Exp. Eye Res. (1955) 21, 251-257
The Effect of Copper on Human and Bovine Lens and on Human Cultured Lens Epithelium Enzymes YOGESH
C. AWASTHI, STEVEN P. MILLER, SATISH
D. W. ARYA AND
K. SRIVASTAVA
The University of Texas Medical Byan& at Galveston, Texas, and Doheny Eye Foundation, Los Angeles, Californiu, U.X.A. (Received 20 Junuury 1975, asadin revised form 25 March 1975; Boston) The effect of physiological levels of copper on the glycolytic and shnnt pathway enzymes of glucose metabolism in human and bovine lens and on cultured lens epithelium ha,s been hexokinase, phosphofructokinase, studied. In human and bovine lens homogenates, pyruvate kinase, glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase and A-phosphoglucona,te dehydrogenase were almost completely inhibited in the presence of dehydrogenase and triose phosphate 50 piw-copper. -4ldolase, glucose-6-phosphate isomerase were inhibited 78, 50 and 36% respectively. The inhibition of most of the enzymes was not significantly reversed by the addition of EDTA. The inhibition of enzyme activity was also not reversed by dialysis of the copper-treated enzyme against phosphate buffer. Similar results mere obtained when human cultured lens epithelium was used as the enzyme source. The studies indicate that if the increased amount of copper present in senile cataractons lenses is in the free form, it can significantly impair glucose metabolism in t’he lens.
1. Introduction Impaired copper metabolism has in the past been linked to the development of senile cataract (Shlopak, 1962a and b). Almost a 12-fold increase in the lens copper content of the mature senile cataract patients has been reported by Shlopnk (1962a). Nath and Srivastava (1969) have also shown decreased activities of lactate dehydrogena.se along with increased copper content in the mature senile cataractous lenses. There are conflicting reports on the content of copper in normal as well as catasactous lenses. Galin, Nano and aall (1962), Deazevedo and Dejorge ,(1965) and Swanson and Truesdale (1971) have reported either unaltered or decreased levels of copper in some forms of senile cataract. Although the effect of the physiological levels of copper ions on the red cell enzymes has been studied (Boulard, Blume and Beutler, 1972) in order to study the hemolytic process induced by the increased copper levels (Brinton, 1947; McIntyre, Clink, Levi, Cummings and Sherlock, 1967), a systematic study of the effect of copper ions on various glycolytic enzymes of the lens has not been carried out. The present report deals with the studies on the effect of copper on the glycolytic enzymes of human and bovine lens and on the enzymes of cultured human lens epithelium. Besides, an attempt has been made to determine the mean values for the activity of various eizzymes of normal human and bovine lens which could, possibly, be used for reference in future investigations. 2. Materials and Methods Sormal human lenses were obtained from the eye bank of Doheny Eye Foundation, Los Angeles, California. The lenses with intact capsules were used either immediately after the removal or were stored at -20°C. Bovine lenses were removed from the eyes of freshly 251
252
Y. C. AWASTHI
ET AL.
slaughtered cattle by a posterior approach. NAD, NADH, NADP, and NADPH and all the auxiliary enzymes used in this study were purchased from Sigma Chemical Co. U.S.A. The enzyme assays were conducted according to the method described by Beutler (19’71) using a Gilford recording spectrophotometer. The enzyme activities are expressed as international units: 4 iu of enzyme brought about utilization of 1 /Jrnol of substrate per min at 37°C under the assay conditions described by Beutler (1971). Since EDTA is a constituent of Solution “G” recommended by Beutler (1971) for making red cell hemolysate, only phosphate buffer 10 mM, pH 7.0, was used for ma,lting the lens and cultured epithelium homogenates. Tissues were homogenized using Potter Elvehjem tissue homogenizer in potassium phosphate buffer, 10 m&f, pH 7.0 to make a 10% homogenate. The homogenate was centrifuged at 10 000 xg for 30 min. Tbe sediment was discarded and the clear supernatant was used on the same day for enzyme assays. Protein in the supernatant was determined by the method of Lowry, Rosebrough, Parr and Ra,ndall (1951). Culture of human less epitkelium The lenses were removed from the donor eye by a posterior approach, keeping the capsule intact. The lens was incised at the equator and the epithelium along with capsule was gently removed from the cortex under a dissecting microscope. It was then placed in a Rose chamber (Rose, 1954) under a perforated cellophane strip. Eagle’s minimum essential medium containing 15ye fetal bovine serum, fortified with 0.5:/, dextrose, 2 ma{-glutamine a,nd twice the original concentration of amino acids and vitamins was used as the culture medium. The medium was exchanged two to three times a week. When the cells formed a monolayer, they were subcultured by trypsinization. The human lens epithelial cells used in these experiments were from