HoOo-ModificQtion of,k-atpase Alterations in External and K Stimulation of K Influx Margaret H. Garner, William H. Garner, and Abraham Specror Studies, at steady state, of the,k-atpase dependent influx of K + into es in organ culture are used to characterize further the H 2 0 2 -modification of the + pump. Control lenses display constants for interaction with external + and K + similar to those obtained for the erythrocyte. H 2 O 2 treatment of the leads to total loss of external + stimulation and alteration of external K + stimulation. Invest Ophthalmol Vis Sci 27:103-107, 1986 In human senile cataract, intracellular concentrations of + and K + are altered (1), the (,K)-ATPase is increasingly inhibited (2), the sulfur containing amino acid side chains of the lens proteins are oxidized (3), and in a certain population of cataract patients, abnormally high concentrations of H 2 O 2 are found in the aqueous humor and the lens (4). H 2 O 2 inhibits the influx of 86 Rb + (K + ) into cultured lenses. 5 " 8 In the cultured, H 2 O 2 inhibits the influx of 86 Rb + by direct modification of the (,K)- ATPase. 8 The H 2 O 2 -modified enzyme would appear to have two binding sites for eosin maleimide 8 and two p-nitrophenylphosphate hydrolysis sites as indicated by a twofold increase in the maximal velocity (V max). 9 ATP hydrolysis of the H 2 O 2 -modified enzyme is positively cooperative, unlike the functionally unmodified enzyme for which the* hydrolysis of ATP is negatively cooperative. 9 While K + stimulation and inhibition of ATP hydrolysis is normal in the H 2 O 2 -modified enzyme, + stimulation of ATP hydrolysis is altered (K 50 + = 20 mm for the unmodified enzyme and 34 mm for the H 2 O 2 -modified enzyme). 9 In order to relate the observed changes in substrate hydrolysis kinetics and allosteric effector control of substrate hydrolysis to cation flux, the effect of extracellular [ + ] and [K + ] upon K + influx has been studied in H 2 O 2 -treated and untreated, cultured bovine lenses. The results of this study confirm the earlier observations 5 " 8 that H 2 O 2 modification inhibits K + in- From the Biochemistry and Molecular Biology Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, New York. This work was supported by the tional Eye Institute, tional Institutes of Health and the use of the Prophet Computer facility supported by a Cooperative Cataract Research Group Grant. Submitted for publication: January 17, 1985. Reprint requests: Abraham Spector, PhD, Department of Ophthalmology, Research Division, College of Physicians & Surgeons of Columbia University, 630 West 168th Street, New York, NY 10032. flux. The results demonstrate that, for the untreated lenses, K + influx is dependent upon both extracellular K + and +. After H 2 O 2 treatment, as demonstrated in this paper, external + no longer affects (,K)- ATPase dependent K + influx. Materials and Methods TCI99 and a penicillin, fungizone, streptomycin solution were obtained from DIFCO (Detroit, MI). Glucose oxidase, catalase, ouabain, and choline chloride were obtained from Sigma Chemical; St. Louis, MO 63178. The 86 RbC 1 (1.86 mci mg" 1 ) was obtained from New England Nuclear (Boston, MA). All other chemicals were reagent grade. Experimental Procedure Paired bovine eyes were processed within 4 hr of death. Lenses (1.4-1.7 g) were removed by posterior extraction. Preincubation of the lenses in bicarbonate modified TCI99 medium and incubation of contralateral lenses in the presence and absence of H 2 O 2 were performed as described previously. 8 ' 9 The 3-hr post-incubations, after the H 2 O 2 had been destroyed, were performed in modified Tyrode's media 10 containing appropriate concentrations of + and K +. Choline chloride was used to replace either + or K + in media which were prepared deficient in either one of the two cations." The total osmolarity of these media was kept at 290 ± 5 mosm as measured using an Osmette A osmometer (Precision Systems, Inc.; Sudbury, MA). The [ + ] and [K + ] in the media were measured with ion specific electrodes using an Ion 85 meter (Radiometer America; West Lake, OH). Incubation in an atmosphere of 5% CO 2 was not necessary during the interval in which the lenses were in the Tyrode's media. During the 3-hr, post-incubation period, 86 Rb influx was measured. Five /id of 86 Rb were added to 10 ml 103
