Exp. Eye Res. (1989) 49, 887-899 Disulfide Cross-linking of Urea-insoluble Proteins in Rabbit Lenses Treated with Hyperbaric Oxygen VANITA PADGAONKAR, FRANK J. GIBLIN* AND V. N. REDDY Eye Research Institute of Oakland University, Rochester, MI 48309-4401, U.S.A. (Received 16 March 1989 and accepted in revised form 8 June 1989) In vivo exposure of human patients and experimental animals to hyperbaric O 2 has been shown by other investigators to lead to opacification of the lens especially in the nuclear region. In the present study, cultured rabbit lenses were treated with hyperbaric O 2 in order to investigate possible formation of disulfide-cross-linked proteins in the urea-insoluble fraction of lens cortex and nucleus. When lenses were treated with 100 atmospheres of 100 % 02 for 24 hr, intermolecular disuIfide-linked proteins formed iN both the cortical and Nuclear regions. Under these conditions the level of reduced glutathione and the activity of glyceraldehyde-3 phosphate dehydrogenase (G-3PD) were depleted by > 95 % in both regions. The lenses were hazy in appearance but not opaque. Two-dimensional diagonal eleetrophoresis followed by immunoblotting indicated that the majority of the cross-linked proteins were/~- and ?~-crystallins. Also involved in the cross- linking was the enzyme G-3PD but not the main intrinsic membrane protein, MIP2nkD~. Treatment of the nuclear ureadnsoluble fraction of 02-treated lenses with sodium borohydride showed a nearly fourfold increase in the level of protein disulfide compared to that present in the same fraction of either fresh lenses or N2-treated controls. It was determined that an increase of approximately one disulfide group per 10n Da molecular weight corresponded to cross-linking of nearly 20 % of the urea-insoluble protein present in the O~-treated lenses. Experiments carried out at 8 atmospheres O2 were used to determine the region of the lens in which urea-insoluble disulfide first formed after exposure to O~. After 8 hr of treatment of lenses with 8 atmospheres 02 an increase in protein disulfide was observed in the urea-insoluble proteins of the lens nucleus but not of the cortex. Under these conditions, the level of glutathione had decreased by 62 % in the nucleus compared to only 13% in the cortex. Increasing the culture time to 16 hr under 8 atmospheres 0~ produced a further increase in protein disulfide in the nuclear region, the formation of a small amount of cross-linked protein in the cortex and a significantly greater decrease of G-3PD activity in the lens nucleus compared to the cortex. The overall results of the study demonstrate that exposure of lenses to hyperbaric 02 leads to disulfide-cross-linking of crystallins in the urea-insoluble fraction and that the initial formation of protein disulfide as well as the initial loss of glutathione occurs first in the lens nucleus. A similar type of cross-linking has been shown to occur in X-ray-induced and human senile cataract, and the phenomenon may also be related to the nuclear opacification which develops in lenses of human patients who have been treated with hyperbaric O2. Key words: hyperbaric oxygen ; lens ; rabbit ; glutathione ; protein disulfide ; membrane proteins; crystallins; glyceraldehyde-3-phosphate dehydrogenase. 1. Introduction Oxidation of lens erystallins with formation of high molecular weight protein aggregates has been reported in a number of studies involving human and experimental cataracts (Harding, 1973; Truscott and Augesteyn, 1977; Giblin, Chakrapani and Reddy, 1978; Lohmann, Schmel and Strobel, 1986). The aggregation of proteins in lens cataract has been shown to be associated with intermolecular covalent bonding of both a disulfide and non-disulfide nature (Buckingham, 1972; Harding, 1973; Dilley, 1975; Takemoto and Azari, 1977). Lens membrane proteins have also been found to participate in the aggregation process through intermolecular disulfide links with low molecular weight polypeptides (Spector etal., 1979; Garner, * To whom all correspondence should be addressed. 0014-4835/89/110887+ 13 $03.00/0 9 1989 Academic Press Limited
Transcript
Exp. Eye Res. (1989) 49, 887-899
Disulf ide Cross- l inking of Urea- inso luble Proteins in Rabbit Lenses Treated with Hyperbaric Oxygen
VANITA PADGAONKAR, FRANK J. GIBLIN* AND V. N. R E D D Y
Eye Research Institute of Oakland University, Rochester, MI 48309-4401, U.S.A.
