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1 Activators and inhibitors of lens aldose reductase /. A. Jedziniak and J. H. Kinoshita Aldose reductase in a highly purified state is unstable. It requires the presence of thiol groups to maintain it in an active form. The enzyme apparently exists in 3 forms, only one of which is active. Tetramethylene glutaric acid (TMG) is an effective aldose reductase inhibitor. However, a relatively high level of TMG is needed to depress dulcitol synthesis in the lens incubated in a galactose containing medium. TPN+, a product of the reaction, also inhibits the enzyme. The action of these inhibitors appears to transform the active into inactive enzyme. Key words: aldose reductase, aldose reductase inhibitor, tetramethylene glutaric acid, sugar cataracts, lens enzyme. R.'ecent evidence strongly suggests that the enzyme aldose reductase plays a primary role in the initiation of the cataractous process in experimental galactosemia and diabetes. 2 Moreover, the studies concerning lens aldose reductase have stimulated interest in the possibility that this enzyme may be involved in diabetic complications affecting such tissues as the kidney and nerve. 3 ' 4 Recently, attempts have been made to seek specific inhibitors that can control the activity of the enzyme so that the cataractous process can be prevented or at least delayed. In the course of these studies it was necessary to purify the enzyme and study its properties. This report describes (1) the de- From the Howe Laboratory of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Mass. Manuscript submitted Mar. 3, 1971; manuscript accepted Mar. 24, Reprint requests: Dr. Jin H. Kinoshita, Howe Laboratory of Ophthalmology, 243 Charles St., Boston, Mass. Q2U4, 357 pendency of the enzyme on sulfhydryl compounds, (2) the inhibition by 3,3'-tetramethylene glutaric acid (TMG), (3) product inhibition by TPN +, and (4) changes in the electrophoretic patterns of the enzyme in the presence of these inhibitors. Materials and methods Calf eyes were obtained from a local abattoir soon after slaughtering and the lenses were removed and frozen until needed. TMG and analogues of TMG were made available through Dr. D. Dvornik, Ayerst Chemical Company, Montreal, Canada. The other materials used were purchased from the following sources: TPN+ and TPNH, Sigma Chemical Company; DL-glyceraldehyde, Nutritional Biochemical Company and Sigma Chemical Company; and beta-mercaptoethanol (/J-mETOH), Eastman Kodak Company. Lens aldose reductase was purified according to the method of Hayman and Kinoshita 7 with the following modifications: (1) the enzyme was eluted from DEAE-cellulose between phosphate concentrations of 0.1M and 0.25M and, in all experiments except where stated to the contrary, enzyme was collected in 5 nim. /J-mETOH and (2) further enzyme purification was achieved by successive passes of aliquots of the pooled DEAE fractions through Sephadex G-75 and then Sephadex G-100 columns equilibrated with 0.01M
2 358 Jedziniak and Kinoshita Investigative Ophthalmology May 1971 Table I. Effect of dialysis on enzyme activity Sample Predialysis Postdialysis Specific activity, LuSOi, 2, ,348 1,064 Per cent activity remaining, LuSO ( H LuSO t stimulation Postdialysis + S 4,493 7, Aliquots of enzyme pooled after DEAE-chromatography were assayed as described in Methods. Dialysis was performed versus 0.01M TO* buffer, ph 6.8, at 5 C, for 24 hours. (8-mETOH (5 mm.) was added to dialyzed enzyme and its effect was measured following at 15 minute incubation period. Concentration of LisSOi used was 0.4M phosphate, ph 6.8, containing 5 mm. /?-metoh. The final purification varied between 2- and 7,000-fold, depending upon the age of enzyme preparation. In determining enzyme activity a sample cuvette containing phosphate buffer, 0.065M, ph 6.8; TPNH, 2.15 x 10-4 M; enzyme solution; DLglyceraldehyde, 1.5 x 10-2 M; and water to a final volume of 1 ml. was read against a reference cuvette containing all components but substrate. The reaction was started by the addition of substrate and was followed in a Gilford recording spectrophotometer at 340 nm. A unit of activity was defined as a change in absorbancy of per 5 minutes per milliliter. Protein was determined by the method of Warburg and Christian 5 and confirmed colorimetrically by the Lowry method. 