104 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1986 Vol. 27 3.0 r k i i i i i i i i i i i i i i + ] mm Both + and K + interact with (,K)-ATPase cooperatively. 911 Therefore, the relative velocity V r (calculated by dividing the observed (,K)-ATPase dependent rate of K + influx by the highest observed rate of pump dependent K + influx for the control lenses) was related to either + or K + as an effector using equation 1: V r = V; + (V r max - V0/(l + Ki/[Mj] ni ) (1) This general expression relates the relative velocity (V r ) to a modified Hill function. 12 The number of sites for the extracellular cation in question ([Mj] = [ + ] or [K + ]) and the monovalent cation concentration for half maximal influx are indicated by nj and K ;, respectively. V; is included to account for residual velocity as the concentration of Mj approaches 0, while the other external monovalent cation is saturating its sites. Presumably, for all these experiments, the internal sites for both monovalent cations are saturated. When no influx is observed in the absence of the metal (Mj) ion in question, equation (1) reduces to the familiar Hill equation: V r = V r max/(l +Ki/[Mj] ni ) (2) ~ 2.0 Q. - i.o L 0 20 40 60 80 [ + ] mm 100 120 140 Fig. 1. Plots to evaluate the effect of external K + (top) at + = 135 mm and external + (bottom) at K + = 3.1 mm upon the non-ouabain sensitive influx of K + into es using 86 Rb + as a tracer. Units for the influx are jimol mr'hr" 1. Units for K + and + are mm. The dashed curves represent the 95% confidence limits for the regression. of culture medium in all cases. Counts in the medium and lenses were determined using the Cerenkov radiation technique 8 on a 1217 Rack Beta liquid scintillation counter, LKB Instruments, Inc.; Gaithersburg, MD. In experiments in which ouabain was used to inhibit (,K)-ATPase dependent K + influx, the ouabain was added to the culture medium at the same time as the radioactive label. Effective K + influx was calculated by multiplying the lens:medium (L/M) ratio 8 by the external [K + ], expressed as ^mol ml" 1. Treatment of the Results Results Experiments were first performed to compare the transport of K + in the Tyrode's medium to transport in the bicarbonate modified TCI99 medium used in the previous study. 8 Lenses (10 pairs) were pre-incubated in the bicarbonate modified TCI99 medium, then one lens of each pair was placed in a Tyrode's medium that contained the same amount of + and K + as the TCI99 medium. After 3 hr, 86 Rb influx was the same in the two media (L/M ratio (Tyrode's) = 0.31 ± 0.05, L/M ratio (TC199) = 0.32 ± 0.4). In order to examine the (,K)-ATPase dependent (or active component) influx of K + into the cultured es, the (,K)-ATPase independent (or passive component) influx of K + had to be determined at varying K + and + concentrations. Using 86 Rb as a tracer, this was accomplished by performing influx experiments in the presence of 2 mm ouabain, a specific inhibitor of (,K)-ATPase. The results, Figure 1, were fit using linear regression analysis. For K + effects on K + influx (Figure 1, top), the slope of the line was 0.104 ± 0.006, with an intercept of 0.0 and a linear correlation coefficient (R 2 ) of 0.97. The results for + (Fig. 1, bottom) indicate that there is no significant correlation between external [ + ] and ouabain insensitive K + influx (multiple R 2 = 0.34 P >.15). For all succeeding experiments, the (,K)-ATPase dependent influx (or net active flux) is reported as the difference between the total measured influx and the ouabain insensitive influx as determined from Figure 1. Since the analysis of + and K + dependent substrate hydrolysis suggested a change in the interaction
No. 1 H 2 O 2 MODIFIED LENS NA,K-ATPose / Gorner er ol. 105 Vr Vr 0 20 40 60 80 I00 I20 I40 [No + ] mm Fig. 2. Plots to evaluate the effect of external + concentrations (mm) upon the ouabain sensitive influx of K + using 86 Rb + as a tracer for the control ( ) and H 2 O 2 treated (O) es. External [K + ] were 3.1 mm. The ouabain sensitive influx was calculated by subtracting the non-ouabain sensitive K + influx at 3.1 mm K + (Fig. 1) from the K + influx in the absence of ouabain. The relative velocity plotted on the y-axis was calculated as the observed ouabain sensitive influx divided by the maximal observed sensitive influx. of + with the (,K)-ATPase modified with H 2 O 2, 9 the effect of H 2 O 2 upon + stimulation of K + influx was determined between 0 mm and 135 mm. The results are graphed in Figure 2. The curve through the experimental points for the untreated lenses was determined using equation 1. The value for V is K 50, and n for the + stimulation of K + influx were 0.46 ± 0.02, 63.9 ± 5.0, and 3.9 ± 1.2, respectively. The multiple R 2 was 0.98. The results for the H 2 O 2 -treated lenses, also plotted in Figure 2, indicate