(Received 16 March 1989 and accepted in revised form 8 June 1989)
In vivo exposure of human patients and experimental animals to hyperbaric O 2 has been shown by other investigators to lead to opacification of the lens especially in the nuclear region. In the present study, cultured rabbit lenses were treated with hyperbaric O 2 in order to investigate possible formation of disulfide-cross-linked proteins in the urea-insoluble fraction of lens cortex and nucleus. When lenses were treated with 100 atmospheres of 100 % 02 for 24 hr, intermolecular disuIfide-linked proteins formed iN both the cortical and Nuclear regions. Under these conditions the level of reduced glutathione and the activity of glyceraldehyde-3 phosphate dehydrogenase (G-3PD) were depleted by > 95 % in both regions. The lenses were hazy in appearance but not opaque. Two-dimensional diagonal eleetrophoresis followed by immunoblotting indicated that the majority of the cross-linked proteins were/~- and ?~-crystallins. Also involved in the cross- linking was the enzyme G-3PD but not the main intrinsic membrane protein, MIP2nkD~. Treatment of the nuclear ureadnsoluble fraction of 02-treated lenses with sodium borohydride showed a nearly fourfold increase in the level of protein disulfide compared to that present in the same fraction of either fresh lenses or N2-treated controls. I t was determined that an increase of approximately one disulfide group per 10 n Da molecular weight corresponded to cross-linking of nearly 20 % of the urea-insoluble protein present in the O~-treated lenses. Experiments carried out at 8 atmospheres O 2 were used to determine the region of the lens in which urea-insoluble disulfide first formed after exposure to O~. After 8 hr of treatment of lenses with 8 atmospheres 02 an increase in protein disulfide was observed in the urea-insoluble proteins of the lens nucleus but not of the cortex. Under these conditions, the level of glutathione had decreased by 62 % in the nucleus compared to only 13% in the cortex. Increasing the culture time to 16 hr under 8 atmospheres 0~ produced a further increase in protein disulfide in the nuclear region, the formation of a small amount of cross-linked protein in the cortex and a significantly greater decrease of G-3PD activity in the lens nucleus compared to the cortex. The overall results of the study demonstrate that exposure of lenses to hyperbaric 02 leads to disulfide-cross-linking of crystallins in the urea-insoluble fraction and that the initial formation of protein disulfide as well as the initial loss of glutathione occurs first in the lens nucleus. A similar type of cross-linking has been shown to occur in X-ray-induced and human senile cataract, and the phenomenon may also be related to the nuclear opacification which develops in lenses of human patients who have been treated with hyperbaric O 2.
Garner and Spector, 1981; Takemoto and Hansen, 1982). In a recent study which involved purified fiber cell membranes from human cataracts, Kodama and Takemoto (1988) showed that/?- and y-crystallins had become bound to membrane proteins by disulfide bonds. In an investigation of X-ray cataract in our laboratory we also determined the presence of disulfide-linked polypeptides in the urea-insoluble, membrane fraction, both in the mature stage of the cataract and in a stage just prior to maturi ty (Garadi, Giblin and Reddy, 1987). However, for both human and experimental cataracts, little is known about the mechanism of the disulfide cross- linking or the nature of the oxidant that may be involved.
Recently we have employed hyperbaric 02 as a model for investigating oxidative processes in the lens. When cultured rabbit lenses were exposed to increased pressures of 02, a rapid decrease occurred in the normal level of reduced glutathione (GSH) in the lens which coincided with accumulation of oxidized glutathione (GSSG) and stimulation in activity of the hexose monophosphate shunt (Giblin, Schrimscher, Chakrapani and Reddy, 1988). These results demonstrated that 02 is potentially reactive in the lens and that the reactivity may be enhanced by the fact that H202 is one of the products of the reaction of O 3 with GSH. A further observation in the study was a drop in GSH level in 02-treated lenses which occurred first in the lens nucleus before any loss of the tripeptide could be detected in the superficial cortex. This suggested that the nucleus is less capable than the rest of the lens of maintaining a reduced environment, vchich may account for the apparent susceptibility of the lens nucleus to loss of transparency following in vivo exposure of human patients and experimental animals to hyperbaric 02 (Schocket, Esterson, Bradford, Michaelis and Richards, 1972; Palmquist, Philipson and Barr, 1984).
The purpose of the present study was to determine whether exposure of cultured lenses to hyperbaric 02 would produce oxidation of protein sulfhydryl (--SH) groups in addition to its oxidative effects on GSH. The technique of two-dimensional diagonal electrophoresis was employed to demonstrate that disulfide cross-linking of proteins does indeed occur in the membrane fraction of the 02-treated lens. Immunoblotting techniques were employed to identify the cross-linked polypeptides and, in addition, methods involving reduction of protein disulfide with sodium borohydride were used to quantitate the amount of disulfide produced in the urea soluble fraction. In view of the observed participation of glyceraldehyde-3-phosphate dehydrogenase (G-3PD) in disulfide cross-linking, it was also of interest to measure activity of this enzyme following exposure of lenses to 02 . Finally, experiments were conducted to determine the region of the lens in which urea-insoluble protein disulfide is first formed following in vitro exposure of lenses to increased pressures of 02 .