6 Polyacrylamide disc gel electrophoresis was performed according to the method of Davis s using the Model 12 apparatus and reagents from Canalco (Canal Industrial Corporation). Standard 7.5 per cent polyacrylamide gels were used. The enzyme sample was applied without sample gel and stacking gel as 40 per cent sucrose solution. The reservoir buffer was Tris-glycine, ph 8.5, and 5 mm. /3-mETOH was added to the system where stated in the text. To estimate activity distribution on the gels, gels were sliced at 0.5 cm. intervals and homogenized manually with 0.01M phosphate buffer, ph 6.8, containing 5 mm. /3-mETOH. After cencrifugation, 0.5 ml. aliquots of sample were assayed as described. The eluted active enzyme free from other protein contaminants was then subjected to the various reagents studied and was reapplied to the electrophoresis system. Enzyme activity was eluted from these gels in the same way as in the preliminary run. To visualize protein, complementary gels from both the first and final electrophoresis were fixed with 12 per cent TCA and stained with Coomassie blue overnight. Excess dye was removed by continuous washings with 7 per cent acetic acid. This method of gel rerun yielded a homogenous enzyme preparation without additives and ensured that the changes described were due Table II. Effect of NEM on enzyme activity NEM (M x 10-*M) (-) (S-mETOH (per cent activity) (+) 5 mm. P-mETOH (per cent activity, Enzyme pooled after DEAE-chromatography (1,600-fold pure) was assayed as described in Methods. The above concentrations of NEM were added to appropriate enzyme solution. The activity of the enzyme was determined after 30 minute incubation with NEM. /3-mETOH (5 mm.) was added to the reaction mixture 5 minutes after the reaction had begun. entirely to the presence of the reagents studied. The method of Weber and Osborn 9 was used for determining molecular weights of proteins observed on polyacrylamide gels. The apparent molecular weights of sodium dodecyl sulfate (SDS) -treated enzyme were extrapolated from a plot of the log of molecular weight of a series of standard proteins versus their mobility on similarly prepared gels. Results Sulfhydryl dependency. The purified lens aldose reductase was exceptionally unstable. Attempts to store or dialyze the enzyme led to rapid inactivation. Before any study on the properties of the enzyme could be initiated, it was necessary to find means of stabilizing it. We found that the presence of sulfhydryl compounds protected and stabilized the enzyme. The dependency of purified lens aldose reductase on reduced thiols was demonstrated in the loss of enzyme activity when /?- metoh was removed by dialysis (Table I). Earlier studies 7 had shown that aldose reductase was stimulated by high con-
3 Volume 10 Number 5 Aldose reductase activators and inhibitors 359 S 0.4 Lithium Sulfate Fig. 1. Effect of LisSO* on enzyme activity. Enzyme prepared without thiol was assayed as described in Materials and Methods in the presence of increased concentrations of Li-SCX (ph 6.2). The closed circles represent the response of enzyme preheated with 5 nim. /3-mETOH. Activity, V, is expressed as change in optical density (O.D.) per 5 minutes per milliliter of enzyme solution. centrations of Li 2 SO 4. This effect was found to be due primarily to the sulfate ion. As shown in Table I, dialysis led to a 74 per cent loss in activity. Treating the dialyzed enzyme with /?-metoh restored the activity. After the dialysis the addition of lithium sulfate increased the specific activity from 726 to 1,064 but this degree of stimulation was much- less than that elicited by treatment with /?-met0h. The LLSO 4 stimulation appeared additive to the sulfhydryl effect. The data suggest that the activation by LiaSO* is enhanced when the enzyme is first pretreated with (3- metoh. The sulfhydryl requirement of aldose reductase was further illustrated in the experiments with N-ethylmaleimide (NEM), a compound known to inhibit SH enzymes. The addition of NEM in concentrations at or above 1 mm. markedly inhibited the enzyme (Table II). When the NEM-inhibited enzyme was treated with /?-metoh there was a partial but substantial restoration of enzyme activity. The striking effect of /?-metoh on the response of aldose reductase to Li 2 SO 4 is demonstrated in greater detail in Fig. 1. As shown in the figure, activity increased linearly when lithium sulfate concentration was increased to 0.3M in the presence of /3-mETOH but showed an erratic re-