that + no longer stimulates K + influx. There is no significant correlation between K + influx and [ + ] (multiple R 2 = 0.28, P > 0.2), since K + influx remains constant at 0.06 ± 0.01 mm hr" 1 (P > 0.001). Analysis of the ion-dependent ATP substrate hydrolysis 9 indicated no apparent change in the interaction of the H 2 O 2 -modified enzyme with K +. To test if this observation was true also for K + influx, the dependence of K + influx upon extracellular K + was determined in modified Tyrode's medium containing 135 mm +. The [K + ] was varied from 0 to 26.1 mm. For the control lenses, external + appeared to be necessary for maximal K + influx, Figure 2. Therefore, an experiment was also designed to study the effect of external K + upon K + influx in the absence of +. (It should be noted that a similar experiment, of course, could not be performed for H 2 O 2 -treated lenses since the data, [K + ]mm Fig. 3. Plots to evaluate the effect of external K + concentrations (mm) upon the ouabain sensitive influx of K + using 86 Rb + as a tracer for the control (O) and H 2 O 2 -treated ( ) es ([ + ] = 135 mm) and for control (A) at [ + ] = 0 mm. Ouabain sensitive K + influx and the relative velocity (V r ) were calculated as described in Figure 2. shown in Figure 2, indicated that [ + ] has no stimulating effect upon the H 2 O 2 -treated lenses). The results for K + stimulation are plotted in Figure 3. Curves through the experimental points were determined by fitting the data to equation 2. The shape of the curves for the control ( ) and H 2 O 2 (O) ( + = 135 mm) indicates that K + is a positively cooperative effector of K + influx. The control lenses in the absence of + (A) display simple Michaelis-Menten behavior. The values for V r max, K 50, and n (equation 2) for K + stimulation are collected in Table 1. The apparent K 50, in the more physiological high + containing medium for the H 2 O 2 -treated lenses is approximately three times greater than the K 50 for the control lenses. The V r max is only 50% of the value for the control lenses. In the + free medium, the V r max for the control lenses is Table 1. Fitted values for K + stimulation of K + influx* V r max v + n R 2 Control lens [ + ] = 0 0.57 ± 0.06 0.73 ±0.18 1.14 ± 0.18 0.998 * See text for description of terms. t Units for K 50 are mm. Control lens [ + ] = 135 mm 0.89 ± 0.04 1.54 ±0.15 2.76 ± 0.59 0.993 H 2 O 2 lens [ + ] = 135 mm 0.47 ±0.01 4.28 ±0.12 2.97 ± 0.23 0.998
106 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1986 Vol. 27 ATP [A) ADP-P T K*Of No* No K ( ) L ( ADP H <J ( Fig. 4. Dimer relaxation model (Swann and Albers") showing three sites for monovalent cations. approximately 50% of that for the control lenses in the high + -containing media. Discussion Since + and K + control of,k-atpase is complex, the monovalent cation sites of the unmodified enzyme must be described if H 2 O 2 's dramatic alteration of pump dependent K + influx is to be fully understood. This goal is best accomplished using the dimer relaxation mechanism of Swann and Albers, Figure 4.'' The mechanism includes a single binding site for ATP between two functional subunits, two different conformations (R and T), and three sets of monovalent cation binding sites (A, B, and C). At each set of binding sites, R wp Table 2. Constants for + and K + control of (,K)-ATPase A sites bovine braint B sites bovine brainf C sites bovine brainf 166-300 166 307 3 20 mm 5.4(15.9)* 5.0 3 (1.5)* 0.1-0.8f 0.14f 0.73* 1.5-4.5 4.5 1.7f 2.0 2.0 2.0f (2.8)* 0.8-1.1 1.0* 1.1* 7-30 26.3 (5.8)* 20.0f 3-10 mm 8.5(11.8)* N.D. 5 58-112* 58* 64* K 2.9-5.0 5.0 2.9t 1.6-2.0 1.6 N.D.H 1.0-3.9* 1.6* 3/9* Units for K 50 are millimolar; the of values were obtained from this report and references. 9 '" 1 ' 3 * Values in the presence of ATP. t Values obtained by Swann and Albers." % Values obtained by Garner et al. 9 Values reported here (see results and Table 1). H N.D. Not determined here or in reference. 9 either + or K + can bind. Each set of monovalent cation binding sites is easily distinguished from the others by its affinity for K +." 13 As demonstrated in Table 2, the A, B, and C sites have low (K 50 > 100 mm), moderate (K 50 > 1 mm), and high (K 50 < 1 mm) affinity, respectively, for K +. The B and C sites are on the extracellular membrane surface; the A sites are on the intracellular surface. 1415 The A and B sites are present on the free enzyme. K + binding to the A and B sites is demonstrable in the phosphatase reaction in the absence of ATP and +. 