2. Mater ia ls and M e t h o d s
New Zealand White rabbits weighing between 1"8 and 2"2 kg were employed ~n the study. The animals were killed by an overdose of sodium pentobarbital and the lenses were removed from excised eyes using a posterior approach. Speciality, custom grade gases were obtained from the Linde Division of the Union Carbide Corporation. Exposure of lenses to various pressures of N 2 or 02 was conducted in an 1850 ml Cell Disruption Bomb (Parr Instrument Company, Moline, IL). A description of the procedures used for culture of lenses under hyperbaric conditions has been presented previously (Giblin et al., 1988). The culture medium employed was TC199 containing 10 mM Hepes buffer, pH 7"4, with an osmolarity of 290 mosmol. For extended periods of culture (16 and 24 hr) streptomycin and penicillin-G (200 units ml 1 each) were added to the medium.
PROTEIN DISULFIDE INO2-TREATED LENSES 889
At the end of each experiment, lenses were immediately frozen in crushed dry ice and, with use of a cork borer, divided into superficial cortex (outer 40 % of total weight) and nucleus (inner 20 % of total weight). The tissues were homogenized (100 mg wet weight of lens per ml buffer) at 0-3~ in a N 2 atmosphere in 5 mM Tris-HC1 buffer, pH 7"9, containing 50 m~ iodoacetamide, 0"1 mM phenylmethyl sulfonylfluoride (PMSF) and 2 mM EDTA (all from Sigma Chemical Co., St Louis, MO). The amount of iodoacetamide used represented an approximate eightfold excess over the total glutathione and protein - - S H present in each sample. Following centrifugation of the homogenate for 20 rain at 12000 g the pellet was washed three times with ice-cold buffer and resuspended in 7"2 M urea. The urea was diluted to 3'5 M with homogenizing buffer and, following centrifugation, the urea-insoluble pellet was washed three times with 7-2 M urea plus homogenizing buffer (1:1), once with 0'1 M NaOH and finally with 5 mM Tris-HC1 buffer, pH 7"9, containing 2 mM EDTA (Russell, Robison and Kinoshita, 1981). The resultant urea-insoluble pellet was weighed and dissolved at 37~ for 16 hr in 0-5 M Tris-HC1 buffer, pH 6'8, containing 1% sodium dodecyl sulfate (SDS) and 10 % glycerol.
Two-dimensional diagonal electrophoresis was carried out according to the modified method of Takemoto and Hansen (1982) and Garadi et al. (1987). Ten per cent polyacrylamide gels were employed in both the first and second dimension. Electrophoresis in the first dimension was carried out in 1-5 mm i.d. tubes, overnight at 50 V following application of 120/,g protein. The gels were removed from the tubes and incubated for 30 min in 50 mM Tris-HC1 buffer, pH 8"0, containing 2 mM EDTA, 2% SDS and 5% fl- mercaptoethanol. Each tube gel was applied on a second dimension slab gel and a solution of 1% agarose in the above incubation buffer was poured over both gels to hold them together. Electrophoresis in the second dimension was for 16 hr at 50 V. Molecular weight standards (Sigma Chemical Co.) included a-lactalbumin (14 kDa), soybean trypsin inhibitor (21 kDa), trypsinogen (24 kDa), carbonic anhydrase (29 kDa), G-3PD (36 kDa), egg albumin (45 kDa) and bovine serum albumin (66 kDa).
Immunoblott ing procedures were carried out following equilibration of electrophoretic gels in 500 ml of transfer buffer (25 mM Tris, 192 mM glycine, 20 % methanol) for 30 min. The proteins were transferred to nitrocellulose sheets using a Bio-Rad transblottmg cell at 30 V and 0-08 A for 3 hr. After transfer, the nitrocellulose membrane was washed with TBS (20 mM Tris buffer, pH 7-5, containing 0-5 M NaC1) for 10 min. The transblot was then treated with a blocking solution (3 % gelatin made in TBS) for 1 hr and then with the primary antibody solution made in 1% gelatin. Lens crystallin antibodies formed in rabbits against calf a-, fl- and 7-crystallins were gifts from Dr J. S. Zigler, Jr, Laboratory of Vision Research, National Eye Institute, G-3PD antibody formed in sheep against rabbit muscle G-3PD was a gift from Dr Donita Garland, Laboratory of Vision Research, National Eye Institute. The G-3PD antibody was activated by precipitation with 40% ammonium sulfate and subsequent dissolving of the precipitate in 1% gelatin made in TBS. Main intrinsic membrane protein (MIP2~ kin) antibody was a gift fro m Dr Larry Takemoto, Kansas State University. After incubation with the primary antibody for 1 hr, the membrane was washed With TBS for 20 rain to remove free antibody and transferred to the secondary antibody solution (goat anti-rabbit IgG-horseradish peroxidase conjugate for crystallin antibodies or rabbit anti-sheep IgG-horseradish peroxidase conjugate for G-3PD). The antisera dilutions employed were 1 : 200 for each antibody except G-3PD which was 1 : 100. The transblot was washed again with TBS and immersed into horseradish peroxidase color development solution containing 0"015 % H202. The immunoblot was washed with deionized water and photographed.