4 360 Jedziniak and Kinoshita Investigative Ophthalmology May 1971 X Glyceraldehyde X1O -4 Fig. 2. Effect of TMG on Lineweaver-Burke plot of enzyme with glyceraldehyde as substrate. The ordinate represents the reciprocal of initial velocity expressed as Mmoles per 5 minutes per milliliter; the abscissa represents the reciprocal of glyceraldehyde concentration between and 50 x 10-4 M. The open circles express uninhibited enzyme; the hatched circles and closed circles represent enzyme inhibited by 1.6 and 4.06 x 10~ 5 M TMG respectively. sponse to the activator in the absence of thiol. These studies of activators indicate that /?-metoh is essential for enzyme activity but Li-jSOj is not. The lithium sulfate effect is not maximally manifested until the enzyme is first activated by /?-met0h. It becomes obvious that the modes of action of these two activators are considerably different. TMG inhibition. Previous studies on aldose reductase 7 demonstrated that the' enzyme is inhibited by a wide variety of organic anions. It was found that TMG, a dicarboxylic acid, was a most effective inhibitor. Preliminary experiments with aldose reductase purified only to the ammonium sulfate stage 7 indicated that TMG is not tightly bound to the enzyme. This enzyme preparation, when mixed with 10" 3 M TMG, showed virtually no activity. When this enzyme-inhibitor mixture was dialyzed overnight, 60 per cent of the activity was recovered. Thus, it appears that TMG is not irreversibly bound to the enzyme. It should be mentioned that dialysis does not cause any loss of activity of this crude enzyme preparation. Even though the TMG inhibitory effect was easily demonstrable with crude aldose reductase, in comparison, TMG did not have a dramatic effect on the purified enzyme. To elicit inhibition by TMG, the enzyme had to be activated by pretreatment with /?-metoh. Thus the thiol requirement for the inhibitory response is similar to the stimulatory effect of lithium sulfate. Kinetic studies on the inhibitory effect of TMG were made. Fig. 2 illustrates the double reciprocal plots of velocity versus glyceraldehyde concentration at various
5 Volume 10 Number 5 Aldose reductase activators and inhibitors TMG, 10" 6 M Fig. 3. Effect of glyceraldehyde concentration on the response of enzyme to TMG concentration. The ordinate represents the reciprocal of initial velocity and the abscissa describes the variation in concentration of TMG up to 4 x 10" 5 M. Open circles represent data obtained at 4.5 x 10~ 4 M glyceraldehyde; closed circles show data x 0.45 x 10~ 4 M glyceraldehyde. Velocity, V, is given as A O.D. per 5 minute per milliliter of enzyme solution. TMG concentrations. The addition of 1.6 x 10-5 M TMG altered both the K m and V,nnx. of the uninhibited enzyme, and a plot parallel to the active enzyme was found. These observations indicate an uncompetitive type of inhibition. However, at high concentrations of TMG (4.06 x 10-5 M), the effect of inhibitor on K m decreased and the reciprocal plot of inhibited versus uninhibited enzyme converged to yield closely similar K m 's to describe noncompetitive inhibition. Table III summarizes the variation in kinetic constants determined from these plots. These studies revealed that TMG is an uncompetitive inhibitor of aldose reductase. Typical of a noncompetitive inhibitor is the change to an uncompetitive type at high inhibitor concentrations. When the reciprocal initial velocity was plotted as a function of TMG concentration at different concen- Table III. Effect of TMG on the Michaelis constants for aldose reductase TMG (M x lo-s) Experimental methods from Fig. 2. l/v were similar to Km and data _ s ~ l/v x 10-5 X 10-5 X 10-5 calculated trations of substrate (0.45 x 10~ 4 M and 4.5 x 10"' J M glyceraldehyde), the uncompetitive nature of TMG inhibition was confirmed. These data are summarized in Fig. 3 again reveal the parallel character of TMG inhibition. This latter series of plots allowed direct measurement of the Ki for TMG inasmuch as the K appnre nt could be defined by the following equation: Y = K, (1 + K./S)
6 362 Jedziniak and Kinoshita Investigative Ophthalmology May 1971 OCH A % INHIBITION OF LENS ALDOSE REDUCTASE % INHIBITION OF DULCITOL SYNTHESIS 1 ] (TMG) Cx. 0 o Fig. 4. Aldose reductase inhibitors. TMG analogues were tested against purified lens aldose reductase for inhibitory activity at 0.1 mm. They were also tested in lens culture in the presence of 30 mm. and their effectiveness at 2.5 mm. in blocking dulcitol synthesis was determined. The conditions of incubation were identical to those previously described. 10 The Ki values obtained at low and high levels of glyceraldehyde were essentially the same, 1.0 and 1.2 x 10" 5 M, and show that TMG is thus a very effective inhibitor of lens aldose reductase. As shown in Fig. 4, TMG analogues, those having a ring structure other than the tetramethylene group, were tested for their effectiveness in inhibiting aldose reductase in the purified preparation and in the intact lens. The effect of these TMG analogues on crude aldose reductase was previously reported by Dvornik. 12 It appears that the tetramethylene group can be substituted for by larger ring structures without significantly altering the inhibitory activity. The ring structure is essential as indicated by the fact that glutaric acid itself is not an inhibitor. In lens culture all compounds that inhibited the enzyme also reduced the level of dulcitol that accumulates in the lens incubated in a