the 175th to 185th passages. Prescription bottles (32 oz, Brockway Glass Co., Inc.) were sealed with 20 ml of 100 000 cells/ml. After 48 hr the medium was exchanged and the cells were allowed to grow to confluency. The confluent monolayers were washed with phosphate buffered saline pH 7.0 and harvested with a rubber policeman. The cells were washed twice with phosphate buffered saline and frozen at -20°C until used. Incubations with copper ions In the case of cultured human lens epithelium 500 ~1 of the homogenate was incubated with CuSO,, final conc,entration 0.5 mM at 4°C for 1 hr. The concentration of CuSO, was 0.5 mM because 500 ~1 homogenate which was used without dilution contained a large amount of protein which could react with copper. The reaction mixture was then dialyzed overnight against 500 volumes of potassium phosphate buffer, 10 mM, pH 7.0, to remove excess copper ions. A control having no CuSO, was incubated alongside and processed similarly to account for any loss of activity during incubation and dialysis. In order to study the effect of EDTA on CuSO, treated and dialyzed enzymes, EDTA, final concentration 400 PM, was added before starting the enzyme reaction. In the case of human and bovine lens enzymes, lo-20 ~1 of homogenate was diluted to 200 ~1 with water. Varying amounts of 1 mniI CuSO,, 5-150 ~1, were added and the volume in each tube was adjusted to 0.35 ml with water. The homogenate was incubated for 1 hr at 4°C after which sufficient EDTA (neutral, pH 7.0, 0.2 MM)was added to make final concentration to 400 PM in 1 ml. Subsequently, the substrate and the auxiliary enzymes were added to make the final volume to 1.0 ml. Thus, the final concentration of CuSO, was 5-150 pM.
3. Results The human lens with its encapsulated epithelium is an ideal source for isolating and studying pure epithelial cells in vitro. Lens epithelium and capsule can be cultured as
EFFECT
OF
COPPER
OK
LEXS
ENZYXES
253
explants or the cells can be enzymatically removed from the lens capsule and established jn culture. We have established three human lens epithelial cell lines. The human lens epithelial cell line ( # D450) used in these studies was derived from a 5%year-old donor and is now in the 197th passage. The cells from 150th to 175th passages were used in these experiments. Morphologically these cells were epithelial and exhibited contact inhibition (Fig. 1). The mitotic index of this line was 7.8% and
FIG. i. Phase-contrast nagnification X 200).
photomicrograph
of human
lens epithelia.1
cell iine in the 164th passage (original
254
Y. C. AWASTHI
ET AL
the generation time 22 hr. The karyotype of this lens epithelial cell line was heteroploid with a chromosome distribution from 50 to 112 and a hypertriploid mode “74”. No evidence of mycoplasmal or fungal contamination was found in these cells. When cells were implanted subcutaneously into the cheek pouches of immunosuppressed hamsters no tumors were induced. The isozyme patterns of glucose-6-phosphate dehydrogenase, malate dehydrogenase and lactate dehydrogenase were found to be similar in the fresh lens epithelium, the cultured lens epithelial cells and the established lens epithelial cell line. The cells of this line synthesize collagen. However, studies on crystallin synthesis have not yet been carried out. As shown in Table I, hexokinase, phosphofructokinase, aldolase, phosphoglycerate kinase, pyruvate kinase and 6-phosphogluconate-dehydrogenase are almost completely inhibited by 50 PM-CuSO,. Inhibition of triosephosphate isomerase and
Effect of copper on glycolytic enzymes of normal laurnan and bovine lens
Enzvme actirity* Enzyme
(u/g protein)
Hexokinase 0.154&0.026 Glucose phosphate isomerase 232-&2.9 Phosphofructokinase 4.1 kO.32 Xldolase 1~16~0~10 Triosephosphate isomerase 921&161 Glyceraldehyde phosphate dehydrogenase 55+3&6~9 213k13.0 Phosphoglycerate kinase Lactate dehydrogenase 134*13,6 88.7k7.4 Pyruvate kinase 19,13*1.3 Enolase 33.2i1.3 Nonophosphoglyceromutase Glucose-6-P-dehydrogenase 3.2650.45 6-Phosphogluconate dehydrogenase 0*716&0~106
Human lens Inhibitiont by 50 pwcu (%)
Enzyme activity*
Bovine iens Inhibition? by 50 pi-Cu
Wit,hout With EDTA EDTA (400 ~$1) (u/g protein)
100 100 78 26 94 98 14 98 0 0 49 100
90 100 5i 0
Without EDTA (%)
0~109~0~010 183-cS.9 3.8&0.103 0.951&0.12 47985298
100 96 92 30
20
677h6.1 34’7-&42 2512.6 62816.6 135h6.5 44.1+3.6 0+349+0~054
95 14 96 0 0 36
63
0.670&0.02
96
96 98 -
* The activity is expressed as mean j, standard error of six different samples of normal human and bovine lenses. t The effect of Cu*+ was studied on one of the samples under the conditions described in the text. The homogenate (lo-20 ~1) was incubated for 10 min at 37°C in a total volume of 0.35 ml which contained respective buffer and 0.05 ml of 1 mar-CuSO,. At the end of incubation 0.1 ml of 4 m&r-ethylene diamine tetraoetate (pH 7.0) was added wherever specified. The enzyme assays were carried out exactly as described by Beutler (1971). Hexokinase, phosphofruotokina,se and pyruvate kinase were almost completely inhibited with 20 PM-CuSO,.