Protein concentrations were determined with Pierce BCA Protein Assay Reagent using bovine serum albumin as a standard. The concentration of GSH and total protein sulfhydryl was determined with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) (Sedlak and Lindsay, 1968). For this assay, each lens cortex or nucleus sample was homogenized in 750 #l of ice- cold 0-05 ~ EDTA followed by addition of an equal volume of 20 % trichloroacetic acid and centrifugation. For determination of levels of protein disulfide, a water-insoluble pellet was obtained from lens nucleus and treated in a manner similar to that described by Garner and Spector (1980). The pellet was homogenized under N 2 in 7 M guanidine HC1--0'2 M Tris HCI, pH 8"6, containing 2 mM EDTA and 50 m i iodoacetamide. The homogenate was incubated
890 V. PADGAONKAR ET AL.
at 37~ for 2 hr and then treated with citraconic anhydride at a concentration of 100 mg m1-1 at room temperature in order to solubilize the water-insoluble nonmembrane fraction. The pH of the solution was kept between 8"5 9-5 during the reaction by addition of 2 N NaOH. The solution was dialyzed at 4~ against 0"2% NH40H with repeated changes and centrifuged at 49 000 g for 1 hr. The pellet containing the membrane protein was washed two times with 5 m~ phosphate buffer, pH 8-0, containing PMSF, once with 0-1 ~ NaOH and then again with 5 mM phosphate buffer. The pellet was finally dissolved at 37~ for 16 hr in 50 mM Tris-HC1 buffer, pH 7-9, containing 1% SDS. Aliquots of the dissolved membrane sample were incubated with sodium borohydride under a stream of ~ at 37~ for 30 min as described previously (Brown, 1960). The reaction was terminated by the addition of acetone. The total level of protein - - S H groups formed as a result of reduction with borohydride was determined by analysis with DTNB. The level of protein disulfide was determined as the difference between the level o f - - S H groups obtained before and after borohydride treatment.
For assay of G-3PD activity, homogenization of lens cortex or nucleus was carried out in 2 ml of 0'1 M triethanolamine buffer, pH 7"6, containing 2 m i EDTA. This was followed by centrifugation of the homogenate for t5 rain at 10000 g. Activity of the enzyme in ,he soluble protein fraction was determined by measuring the change in absorbance at 340 nm of NADH as described by Bergmeyer, Gawehn and Grassl (1974).
3. R e s u l t s
To invest igate possible cross-linking of proteins in lenses exposed to hyperbar ic 02, lenses were t rea ted with 100 atmospheres of 100% 0 2 for 24 hr. Under these condit ions levels of GSH in the lenses were significantly decreased in both the cortical and nuclear regions (97 % in the cortex and 94% in the nucleus compared to N~- t rea ted controls, Table I). GSH concentra t ions in control lenses did no t differ significantly from those for fresh lenses. The O~-treated lenses were hazy in appearance bu t no t opaque while control lenses, exposed similarly to 100% N~, remained clear. Two-dimensional diagonal electrophoresis revealed the presence of disulfide-linked proteins in the urea-insoluble fractions of both the cortex and nucleus of O~-treated lenses [Figs I(B) and (D)]. The evidence for cross-linking is the presence
TABLE I
Effect of hyperbaric N 2 and 02 on the level of GSH in the cortex and nucleus of cultured rabbit lenses*
GSH concentration (mM)
Conditions Cortex Nucleus
100 atmospheres, 24 hr (8) N 2 23"6+2.9 4'7 • O 3 0"8___0.2 0"3•
* Results are expressed as means• The number of experiments is in parentheses. Each of the differences between N 2 and O~-treated samples was significant at a level of P < 0-001.
