Electrophoretic modifications of three enzymes in extracts of human and bovine lens. Posttranslational “aging” of lens enzymes

385

Clinica

Chimica

@ Elsevier

Acta,

Scientific

70 (1976) 385-390 Publishing Company,

Amsterdam

- Printed

in The Netherlands

CCA 7785

ELECTROPHORETIC MODIFICATIONS OF THREE ENZYMES IN EXTRACTS OF HUMAN AND BOVINE LENS POSTTRANSLATIONAL “AGING” OF LENS ENZYMES

HENRIETTE Institut

SKALA-RUBINSON,‘MAGDELEINE

de Pathologie

(Received

January

Moltkulaire,

VIBERT

24 rue du Faubourg

and J.C. DREYFUS

St. Jacques,

75014

*

Paris (France)

9, 1976)

Summary Electrophoretic modifications have been found in extracts from human and bovine lenses for three enzymes: glucose-6-phosphate dehydrogenase, triosephosphate isomerase and nucleoside phosphorylase. Increased anodic mobility is observed in all cases. It is more pronounced than in red cell lysates, also more evident in lenses from adult than from young animals. These results give evidence of posttranslational “aging” of enzyme molecules in lenses.

Introduction Compared to other organs, the lens is regarded as being metabolically inactive: its protein turnover is very slow [1,2]. Three areas can be distinguished in the lens: a layer of epithelial cells (area I); the cells of this layer are nucleated, they differentiate into fibers (area II), which progress to the center of the lens (area III). The cells lose their nucleus during their differentiation into fibers. The center of the lens, having no nuclei, undergoes no protein synthesis, so that its proteins are very long-lived and may even stem from fetal cells. Lens, therefore, is a good material for the study of aging of proteins in vivo. It can be compared on the one hand to actively metabolizing tissues and on the other hand to red blood cells, which in mammals possess no nucleus and display no protein synthesis. In the red cells posttranslational modifications have been demonstrated for various enzymes [ 3,4]. Isozyme studies have been performed for hexokinase [5] and lactate dehydrogenase [6,7] but no abnormality in the position of electrophoretic bands was reported in extracts from bovine lenses. * To **

whom

Universitk

all correspondence Paris V,

II 129

should

be addressed.

de l’I.N.S.E.R.M.,

L.A.

85du

C.N.R.S.

386

In an approach to this problem, we chose three enzymes which undergo electrophoretic modifications in human red cells to a different extent, and which are coded in tissues by only one gene, e.g. glucose-6-phosphate dehydrogen~e (EC 1.1.X49), for which changes are only apparent by using electrofocusing techniques 143 ; triosephosphate isomerase (EC 5.3.1.1), which displays a progressive change between the three normal bands; and nucleoside phosphorylase (EC 2.4.2.1) for which new, more anodic bands appear in old red cells 131. The present work demonstrates electrophoretic changes in extracts from lenses of man and cattle. These changes are much greater than those of red cell enzymes. They are more pronounced in the oldest part of the lens (nucleus). In addition, clear differences are found between lenses of young (calf) and adult animals. eateries

and methods

Material. Substrates for enzymatic reactions were obtained from Boehringer Mannheim and Sigma CC. Other reagents were purchased from Merck and Calbiochem. Ampholines were provided by LKB, acrylamide and bisacrylamide by Eastman Kodak. Human lenses were obtained from men over 60 who had been operated for cataract. Bovine lenses came from the slaughter house. Lenses were dissected from contaminating tissues, especially the iris, and wiped with filter paper to eliminate eye fluids. Methods. Lenses were homogenised in a Potter-Elvehjem apparatus in water at a I : I d~ution~ then centrifuged at 10 000 X g for 15 min. Bovine lenses were dissected into three areas prior to homogenization. Starch gel elelctrophoresis and staining for triosephosphate isomerase were carried out as described earlier [8] and nucleoside phosphorylase according to

a. Human glucose-6-phosphate dehydmhoa?hate de?hydrogcnase. b. Beef and calf ~uc~s~-6-phosphate blood cells; 3, lens extract. beef lens Area I: 3, beef fens Area II; 4, beef white blood cells: 7, calf liver.

387

a

Fig. 2. Ektrophoresis of triosephosphate isomerase. a. Human trlosephosphate isomerase: 1, red blood cells; 2, white blood cells; 3, lens extract. b. Beef and calf triosephosphate isomerase: 1, red blood cells: 2. liver; 3, beef lens extract Area I; 4, beef lens extract Area II: 5, beef lens extract Area III; 6, calf lens extract Area I: 7, calf lens extract Area II; 8, calf lens extract Area III.

Turner et al. [9]. Isoelectric focusing and staining for glucose-6-phosphate dehydrogenase were performed according to Kahn et al. [4]. Results 1. Glu~ose-6-~~osphate de~ydroge~use (GWD) The results of electrofocusing of GGPD are shown in Fig. 1. Human GGPD (Fig. la) undergoes a lowering of the isoelectric point more pronounced than that of red cells. In cattle (Fig. lb) red cell GGPD was not modified compared to other tissues. In the intermediary zone of the lens there was a considerable modification of the isoelectric point in adults, while calf lens extracts showed no difference with liver taken as control. No GGPD activity was found in the nucleus of either beef or calf. 2. Triosephosphate isomerase (Fig. 2) Fig. 2a shows the results in man. Hemolysates and tissue extracts displayed three main bands. In hemolysates the strongest band was the more cathodic in

388

b

Electrophoresis

of

3, lens

Fig.