glucose-6-P dehydrogenase was only partial with 50 JLLM-CUSO~ and increasing the CuSO, concentrations up to 150 PM did not result in any significant increase in inhibition. Hexokinase, phosphofructokinase and pyruvate kinase were almost completely inhibited by 20 PM-CuSO,, whereas 5 PM-CuSO, brought about 80% inhibition of phosphofructokinase. Addition of EDTA to samples incubated with 50 PM-C&O,
EFFECT
OF COPPER
OX LENS
255
EKZYMES
to bring a final concentration of EDTA (in 1.0 ml) to 400 ,uM did not significantly reverse the inhibition of most of the enzymes by CuSO,. EDTA, however, significantly lowered the inhibition of glucose-6-P and 6-phosphogluconate dehydrogenases and triose phosphate isomerase. The activity of various enzymes in the cultured human lens epithelium was significantly higher than that of the whole lens (Table II). A significant loss in the activity of some of the enzymes on dialysis even without copper treatment was observed (Table II). Phosphofructokinase, lactate dehydrogenase, pyruvate kinase; monophosphoglyceromutase and glucose-6-P dehydrogenase lose most of their activity on dialysis alone. Triosephosphate isomerase, phosphoglycerate kinase, enolase and 6-phosphogluconate dehydrogenase also lose some enzyme activity during overnight dialysis without any treatment with CuSO,. However, the activities of t.hese enzymes remain almost unaltered during overnight storage at 4°C without dialysis. Thus the per cent inhibition in various enzyme activities was calculated by comparing the activity in the dialyzed samples with and without incubation with &SO, (Table II). TABLE
Effect of copper
on
glycolytic
enzymes
II
of cultured human lens epithelium
Enzyme activity
Enzyme
Hexokinase Glucose phosphate isomerase Phosphofructokinase Aldolase Triosephosphate isomerase Glyceraldehyde phosphate dehydrogenase Phosphoglycerate kinase Pyruvate kinase Lactate dehydrogenase SIonophosphoglyceromutase Enolase Glucose-6-P-dehydrogenase 6.Phosphogluconate dehydrogenase
Before incubation, no dialysis
54.5 3094 65.0 276
(u/g protein)
Incubat.ion without Cu3+ and dialysis
Inhibition by CUE+ (%I
100
32 561
0 1738 0.99 97.2 21 540
7141 1557 6083
473 3554 125 so2
11.64 46.5 29.1 0
9s 99 77
1103
3S.l
0
1785 654
1113 34.0
86.6 4.3
100 93 58
46.0
0
100
66127
1005
112
37.2 2712 2.3 293
Incubation with Cu2+ and dialysis
36 57 67 33
100
Lens epithelium homogenate, incubated 1 hr with excess of &SO, at 4” (500 pmolar final concentration of CuSO,); dialyzed for 16 hr against 10 rnlw phosphate buffer pH 7.4 to remove unbound CuSO, and wsayed for enzyme activity. Per cent inhibition was calculated against the enzyme activity in the control incubated without CuSO, and dialyzed. Enzyme activities are expressed as pmoles substrate cleaved/min/g protein. Enzyme activities of homogenate were unaltered for at least 8 hr when the homogenate was stored at 4”.
Incubation of the cultured lens epithelium supernatant with an excess of C’USO, (final concentration 500 pM) at 4°C for 1 hr followed by an overnight dialysis resulted in a signifjcant loss of the activity of several enzymes (Table II). Hexokinase,
256
Y. C. AWASTHI
ET AL.
glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, lactate dehydrogenase, enolase and monophosphoglyceromutase activities were almost completely inhibited by copper. On the other hand, phosphofructokinase, aldolase, triose phosphate isomerase and pyruvate kinase were only partially inhibited. Effect of incubation of cultured lens epithelium homogenate with various concentrations of CuSO, (5-150 PM) and reversal of inhibition by the addition of 400 PM-EDTA prior to the addition of auxiliary enzymes was performed as described for lens enzymes. The inhibition of various enzymes was comparable to lens epithelia,l enzymes treated with copper and dialyzed to remove excess of copper (Table III). EDTA significantly reversed the inhibition of aldolase and 6-phosphogluconat,e dehydrogenase by CuSO,.