PROTEIN DISULFIDE IN O2-TREATED LENSES 891
FIG. 1. Coomassie Blue-stained two-dimensional diagonal SDS-PAGE patterns of urea-insoluble protein fractions of lenses exposed to 100 atmospheres of 10O% O~ or 100% N~ for 24 hr. Samples of 120 #g protein were run on tube gels in the first dimension without prior treatment with mercaptoethanol. The tube gels were then incubated for 30 rain in buffer containing mercaptoethalml and applied on the second dimension. The patterns are representative results from at least five experiments. (A) cortex of N~-treated lens. (B) cortex of O~-treated lens. (C) nucleus of N2-treated lens. (D) nucleus of O2-treated lens. Note the presence of off-diagonal spots indicating disulfide-linked proteins.
of off-diagonal spots in the electrophoretic profiles for the experimental lenses. The spots correspond to polypeptides which remained disulfide-linked in the first dimension of electrophoresis, but which became reduced in the second dimension after t r ea tment with mercaptoethanol . All spots located on a vertical line below the diagonal represent proteins which originally had been cross-linked with each other. The major polypeptides involved in the formation of disulfide cross-links in the nuclear region of the O~-treated lens have apparent molecular weights of 60, 53, 50, 35, 32, 30, 28 and 25 kDa [Fig. I(D)]. Al though the nucleus of the N2-treated lens contained a small amount of disulfide-linked protein [Fig. 1(C)], it was significantly less than tha t present in the same fraction of the 02-treated lens and was similar to the amoun t present in a fresh lens nucleus (results not shown). I t can be observed in Figs I(B) and (D) tha t a number of off-diagonal spots are frequently present tha t correspond to a specific molecular weight. This m a y indicate tha t this part icular protein was present in aggregates of a number of different sizes. I t can be est imated quali tat ively from Figs I(B) and (D) tha t approximate ly 20% of the total urea- insoluble protein had become disulfide-linked as a result of the O2- t rea tment .
In order to identify the proteins involved in the disulfide cross-linking,
892 V. PADGAONKAR ET AL.
FIG. 2. hnmunoblots of the two dimensional gel shown in Fig. I(D) for urea-insoluble proteins of the nucleus of lenses exposed to 100 atmospheres Oe for'24 hr. Antibodies to each of the lens crystallins and to G-3PD were employed. (A) a-crystallin. (B) fl-crystallin. (C) ~/-crystallin. (D) G-3PD.
immunoblotting techniques were carried out using antibodies to a, fl- and 7- crystallins as well as to MIP26kD ~ and to the enzyme G-3PD. Results of the immunoblotting study for the nucleus of the 02-treated lens are shown in Fig. 2 and may be compared to the Coomassie Blue-stained gel of Fig. 1 (D). The majority of the cross-linked proteins in the nuclear urea-insoluble fraction corresponded to fl- and 7- crystallins [Figs 2(B) and (C)]. Although a-crystallin antibody was observed to react with protein present on the diagonal of Fig. 2(A) there was very little off-diagonal reaction. Use of higher concentrations ofa-crystallin antibody did not produced more intense immunostain, fl-crystallin antibody was seen to react with high molecular weight polypeptides of 50, 53 and 60 kDa [Figs 2(B) and I(D)] even though subunits of fl-crystallin of this size are not present in the normal rabbit lens. I t was of interest that one of the disulfide-linked proteins, with an apparent molecular weight of 35 kDa, was unique in that it did not immunostain with any of the crystallin antibodies but did react with G-3PD antibody [Fig. 2(D)]. MIP26 kD~ antibody did not cross-react with any of the off-diagonal spots (results not shown), indicating that MIP26 kD~ did not participate in the disulfide cross-linking. Studies carried out with the cortical membrane fraction also indicated that the majority of the cross-linked proteins were fl- and 7-crystallins (results not shown).
I t was also of interest to determine the region of the lens in which oxidation of urea- insoluble protein was initiated following exposure to hyperbaric 02. For these
PROTEIN D I S U L F I D E 1N 02-TREATED LENSES 893
@
FIG. 3. Coomassie Blue-stained two-dimensionM diagonal SDS-PAGE patterns of urea-insoluble protein fractions of lenses exposed to 8 atmospheres of 100 % 02 for either 8 or 16 hr. For experimental details see Fig. 1. The patterns are representative results from at least five experiments. (A) cortex of O~-treated lens (8 hr). (B) nucleus of O2-treated lens (8 hr). (C) cortex of Oe-treated lens (16 hr). (D) nucleus of O2-treated lens (16 hr). The controls for these experiments (lenses treated with N 2 under identical conditions) exhibited patterns identical to those shown in Figs I(A) and (C) for cortex and nucleus, respectively.