91113 The C sites are demonstrable only after a conformational change which occurs either by formation of the phosphoenzyme or binding of K + or + plus K + to the A and B sites (Fig. 4)." The C sites are demonstrable in the phosphatase reaction only when micromolar quantities of ATP and millimolar quantities of + are present. 1113 In the transport experiments with the control bovine lenses, sites with moderate affinity for K + (K 50 = 1.5 mm), the B sites were observed (Fig. 3). When + was removed from the incubation medium, sites with high affinity for K + (K 50 = 0.73 mm), the C sites were observed. In the absence of extracellular +, binding of K + at the B sites does not occur. Binding of external + to the C sites (K 50 = 64 mm, Fig. 2) appears to be necessary for maximal K + influx. Previously, it was demonstrated that + binding to these sites is necessary for + efflux from red blood cells. 16 K + and + binding to the A sites and + binding to the B sites were not observed in the K + influx experiments. Presumably, these sites will become apparent in future studies to elucidate intracellular monovalent cation control of + and K + transport in es. After H 2 0 2 -modification, binding of + or K + to the C sites no longer stimulates K + influx. External + control is totally lost. There is no difference in K + binding at 3.1 mm K + in the presence or absence of +, indicating a loss of K + binding at the C sites. After H 2 O 2 -modification, external K + binding at the B sites still stimulates K + influx. This value is similar to the value obtained for the bovine brain enzyme and similar to the value obtained for K + stimulation of the phosphotase reaction for (,K)-ATPase isolated from lenses incubated in the presence and absence of H 2 O 2. 9U Although V r max for H 2 O 2 -treated lenses is 47% of the V r max for the control lenses, at physiological concentrations of external K + (2-5 mm), the H 2 O 2 - treated lens (,K)-ATPase pumps at 5 to 25% of the control enzyme. At similar external K + concentrations, the control enzyme is pumping at 50-90% of its maximal velocity. Key words: lens, H2O2,,K-ATPase, transport
No. 1 H 2 O 2 MODIFIED LENS NA,K-ATPose / Garner er ol. 107 Acknowledgments We acknowledge with thanks the technical assistance of Ms. Gloria Jenkins and Ms. Ruey-Ruey Huang. References 1. Patmore L and Duncan G: editors. Mechanism of cataract formation. In Human Lens. New York, Academic Press, 1981, pp. 193-236. 2. Kobayashi S, Roy D, and Spector A: Sodium/potassium ATPase in normal and cataractous human lenses. Curr Eye Res 2:327, 1983. 3. Garner MH and Spector A: Selective oxidation of cysteine and methionine in normal and cataractous lenses. Proc tl Acad Sci USA 77:1274, 1980. 4. Spector A and Garner WH: Hydrogen peroxide and human cataract. Exp Eye Res 33:673, 1981. 5. Giblin FJ, McCready JB, and Reddy VN: The role of glutathione metabolism in the detoxification of H 2 O 2 in rabbit lens. Invest Ophthalmol Vis Sci 22:330, 1982. 6. Giblin FJ, Chakrapani B, and Reddy VN: Glutathione and lens epithelial function. Invest Ophthalmol 15:381, 1976. 7. Fukui HN, Epstein DL, and Kinoshita JH: Ascorbic acid effects on lens rubidium-86 transport. Exp Eye Res 15:249, 1973. 8. Garner WH, Garner MH, and Spector A: H 2 O 2 -induced uncoupling of (,K)-ATPase. Proc tl Acad Sci USA 80:2044, 1983. 9. Garner MH, Garner WH, and Spector A: Kinetic cooperativity change after H 2 O 2 modification of (,K)-ATPase. J Biol Chem 259:7712, 1984. 10. Garrahan PJ and Glynn IM: The sensitivity of the sodium pump to external sodium. J Physiol 192:175, 1967. 11. Swann AC and Albers RW: Sodium and potassium-activated ATPase of mammalian brain regulation of phosphatase activity. Biochim Biophys Acta 382:456, 1975. 12. DeLean A, Manson PJ, and Rodbard P: Simultaneous analysis of families of sigmoidal curves: Application to bioassay, radioligand assay and physiological dose response curves. Am J Physiol 235:E97, 1978. 13. Robinson JD: Functionally distinct classes of K + sites on the ( + K) dependent ATPase. Biochim Biophys Acta 384:250, 1975. 14. Rega AF, Garrahan PJ, and Pauchan MI: Potassium activated phosphatase from human red blood cells, the asymmetrical effects of K +, +, Mg ++ and adenosine triphosphate. J Memb Biol 3: 14, 1970. 15. Glynn IM: Activation of adenosine triphosphate activity in a cell membrane by external potassium and internal sodium. J Physiol 160:18P, 1961. 16. Garay RP and Garrahan PJ: The effect of external + on the different types of + extrusion catalyzed by the -pump. In Human Red Cells in (,K)-ATPase Structure and Kinetics, Skou JC and Norby JG, editors. New York, Academic Press, 1979, pp. 247-259.