7 Volume 10 Number 5 Aldose reductase activators and inhibitors x TPNH Fig. 5. The effect of TPN+ on Lineweaver-Burke plots of initial velocity versus TPNH concentration. Standard assay conditions were used. The ordinate represents the reciprocal of initial velocity expressed as change in O.D. per 5 minutes per milliliter and the abscissa represents the reciprocal of TPNH concentration. Measurements were done with no TPN+ (open circles), 1.72 x 1(HM (hatched circles), and 3.44 x 1(HM TPN+ (closed circles). high-galactose medium. The data in Fig. 4 indicate that TMG and its analogues do not enter the lens rapidly. The incubation of the lens in 2.5 mm. of TMG caused about the same degree of inhibition as that observed when the isolated enzyme was exposed to 0.1 mm. of inhibitors. Product inhibition of TPN +. Enzyme activity in the absence and presence of /?- metoh displayed a final velocity that was lower than the initial velocity. In seeking an explanation for this we examined the possibility of inhibition by the products of the following aldose reductase reaction. Glyceraldehyde + TPNH > glycerol + TPN+ A.R. Addition of various levels of glycerol did not effect the forward reaction. However, TPN + had a pronounced inhibitory effect. The kinetics of the reaction with and without TPN + was analyzed to determine the nature of inhibition. Fig. 5 shows the TPN + effects on the reaction as analyzed by the Lineweaver-Burke plots. The intercepts of the Y axis were identical both in the absence and presence of 1.7 x 10~' J M and 3.4 x 10" 4 M of TPN +, indicating that the V max. does not change in the presence of this inhibitor. On the other hand, the K m of the reaction is increased in the presence of TPN +. Thus, in the presence of TPN +, the V max. remains unchanged and K m is increased. This description is
8 Invcxtinalive Ophthalmology May Jedziniak and Kinoshita I e D Fig. 6. protein distribution on polyacrvlamide gels. The general procedure of Davis" was followed. Samples were applied to gel columns (0.5 cm. in diameter) in dilute Tris butter (ph 8.6) containing 40 per cent sucrose. A current of 4 ma. per gel was applied at 5 C. until the tracking dye was approximately 0.5 cm. from the bottom of the gel. The patterns shown are: (A) native enzyme containing 5 mm. /3-mETOH, (B) enzyme in 5 nim. NEM, (C) enzyme in 5 mm. TMG, and (D) enzyme in 5 mm. TPN+. In all gels except B, both the sample and electrophoresis buffer contained 5 mm. /3-mETOH. typical of a competitive type of inhibitor. The effectiveness of TPN+ is given by its K, values of 7.5 x 10 "M. Polyacrylamide gel electrophoresis. The effect of inhibitors on the protein and enzyme patterns of aldose reductase preparations after electrophoresis on polyacrylamide gel was examined. To determine the location of the enzyme on the gel it was necessary to section the gel and to elute each section for enzyme assay. Duplicate gels ran simultaneously were stained for proteins as described in Methods. Electrophoretic patterns developed in the presence of /3-mETOH showed that the purified enzyme was made up of three protein bands as shown in Fig. 6. Enzyme activity was associated only with protein Band 1. After electrophoresis, when the enzyme was recovered by edition from a position on an unstained gel corresponding to Band 1 and rerun on another gel, the same three protein bands were observed. It appears that the enzyme exists in three protein forms, one enzymatically active and two inactive forms. /8-mETOH is essential to preserve the active form. The action of the inhibitors is to convert the active to the inactive forms of the enzyme. Fig. 6, B shows that treatment of the active enzyme with NEM leads only to inactive Band 3. Despite the presence of /?