exper iments , lenses were chal lenged wi th 8 a tmospheres 0 2 for 8 hr which p roduced a 62 % decrease of G S H in the lens nucleus compared to a 13 % loss of the t r i pep t ide in the cor tex (Table I), Two-dimens iona l d iagona l e lec t rophores is of the urea- insoluble pro te ins of bo th regions revea led the presence of disulf ide- l inked pro te ins in the nucleus b u t not in the cor tex [Figs 3(B) and 3(A), respect ively] . The a m o u n t of p ro te in disulfide observed for the O2-treated nuc lear sample was s ignif icant ly higher than the control [Fig. I(C)]. The po lypep t ides involved in the fo rmat ion of the disulfide cross-l inks have a p p a r e n t molecular weights of 35, 32, 28 and 25 kDa. Increas ing the cul ture t ime to 16 hr a t 8 a tmospheres 02 p roduced an increase of p ro te in disulfide in the nucleus of the lenses [Fig. 3(D)] and the appea rance of a small a m o u n t of cross- l inked p ro te in in the cor tex [Fig. 3(C)]. Unde r these condi t ions the level of GSH in the cor tex was decreased by 2 8 % compared to 8 5 % in the lens nucleus (Table I).
P ro t e in concent ra t ions presen t in the lens nucleus and cor tex following t r e a t m e n t wi th 02 and N 2 are shown in Table I I . Exposure of lenses to 100 a tmospheres 02 for 24 hr p roduced losses of water -so luble pro te in bo th in the cor tex (24 %) and in the nucleus (39%) compared to N~-t reated controls . This t r e a t m e n t also resul ted in
894 V. P A D G A O N K A R E T AL.
TABLE II
Effect of hyperbaric N 2 and 0 2 on the concentrations of water-soluble protein (WSP) and urea-insoluble protein (UIP) in the cortex and nucleus of cultured rabbit lenses*
* Resul ts are expressed as mean+_s.D, for eight experiments . The uni t s are mg protein per g wet weight of tissue.
increases in the concentrations of urea-insoluble protein present in the two regions (33 % in the cortex and 30% in the nucleus). In contrast, exposure of lenses to 8 atmospheres O 2 for 16 hr had little effect on the distribution of proteins in the two regions. The level of soluble protein in the cortex was unaffected by 8 atmospheres 02 (P > 0"5) while soluble protein concentration in the nucleus decreased by 10% (P < 0"05). Levels of urea-insoluble protein were not significantly affected in either the cortex or nucleus after 8 atmospheres 02-treatment for 16 hr (P > 0"5).
Sodium-borohydride reduction was employed to quanti tate levels of protein disulfide present in the urea-insoluble protein fraction of the nucleus of lenses treated with 100 atmospheres 02 for 24 hr. I t was observed that, under these conditions, there was nearly a fourfold increase in the level of protein disulfide in the nucleus of 02- treated lenses compared to fresh lenses or Ne-treated controls (Table I I I ) . In the O~- treated lenses, 17 % of the urea-insoluble protein - - S H groups in the nucleus existed
TABLE III
Effect of hyperbaric N 2 and 02 on the level of protein disulfide (PSSP) in the urea- insoluble protein fraction of the nucleus of cultured rabbit lenses*
protein - S H (PSH) PSSP as PSH PSSP (% fresh Conditions (#mol mg -1 protein) {/*mol mg -1 protein) lens nucleus PSH)
* Resul ts are expressed as means + S.D. The n u m b e r of exper iments is in parentheses. The difference between the N 2 and O~-treated samples was significant at a leve] of P < 0"01.
~.D. : Not determined.
PROTEIN D I S U L F I D E IN O~-TREATED LENSES 895
TABLE IV
Effect of hyperbaric N2 and O~ on the activity of glyceraldehyde-3-phosphate dehydrogenase in the soluble fraction of cortex and nucleus of cultured rabbit lenses*
as pro te in disulfide compared to 4"5 % in the controls. I t can be ca lcu la ted from the d a t a of Table I I I t h a t the level of p ro te in disulfide in the O2-treated lens was 1"5 disulfide groups per 105 Da molecular weight* compared to a value of 0"4 in the controls.