Inhibitim epithelium
of the glycolytic erqpes of cultwed human lens by copper and reversnl of inlzibition by EDTA Inhibition
Enzyme
Hexokinase Phosphofructokinase dldolase Triosephosphate isomerase Glyoeraldehyde phosphate dehydrogeuase Phosphoglycerate kinase Pyruvate kinese Glucose-A-P-dehydrogenase 6-Phosphogluconate dehydrogenase
20 5 1.50 150 100 100 50 150 50
(‘$&)
Ko EDTA
With EDTA (400 pm)
100 72 94 62 4s 100 99 56 100
100 60 26 48 44 100 96 44 2s
The cultured lens epithelium homogenate (supernatant) was incubated with various concentrations of copper and after first incubation, 400 pnr-EDTA was added to chelate excess copper and the enzyme a&i&es mere determined as in Table I.
4. Discussion Copper, in very small concentrations as present in the serum, has been found to he inhibit,ory to various glycolytic enzymes of the red cells (Boulard et al., 1972). At more nearly physiological levels of copper, we find that several glycolytic enzymes of human and bovine lens; and human lens epithelium are significantly inhibited. In human and bovine lens homogenates, several key enzymes such as hexokinase, phosphofruct,okinase, pyruvate kinase, glyceraldehyde phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase which regulate glycolysis and hexosemonophosphate shunt, pathway were almost completely inhibited in the presence of 50 PM-copper. Aldolase and glucose-6-P dehydrogenase were also significantly inhibited in the presence of 50 PM-CuSO,. The inhibition of most of the enzymes was not significant.ly reversed by the addition of EDTA prior to addition of auxiliary enzymes used in the assay of various enzymes. The auxiliary enzymes should not be inhibited because addition of an eight-fold excess of EDTA should bind all the free This would indicate that the inhibition of various copper in the reaction mixture.
EFFECT
OF COPPER
ONLENS
357
ENZYMES
enzymes is due to a chemical binding of copper to enzymes. Similar results were obtained when the effect of CuSO, was studied on the cultured lens epithelium. Since, the inhibition of various enzymes is not reversed either by the addition of an excess of EDTA or by d’la 1ysis of &SO,-treated samples to remove unbound copper, it appears that the inhibition of most of the glycolytic enzymes is by binding of copper to proteins. It is possible the inhibition of at least some of t,he enzymes which require sulfhydryl groups is by way of direct binding of copper to these groups. It is important to note that dialysis of the cultured lens epithelium homogenate against phosphate hufYer, 10 m&l, pH 7.0, even without the treatment with CuSO, leads to a significant loss in the a&i&es of various enzymes. These studies clearly indicate that if the increased amount of copper present in the cataractous lenses is in the free form, it can significantly impair the glycolytic pathway of the lens; especially when hexokinase which is the first enzyme of the glycolytic pathway of glucose metabolism is almost completely inhibited in the presence of .50PII-copper. However, in view of the conflicting reports on the level of copper in normal
and senile
cataractous
lenses
and the absence
of data
on the ratio
of free
Cu
to protein-bound Cu in the lens, the role of copper in senile cataract cannot be precisely defined.
This work
was supported
ill l:iirt
i:i- the Xational
Institute
of He&h
grants GL-216%
and EY 01677. REFERENCES Keutler, E. (1971). A Manual of Biochemical Mefhods. Grune &: Stratton, Inc., New York. Boula,rd, M., Blume, K. CT. and Beutler, E. (1972). The effect of copper on red cell enzyme activities. J. Clin. I,nvest. 51, 459. Grinton, D. (1947). Wilson’s disease. Proc. Roy. Xoc. Med. 40, 556. De Azevedo and De Jorge, F. B. (1965). Some mineral constituents of normal human eye tissues (Na-K-b:Zg-Ca-P-Cu). Ophthnlmologica 119, 43. Galin, M. A., Nano, H. D. and Hall, T. (1962). Ocular zinc concentration. Invest. Ophthalmol.
1, 142. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with Folin phenol reagent. J. BioZ. Chem. 193, 265. McIntyre, N., Clink, H. M., Levi, A. J., Cummings, J. iX. and Sherlock, S. (1967). Hemolytic anemia in Wiison’s disease. New Eng. J. Med. 276, 439. Xath, R., Srivastava, 8. K. and Singh, K. (1969). Accumulation of copper and inhibition of lactate dehydrogenase in senile cataract.. Ind. J. Exp. Biol. 7, 25,. Rose, G. (1954). A separable and multipurpose tissue culture chamber. Texas Rep. Biol. Xed. 12, 1074. Shlopak, T. V. (1962a). Chemistry of the crystalline lens in the normal and pathological state. II. Chemical contents in the blood and crystalline lenses of patients with cataract. OftaZmoZ. 2h.. 17, 347; Chem. Abstracts (1964) 60, 11196f. dhlopak, T. V. (1962b). Peculiarities of crysta,lline lens chemistry, norma, and pathologica,l. Oftalmol. Zh. 17, 273; Chem. Abstracts (1973) 58, 4879d. dwanson, A. A. and Truesdale, A. W. (1971). Elemental analysis in normal and cataractous human lens tissue. Biochem. Biophys. Res. Commun. 45, 1488.