3.

extract.

(the

4. lens

extract

b. Area

Beef

nucleoside

II; 5, lens

calf

gives

extract

phosphorylase. the Area

same

a.

pattern)

Human:

1, red

1, red blood

blood

cells;

cells;

2. liver;

2,

white

3, lens

blood

extract

cells; Area

I;

III.

young cells, the more anodic in old cells [3]. Lens extracts demonstrated a very different pattern: several new bands appeared and extended far in the anodic direction. In cattle (Fig. 2b) lens extracts showed more anodic bands than tissue extracts. Anodisation was more apparent in the nucleus than in intermediary bands, and more in adult than in calf lens.

389

3. Nucleoside phosphorylase (Fig. 3) In human extracts (Fig. 3a) the modifications which were very apparent in hemolysates were even more pronounced in lens extracts. The most cathodic band which is the major band in tissues has completely disappeared and the mobility towards the anode was more increased. In cattle extracts (Fig. 3b) all tissues show the same pattern, including hemolysate, the activity of which was very low in agreement with the work of Ansay and Hanset [lo]. By contrast, the enzyme from lens extract had a greater anodic mobility, increasing from periphery to center, but was the.same in adult beef and in calves. Discussion Our electrophoretic experiments performed on extracts from human and bovine lenses on three enzymes demonstrated the following facts: 1. All three enzymes showed an increased anodic mobility or a lowered isoelectric point, compared to tissues with an active turnover, in man and adult cattle. 2. Compared to hemolysate, changes were greater in human lens than in red blood cells. They showed up in cattle lenses, contrasting with the lack of any apparent modification in hemolysate. 3. Changes were clearly related to the “age” of the molecule. They were greater in the nucleus than in the intermediary zone. In membrane layer extracts electrophoretic mobility was normal except for a slightly more anodic migration in the case of nucleoside phosphorylase. 4. Changes were also related to the age of the animal, but in a different way according to the enzyme. The pattern was the same in the young and the adults for nucleoside phosphorylase. Anodisation was greater for adult than young lenses as regards triosephosphate isomerase. Glucose-6-phosphate dehydrogenase was modified only in the adult. It is to be noted that GGPD was entirely inactivated before entering the nucleus, where no activity could be detected even in the calf. Aging of the molecule, therefore, results in an increase of the anodic mobility of the three enzymes which were studied. This is in agreement with the changes which have been described in red cells. The changes, however, are much more pronounced in lens than in red cells and, in addition, appear even in the bovine species, for which no changes are found in hemolysates. Modifications which can be attributed to posttranslational effects have been described for the specific proteins of the lens, the crystallins [ll].They involve essentially deamidation and shortening of the chains [12,13]. In our case deamidation is very likely to take place. Shortening of the chains, resulting in the loss of aminoacid residues, is less likely to play a major role, since it would in some cases end in more positively charged proteins, which have not been found up to now. Work is now in progress to test these hypotheses. Acknowledgement We wish to thank Dr Y. Courtois

for helpful

suggestions.

390

References 1 Young, R.W. and Fulborst, H.W. (1966) Invest. Ophtalmol. 5. 288-297 2 Waley. S.G. (1964) Biochem. J. 91, 576-583 3 Turner, B.M., Fisher, R.A. and Harris, H. (1975) in Isozymes (Marker& ed.), Vol. 1, PP. 781-795. Academic Press, New York 4 Kahn, A., Boivin. P., Vibert. M., Cottereau. D. and Dreyfus. J.C. (1974) Biochimie 56, 1395-1407 5 Chylack, L.T.J., Kinosbita, J.H. and Kasabian, R. (1970) Expt. Eye Res. 10, 250-262 6 Bernstein, L., Kerr&an, M. and Maisel, H. (1966) Expt. Eye Res. 5,309-314 7 Stewart, J.A. and Papaconstantinou, J. (1966) Biochim. Biophys. Acta 121,69-78 8 Rubinson, H., Vodovar, M., Meienhofer, M.C. and Dreyfus, J.C. (1971) FEBS L&t. 13. 290-292 9 Turner, B.M., Fisher. R.A. and Harris, H. (1971) Eur. J. Biochem. 24, 288-295 10 Ansay, M. and Hanset. R. (1972) Anim. Blood Groups Biochem. Genet. 3.219-227 11 Schoenmakers, J.G.G. and Bloemendal. H. (1968) Nature 220.790-791 12 Bloemendal. H., Berns, A.J.M., Van der Ouderaa, F.J. and De Jong, W.W. (1972) Expt. Eye Res. 14, 80-81 13 Van Kleef. F.S.M., De Jong, W.W. and Hoenders, H.J. (1975) Nature 258. 264-266