T r e a t m e n t of lenses wi th 100 a tmospheres 02 for 24 hr p roduced a nea r ly comple te loss of G-3PD ac t iv i t y in bo th the lens cor tex and nucleus (Table IV). Exposure of lenses to 8 a tmospheres O~ for 16 hr showed less effect on the a c t i v i t y of the enzyme wi th a 15 % decrease in the cor tex and a near ly 40 % decrease in the nucleus compared to N~-t reated controls. T r e a t m e n t of lenses wi th N 2 a t 8 a tmospheres had no effect on the ac t i v i t y of G-3PD compared to t h a t p resen t in fresh lenses. However , there appea red to be a s l ight increase in enzyme ac t i v i t y in lenses t r e a t e d wi th 100 a tmospheres N 2 for reasons which are no t clear.
4. D i s c u s s i o n
This s t u d y has shown t h a t in v i t ro t r e a t m e n t of r a b b i t lenses wi th hype rba r i c 02 leads to fo rma t ion of disulfide cross- l inks in the urea- insoluble p ro te in f ract ion. The presence of p ro te in disulfide has also been repor ted in the m e m b r a n e f ract ion of h u m a n and X - r a y induced ca t a rac t s (Spector e t al., 1979; T a k e m o t o and Hansen , 1982; Garad i e t al., 1987, K o d a m a and Takemoto , 1988): However , in con t ras t to resul ts for the h u m a n ca ta rac t , (Spector et al., 1979; T a k e m o t o and Hansen, 1982), the presen t s t u d y for r a b b i t lens did no t indica te invo lvemen t of a disulf ide- l inked 18 or 43kDa component . W i t h regards to pub l i shed s tudies of X - r a y induced c a t a r a c t (Garadi e t al., 1987) which were also carr ied out in r abb i t , a compar ison of the molecular weights of po lypep t ides involved in the disulfide cross- l inking indica tes d i s t inc t s imilar i t ies in the resul ts of chal lenges wi th e i ther 02 or X- ray . These include p r e d o m i n a n t invo lvemen t of the /?-crystalline, pa r t i c i pa t i on of a non-c rys ta l l in 35 k D a pro te in (identified as G-3PD in this s tudy) and lack of disulfide cross- l inking of MIP~6 kDa"
* Results are expressed as means +__ S.D. The number of experiments is in parentheses. Each unit of activity corresponds to the conversion of 1/~mol substrate per min. Calculated as follows : 0"030/~mol PSSP (as PSH) per mg protein is equivalent to 0"015/zmol PSSP per mg protein. By multiplying numerator and denominator by 10 s, this can be converted to 1"5 tool PSSP per 105 g protein.
896 V. PADGAONKAR ET AL.
In order to characterize the covalent interactions occurring between the 02-treated lens proteins, two-dimensional diagonal electrophoresis was used in combination with probing of Western blots with polyclonal antisera to a-, /?- and ~,-crystallins. The results of the analyses demonstrated that mainly fl- and y-crystallins and not a- crystallin are associated with the intermolecular disulfide bonding in O2-treated lenses. These findings are similar to those of Kodama and Takemoto (1988) for investigation of human cataracts, although cross-linking of ~,-crystallin was found to predominate in that study. In a separate investigation of human cataracts (Garner et al., 1981) it was also reported that gamma crystallin was involved in a disulfide-linked membrane-cytosol polypeptide complex. Previous studies (Driessen, Herbrink, Bloemendal and DeJong, 1980) have demonstrated that sequence homologies exist between the /?- and 7-crystallins, so it is possible that certain structural features common to the two classes of crystallin may take part in the intermolecular disulfide bonding. Both/?- and ~-crystallins contain significant levels of cysteine (Bloemendal and Herbrink, 1974; Blundell et al., 1981) and it is well-established that the - - S H groups of fl-crystallin are particularly susceptible to oxidation (Dische and Zil, 1951).
The reaction of/?-crystallin antibody with high molecular weight polypeptides of 50, 53 and 60 kDa [Figs 2(B) and I(D)] was unexpected since/?-crystallin subunits of this size are not present in the normal rabbit lens. There are at least two possible explanations for this result. One possibility is that the proteins are non-disulfide- linked aggregates of either two/?-crystallin subunits or a/~-crystallin subunit plus a non-crystallin protein. The fact that the proteins are located off of the diagonal indicates, however, that they were, at one time, also cross-linked to other polypeptides by disulfide bonds. A second possibility is that the proteins are non-crystallin, urea- insoluble components which were able to immunoreact non-specifically with the polyclonal/?-crystallin antibody.