The Effect of Copper on Human and Bovine Lens and on Human Cultured Lens Epithelium Enzymes YOGESH
C. AWASTHI, STEVEN P. MILLER, SATISH
D. W. ARYA AND
K. SRIVASTAVA
The University of Texas Medical Byan& at Galveston, Texas, and Doheny Eye Foundation, Los Angeles, Californiu, U.X.A. (Received 20 Junuury 1975, asadin revised form 25 March 1975; Boston) The effect of physiological levels of copper on the glycolytic and shnnt pathway enzymes of glucose metabolism in human and bovine lens and on cultured lens epithelium ha,s been hexokinase, phosphofructokinase, studied. In human and bovine lens homogenates, pyruvate kinase, glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase and A-phosphoglucona,te dehydrogenase were almost completely inhibited in the presence of dehydrogenase and triose phosphate 50 piw-copper. -4ldolase, glucose-6-phosphate isomerase were inhibited 78, 50 and 36% respectively. The inhibition of most of the enzymes was not significantly reversed by the addition of EDTA. The inhibition of enzyme activity was also not reversed by dialysis of the copper-treated enzyme against phosphate buffer. Similar results mere obtained when human cultured lens epithelium was used as the enzyme source. The studies indicate that if the increased amount of copper present in senile cataractons lenses is in the free form, it can significantly impair glucose metabolism in t’he lens.
1. Introduction Impaired copper metabolism has in the past been linked to the development of senile cataract (Shlopak, 1962a and b). Almost a 12-fold increase in the lens copper content of the mature senile cataract patients has been reported by Shlopnk (1962a). Nath and Srivastava (1969) have also shown decreased activities of lactate dehydrogena.se along with increased copper content in the mature senile cataractous lenses. There are conflicting reports on the content of copper in normal as well as catasactous lenses. Galin, Nano and aall (1962), Deazevedo and Dejorge ,(1965) and Swanson and Truesdale (1971) have reported either unaltered or decreased levels of copper in some forms of senile cataract. Although the effect of the physiological levels of copper ions on the red cell enzymes has been studied (Boulard, Blume and Beutler, 1972) in order to study the hemolytic process induced by the increased copper levels (Brinton, 1947; McIntyre, Clink, Levi, Cummings and Sherlock, 1967), a systematic study of the effect of copper ions on various glycolytic enzymes of the lens has not been carried out. The present report deals with the studies on the effect of copper on the glycolytic enzymes of human and bovine lens and on the enzymes of cultured human lens epithelium. Besides, an attempt has been made to determine the mean values for the activity of various eizzymes of normal human and bovine lens which could, possibly, be used for reference in future investigations. 2. Materials and Methods Sormal human lenses were obtained from the eye bank of Doheny Eye Foundation, Los Angeles, California. The lenses with intact capsules were used either immediately after the removal or were stored at -20°C. Bovine lenses were removed from the eyes of freshly 251
252
Y. C. AWASTHI
ET AL.
slaughtered cattle by a posterior approach. NAD, NADH, NADP, and NADPH and all the auxiliary enzymes used in this study were purchased from Sigma Chemical Co. U.S.A. The enzyme assays were conducted according to the method described by Beutler (19’71) using a Gilford recording spectrophotometer. The enzyme activities are expressed as international units: 4 iu of enzyme brought about utilization of 1 /Jrnol of substrate per min at 37°C under the assay conditions described by Beutler (1971). Since EDTA is a constituent of Solution “G” recommended by Beutler (1971) for making red cell hemolysate, only phosphate buffer 10 mM, pH 7.0, was used for ma,lting the lens and cultured epithelium homogenates. Tissues were homogenized using Potter Elvehjem tissue homogenizer in potassium phosphate buffer, 10 m&f, pH 7.0 to make a 10% homogenate. The homogenate was centrifuged at 10 000 xg for 30 min. Tbe sediment was discarded and the clear supernatant was used on the same day for enzyme assays. Protein in the supernatant was determined by the method of Lowry, Rosebrough, Parr and Ra,ndall (1951). Culture of human less epitkelium The lenses were removed from the donor eye by a posterior approach, keeping the capsule intact. The lens was incised at the equator and the epithelium along with capsule was gently removed from the cortex under a dissecting microscope. It was then placed in a Rose chamber (Rose, 1954) under a perforated cellophane strip. Eagle’s minimum essential medium containing 15ye fetal bovine serum, fortified with 0.5:/, dextrose, 2 ma{-glutamine a,nd twice the original concentration of amino acids and vitamins was used as the culture medium. The medium was exchanged two to three times a week. When the cells formed a monolayer, they were subcultured by trypsinization. The human lens epithelial cells used in these experiments were from the 175th to 