)rechsler, G. (1961). Arch. 029hthalmoL 164, 156. Devi, A. (1962). XI~.Oonc. Ophthal. vol. 2, p. 1149. Friedburg, D. (1966). Arch. Ophthalmol. 170, 365. :H[oekwin, O. and Berghoff, 1~. (1966). Klin. J~[onatsbl. Augenheilk. 149, 87. Lowry, O. ~., Rosebrough, ~N. J., Farr, A. L. and Randall, 1%. J. (1951). J. Biol. Chem. 193, 265.] Maeh, H. (1963). l(lin, i~lonatsbl. Augenheilk. 143, 689. Biaione, hi., ~a;raini, G. ~nd Carla, F. (1967). Ann. Ottalmol. Clin. Oculist. 93, I125. ~raini, G., Sant0ri, Bi. and Carta, F. (1967). JF,xptl Eye Res. ~, 126. Rabaey, ~I. (1959). Over de eiwitsaraenstelling der ooglens. Thesis, Ghent, ]3e]gium. Swanson, A. A., J e t e r , J. and R~ley, K . ' W . (i967). Exptl Eye ires. ~, 351. Zigman, S., Burton, M., Font~ine, J. ~nd Lerman, S. (1963). Invest. Ophthalmol. 2, 621.