I t is not clear from this study whether the disulfide cross-linking of G-3PD observed in the O~-treated lenses contributed significantly to the loss of activity of the enzyme which was also noted. I t is known that G-3PD requires an unblocked - - S H group for activity (Jedziniak, Meys and Arredondo, 1985) and that the enzyme is susceptible to oxidative insult (Spector et al., 1987). GSH may also have a role in maintaining activity of the enzyme (Udvardy, Balogh and Farkas, 1982). While activity of G-3PD has been reported to decline in the aging lens (Ohrloff, Berdjis, Hockwin and Bours, 1983; Dovrat, Schart and Gershon, 1984) it is not known whether the decrease is related to oxidative effects. The possibility that lens G-3PD is' loosely associated with the fiber membrane (Lenstra, Van Raaij and Bloemendal, 1982) may make the enzyme more susceptible to disulfide cross-linking to membrane proteins. However, linkage of the enzyme to crystallins or to GSH may also contribute to its loss of activity.
Formation of urea-insoluble protein disulfide was found to occur first in the nucleus of the 02-treated lens. This was demonstrated by experiments carried out at 8 atmospheres O 3 in which protein disulfide was significantly increased in the nucleus of the O~-treated lens but did not appear in the cortex. I t was shown previously in our laboratory (Giblin et al., 1988) that a fall in GSH level with corresponding accumulation of GSSG also occurs initially in the nuclear region of the 02-treated lens. The nucleus is known to possess a relatively low activity of the glutathione redox cycle including low activities of glutathione reductase, glucose-6-phosphate de- hydrogenase and the hexose monophosphate shunt (Giblin, Nies and Reddy, 1981; Giblin and Reddy, 1980). These results suggest a relationship between a high level of
PROTEIN DISULFIDE IN O~-TREATED LENSES 897
GSH and protection against formation of membrane protein disulfide. A similar correlation was found in studies of the X-irradiated lens (Garadi et a]., 1987).
The present study demonstrates that the formation of a relatively low number of disulfide cross-links is able to produce a substantial proportion of aggregated urea- insoluble protein. An increase of only one disulfide group per 105 Da molecular weight produced cross-linking of approximately 20 % of the total urea-insoluble protein. This indicates tha t extensive oxidation of lens protein - - S H groups may not be required to produce significant aggregation of protein. However, since dense opacification was not observed in any of the O~-treated lenses, it is possible tha t the degree of oxidation was insufficient to produce a loss of transparency. Garner and Spector (! 980) reported tha t 57 % of cysteine was present as disulfide in the intrinsic membrane fraction of normal aged human lenses and tha t this percentage increased in cataraetous lenses. In the present study we report a value of 17% for the oxidation of the total membrane - - S H groups of the O2-treated lenses, a level which may be insufficient to produce opacification. I t is possible that for more extensive oxidation of membrane protein - - S H groups to occur in the O2-treated lens, longer time periods may be required (relative to the 24 hr of culture time employed in the present study) in order to permit appropriate conformational changes of proteins and resultant exposure Of a larger percentage o f - - S H groups to the oxidant.
I t was not possible in this s tudy to determine the percentage of the urea-insoluble material which was comprised of either membrane~crystallin or crystallin-crystallin, disulfide-linked complexes. I t is most likely tha t the urea-insoluble fraction consisted of a mixture of the two types of complexes since some covalently linked crystallins present in the water-insoluble material may have been able to resist the extensive washing which was carried out with both urea and NaOH. Indeed, for experiments conducted at 100 atmospheres 0 2, there was evidence for a significant loss of water- soluble protein and increase in urea-insoluble protein in the lenses. However, experiments carried out at 8 atmospheres 02 indicated only slight changes in the content of water-soluble and urea-insoluble protein and under these conditions a higher percentage of the observed cross-linking may have taken place between crystallin and membrane components.
In conclusion, the study has shown that 02 is potentially reactive in the lens and is capable of producing protein-disulfide in the urea-insoluble protein fraction. The lens nucleus appears to be more susceptible to formation of protein disulfide than the cortex. O2-induced disulfide formation may be related to the nuclear opacification which occurs in lenses of human patients who have been treated with hyperbaric 02 (Palmquist et al., 1984).
A C K N O W L E D G M E N T S
We thank Drs J. S. Zigler, Jr. and Donita Garland, Laboratory of Vision Research, National Eye Institute, for gifts of lens crystallin antibodies and G-3PD antibody, respectively. The gift of MIP2~ko a antibody from Dr Larry Takemoto, Kansas State University, is also appreciated. We thank Lisa Schrimscher, Victor Leverenz and B. Chakrapani for technical assistance and Stan Susan for assistance with photography of gels. The work was supported by a National Research Service Award, EY 05997, from the National Eye Institute, NIH (to V.P.) and grants from the National Eye Institute, EY 02027, EY 00484 and EY 05230 (Core Grant for Vision Research). This study was part of the Cooperative Cataract Research Group consortium grant.
898 V. PADGAONKAR ET AL.
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