185th passages. Prescription bottles (32 oz, Brockway Glass Co., Inc.) were sealed with 20 ml of 100 000 cells/ml. After 48 hr the medium was exchanged and the cells were allowed to grow to confluency. The confluent monolayers were washed with phosphate buffered saline pH 7.0 and harvested with a rubber policeman. The cells were washed twice with phosphate buffered saline and frozen at -20°C until used. Incubations with copper ions In the case of cultured human lens epithelium 500 ~1 of the homogenate was incubated with CuSO,, final conc,entration 0.5 mM at 4°C for 1 hr. The concentration of CuSO, was 0.5 mM because 500 ~1 homogenate which was used without dilution contained a large amount of protein which could react with copper. The reaction mixture was then dialyzed overnight against 500 volumes of potassium phosphate buffer, 10 mM, pH 7.0, to remove excess copper ions. A control having no CuSO, was incubated alongside and processed similarly to account for any loss of activity during incubation and dialysis. In order to study the effect of EDTA on CuSO, treated and dialyzed enzymes, EDTA, final concentration 400 PM, was added before starting the enzyme reaction. In the case of human and bovine lens enzymes, lo-20 ~1 of homogenate was diluted to 200 ~1 with water. Varying amounts of 1 mniI CuSO,, 5-150 ~1, were added and the volume in each tube was adjusted to 0.35 ml with water. The homogenate was incubated for 1 hr at 4°C after which sufficient EDTA (neutral, pH 7.0, 0.2 MM)was added to make final concentration to 400 PM in 1 ml. Subsequently, the substrate and the auxiliary enzymes were added to make the final volume to 1.0 ml. Thus, the final concentration of CuSO, was 5-150 pM.
3. Results The human lens with its encapsulated epithelium is an ideal source for isolating and studying pure epithelial cells in vitro. Lens epithelium and capsule can be cultured as
EFFECT
OF
COPPER
OK
LEXS
ENZYXES
253
explants or the cells can be enzymatically removed from the lens capsule and established jn culture. We have established three human lens epithelial cell lines. The human lens epithelial cell line ( # D450) used in these studies was derived from a 5%year-old donor and is now in the 197th passage. The cells from 150th to 175th passages were used in these experiments. Morphologically these cells were epithelial and exhibited contact inhibition (Fig. 1). The mitotic index of this line was 7.8% and
FIG. i. Phase-contrast nagnification X 200).
photomicrograph
of human
lens epithelia.1
cell iine in the 164th passage (original
254
Y. C. AWASTHI
ET AL
the generation time 22 hr. The karyotype of this lens epithelial cell line was heteroploid with a chromosome distribution from 50 to 112 and a hypertriploid mode “74”. No evidence of mycoplasmal or fungal contamination was found in these cells. When cells were implanted subcutaneously into the cheek pouches of immunosuppressed hamsters no tumors were induced. The isozyme patterns of glucose-6-phosphate dehydrogenase, malate dehydrogenase and lactate dehydrogenase were found to be similar in the fresh lens epithelium, the cultured lens epithelial cells and the established lens epithelial cell line. The cells of this line synthesize collagen. However, studies on crystallin synthesis have not yet been carried out. As shown in Table I, hexokinase, phosphofructokinase, aldolase, phosphoglycerate kinase, pyruvate kinase and 6-phosphogluconate-dehydrogenase are almost completely inhibited by 50 PM-CuSO,. Inhibition of triosephosphate isomerase and
Effect of copper on glycolytic enzymes of normal laurnan and bovine lens
Enzvme actirity* Enzyme
(u/g protein)
Hexokinase 0.154&0.026 Glucose phosphate isomerase 232-&2.9 Phosphofructokinase 4.1 kO.32 Xldolase 1~16~0~10 Triosephosphate isomerase 921&161 Glyceraldehyde phosphate dehydrogenase 55+3&6~9 213k13.0 Phosphoglycerate kinase Lactate dehydrogenase 134*13,6 88.7k7.4 Pyruvate kinase 19,13*1.3 Enolase 33.2i1.3 Nonophosphoglyceromutase Glucose-6-P-dehydrogenase 3.2650.45 6-Phosphogluconate dehydrogenase 0*716&0~106
Human lens Inhibitiont by 50 pwcu (%)
Enzyme activity*
Bovine iens Inhibition? by 50 pi-Cu
Wit,hout With EDTA EDTA (400 ~$1) (u/g protein)
100 100 78 26 94 98 14 98 0 0 49 100
90 100 5i 0
Without EDTA (%)
0~109~0~010 183-cS.9 3.8&0.103 0.951&0.12 47985298
100 96 92 30
20
677h6.1 34’7-&42 2512.6 62816.6 135h6.5 44.1+3.6 0+349+0~054
95 14 96 0 0 36
63
0.670&0.02
96
96 98 -
* The activity is expressed as mean j, standard error of six different samples of normal human and bovine lenses. t The effect of Cu*+ was studied on one of the samples under the conditions described in the text. The homogenate (lo-20 ~1) was incubated for 10 min at 37°C in a total volume of 0.35 ml which contained respective buffer and 0.05 ml of 1 mar-CuSO,. At the end of incubation 0.1 ml of 4 m&r-ethylene diamine tetraoetate (pH 7.0) was added wherever specified. The enzyme assays were carried out exactly as described by Beutler (1971). Hexokinase, phosphofruotokina,se and pyruvate kinase were almost completely inhibited with 20 PM-CuSO,.
glucose-6-P dehydrogenase was only partial with 50 JLLM-CUSO~ and increasing the CuSO, concentrations up to 150 PM did not result in any significant increase in inhibition. Hexokinase, phosphofructokinase and pyruvate kinase were almost completely inhibited by 20 PM-CuSO,, whereas 5 PM-CuSO, brought about 80% inhibition of phosphofructokinase. Addition of EDTA to samples incubated with 50 PM-C&O,
EFFECT
OF COPPER
OX LENS
255
EKZYMES
to bring a final concentration of EDTA (in 1.0 ml) to 400 ,uM did not significantly reverse the inhibition of most of the enzymes by CuSO,. EDTA, however, significantly lowered the inhibition of glucose-6-P and 6-phosphogluconate dehydrogenases and triose phosphate isomerase. The activity of various enzymes in the cultured human lens epithelium was significantly higher than that of the whole lens (Table II). A significant loss in the activity of some of the enzymes on dialysis even without copper treatment was observed (Table II). Phosphofructokinase, lactate dehydrogenase, pyruvate kinase; monophosphoglyceromutase and glucose-6-P dehydrogenase lose most of their activity on dialysis alone. Triosephosphate isomerase, phosphoglycerate kinase, enolase and 6-phosphogluconate dehydrogenase also lose some enzyme activity during overnight dialysis without any treatment with CuSO,. However, the activities of t.hese enzymes remain almost unaltered during overnight storage at 4°C without dialysis. Thus the per cent inhibition in various enzyme activities was calculated by comparing the activity in the dialyzed samples with and without incubation with &SO, (Table II). TABLE
Effect of copper
on
glycolytic
enzymes
II
of cultured human lens epithelium
Enzyme activity
Enzyme
Hexokinase Glucose phosphate isomerase Phosphofructokinase Aldolase Triosephosphate isomerase Glyceraldehyde phosphate dehydrogenase Phosphoglycerate kinase Pyruvate kinase Lactate dehydrogenase SIonophosphoglyceromutase Enolase Glucose-6-P-dehydrogenase 6.Phosphogluconate dehydrogenase
Before incubation, no dialysis
54.5 3094 65.0 276
(u/g protein)
Incubat.ion without Cu3+ and dialysis
Inhibition by CUE+ (%I
100
32 561
0 1738 0.99 97.2 21 540
7141 1557 6083
473 3554 125 so2
11.64 46.5 29.1 0
9s 99 77
1103
3S.l
0
1785 654
1113 34.0
86.6 4.3
100 93 58
46.0
0
100
66127
1005
112
37.2 2712 2.3 293
Incubation with Cu2+ and dialysis
36 57 67 33
100
Lens epithelium homogenate, incubated 1 hr with excess of &SO, at 4” (500 pmolar final concentration of CuSO,); dialyzed for 16 hr against 10 rnlw phosphate buffer pH 7.4 to remove unbound CuSO, and wsayed for enzyme activity. Per cent inhibition was calculated against the enzyme activity in the control incubated without CuSO, and dialyzed. Enzyme activities are expressed as pmoles substrate cleaved/min/g protein. Enzyme activities of homogenate were unaltered for at least 8 hr when the homogenate was stored at 4”.
Incubation of the cultured lens epithelium supernatant with an excess of C’USO, (final concentration 500 pM) at 4°C for 1 hr followed by an overnight dialysis resulted in a signifjcant loss of the activity of several enzymes (Table II). Hexokinase,
256
Y. C. AWASTHI
ET AL.
glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, lactate dehydrogenase, enolase and monophosphoglyceromutase activities were almost completely inhibited by copper. On the other hand, phosphofructokinase, aldolase, triose phosphate isomerase and pyruvate kinase were only partially inhibited. Effect of incubation of cultured lens epithelium homogenate with various concentrations of CuSO, (5-150 PM) and reversal of inhibition by the addition of 400 PM-EDTA prior to the addition of auxiliary enzymes was performed as described for lens enzymes. The inhibition of various enzymes was comparable to lens epithelia,l enzymes treated with copper and dialyzed to remove excess of copper (Table III). EDTA significantly reversed the inhibition of aldolase and 6-phosphogluconat,e dehydrogenase by CuSO,.
Inhibitim epithelium
of the glycolytic erqpes of cultwed human lens by copper and reversnl of inlzibition by EDTA Inhibition
Enzyme
Hexokinase Phosphofructokinase dldolase Triosephosphate isomerase Glyoeraldehyde phosphate dehydrogeuase Phosphoglycerate kinase Pyruvate kinese Glucose-A-P-dehydrogenase 6-Phosphogluconate dehydrogenase
20 5 1.50 150 100 100 50 150 50
(‘$&)
Ko EDTA
With EDTA (400 pm)
100 72 94 62 4s 100 99 56 100
100 60 26 48 44 100 96 44 2s
The cultured lens epithelium homogenate (supernatant) was incubated with various concentrations of copper and after first incubation, 400 pnr-EDTA was added to chelate excess copper and the enzyme a&i&es mere determined as in Table I.
4. Discussion Copper, in very small concentrations as present in the serum, has been found to he inhibit,ory to various glycolytic enzymes of the red cells (Boulard et al., 1972). At more nearly physiological levels of copper, we find that several glycolytic enzymes of human and bovine lens; and human lens epithelium are significantly inhibited. In human and bovine lens homogenates, several key enzymes such as hexokinase, phosphofruct,okinase, pyruvate kinase, glyceraldehyde phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase which regulate glycolysis and hexosemonophosphate shunt, pathway were almost completely inhibited in the presence of 50 PM-copper. Aldolase and glucose-6-P dehydrogenase were also significantly inhibited in the presence of 50 PM-CuSO,. The inhibition of most of the enzymes was not significant.ly reversed by the addition of EDTA prior to addition of auxiliary enzymes used in the assay of various enzymes. The auxiliary enzymes should not be inhibited because addition of an eight-fold excess of EDTA should bind all the free This would indicate that the inhibition of various copper in the reaction mixture.
EFFECT
OF COPPER
ONLENS
357
ENZYMES
enzymes is due to a chemical binding of copper to enzymes. Similar results were obtained when the effect of CuSO, was studied on the cultured lens epithelium. Since, the inhibition of various enzymes is not reversed either by the addition of an excess of EDTA or by d’la 1ysis of &SO,-treated samples to remove unbound copper, it appears that the inhibition of most of the glycolytic enzymes is by binding of copper to proteins. It is possible the inhibition of at least some of t,he enzymes which require sulfhydryl groups is by way of direct binding of copper to these groups. It is important to note that dialysis of the cultured lens epithelium homogenate against phosphate hufYer, 10 m&l, pH 7.0, even without the treatment with CuSO, leads to a significant loss in the a&i&es of various enzymes. These studies clearly indicate that if the increased amount of copper present in the cataractous lenses is in the free form, it can significantly impair the glycolytic pathway of the lens; especially when hexokinase which is the first enzyme of the glycolytic pathway of glucose metabolism is almost completely inhibited in the presence of .50PII-copper. However, in view of the conflicting reports on the level of copper in normal
and senile
cataractous
lenses
and the absence
of data
on the ratio
of free
Cu
to protein-bound Cu in the lens, the role of copper in senile cataract cannot be precisely defined.
This work
was supported
ill l:iirt
i:i- the Xational
Institute
of He&h
grants GL-216%
and EY 01677. REFERENCES Keutler, E. (1971). A Manual of Biochemical Mefhods. Grune &: Stratton, Inc., New York. Boula,rd, M., Blume, K. CT. and Beutler, E. (1972). The effect of copper on red cell enzyme activities. J. Clin. I,nvest. 51, 459. Grinton, D. (1947). Wilson’s disease. Proc. Roy. Xoc. Med. 40, 556. De Azevedo and De Jorge, F. B. (1965). Some mineral constituents of normal human eye tissues (Na-K-b:Zg-Ca-P-Cu). Ophthnlmologica 119, 43. Galin, M. A., Nano, H. D. and Hall, T. (1962). Ocular zinc concentration. Invest. Ophthalmol.
1, 142. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with Folin phenol reagent. J. BioZ. Chem. 193, 265. McIntyre, N., Clink, H. M., Levi, A. J., Cummings, J. iX. and Sherlock, S. (1967). Hemolytic anemia in Wiison’s disease. New Eng. J. Med. 276, 439. Xath, R., Srivastava, 8. K. and Singh, K. (1969). Accumulation of copper and inhibition of lactate dehydrogenase in senile cataract.. Ind. J. Exp. Biol. 7, 25,. Rose, G. (1954). A separable and multipurpose tissue culture chamber. Texas Rep. Biol. Xed. 12, 1074. Shlopak, T. V. (1962a). Chemistry of the crystalline lens in the normal and pathological state. II. Chemical contents in the blood and crystalline lenses of patients with cataract. OftaZmoZ. 2h.. 17, 347; Chem. Abstracts (1964) 60, 11196f. dhlopak, T. V. (1962b). Peculiarities of crysta,lline lens chemistry, norma, and pathologica,l. Oftalmol. Zh. 17, 273; Chem. Abstracts (1973) 58, 4879d. dwanson, A. A. and Truesdale, A. W. (1971). Elemental analysis in normal and cataractous human lens tissue. Biochem. Biophys. Res. Commun. 45, 1488.