-metoh, TMG produces only Band 2 (Fig. 6, C) and TPN+ results in Bands 2 and 3 (Fig. 6, D). The TPNMreated enzyme was recovered by sectioning and eluting that portion of the gel containing the two inactive forms. Because of the dilution of TPN+ in this process, assay of the eluant revealed an active enzyme. When the eluant was run on gel electrophoresis, active Band 1 appeared along with two other bands. These findings suggest that the three different forms of aldose reductase are in some kind of equilibrium. Band 1 and combined Bands 2 and 3 of aldose reductase were recovered from the gels and were treated separately with SDS. These SDS samples were run on gel electrophoresis according to the method of Weber and Osborn.9 The resulting protein distribution patterns from Band 1 and that from Bands 2 and 3 were analyzed by densitometer tracings. Table IV summarizes the data. Band 1, the enzymatically active form, yielded five protein fractions following SDS treatment. Bands 2
9 1 I Volume 10 Number 5 Aldose reductase activators and inhibitors 365 Table IV. Molecular weight determinations by SDS acrylamide gel electrophoresis Peak No Molecular iceight Band 1 Bands 2 and 3 64,000 58,000 40,000 23,700 18,800 68,600 58,200 50,000 28,000 19,000 and 3, the enzymatically inactive forms, also yielded the same five protein fractions. As shown in Table IV, the molecular weights determined from both active and inactive forms were essentially identical within a range of 10,000 M.W. accuracy. Thus aldose reductase can be dissociated by SDS into five fractions varying in molecular weight from 70,000 to 20,000. The fact that both active and inactive forms of enzyme yield identical SDS protein patterns also supports the contention that an equilibrium exists between these forms. Discussion. The effectiveness of TMG in delaying the occurrence of early changes in sugar cataracts was previously demonstrated in lens culture. 10 To be effective, TMG concentrations in the 1 to 10 mm. range had to be used. It would be most difficult to achieve this level of inhibitor in the eye of intact animals. Therefore, although TMG was important in providing support for the concept that aldose reductase initiates the cataractous process, it would not be a satisfactory agent to alter the course of cataract in animals. Thus other more potent aldose reductase inhibitors are needed. From this and other studies 7 we are beginning to recognize what substituent groups are necessary to make a compound effective as an aldose reductase inhibitor. The compound must have at least one carboxyl group. It is possible that some other negative group may substitute for this acidic group. Secondly, a prominent aliphatic portion must be present on the molecule. This portion may be a long aliphatic chain, as in fatty acids, 7 or it could be in the form of a ring structure as in TMG. The ring structure can be of any size without appreciably altering inhibitory activity. However any side chain off the ring structure destroys the inhibitory activity. A new finding in this study was that TPN + is an inhibitor of the aldose reductase reaction. This coenzyme also has a ring structure and negative groups. However, the action of TPN + is probably quite different from TMG. It appears that TPN + attaches to a different site on the enzyme. It produces a competitive type of inhibition whereas TMG causes an uncompetitive inhibition. It is also pertinent that even though TPN + is an effective inhibitor, DPN + is not. Inhibition of aldose reductase by TPN + may represent a highly sensitive physiological control mechanism. Its inhibitory action occurs at a TPN + concentration that is found in the lens. 11 The action of TPN +, as well as other inhibitors, appears to dissociate the active enzyme into inactive forms. Preliminary estimates have shown that Band 1 is a protein of a higher molecular weight than those proteins of Bands 2 and 3. The findings suggest that the rate of aldose reduction by the enzyme in situ may depend on the prevailing TPNH:TPN ratio. This ratio, in turn, most likely determines the ratio of active to inactive forms of the enzyme. Aldose reductase is a difficult enzyme to study because of its instability during the purification process and storage. This SH enzyme requires the protection by other thiols for its activity. In the impure preparation there are sufficient SH groups provided by other lens proteins. In the purified enzyme, exogenous thiols are required. Even in the presence of thiol groups some of the enzyme exists in the inactive forms as shown by the 3 protein bands on gel electrophoresis (Fig. 6). Other evidence, such as SDS experiments and sephadex chromatography, is available also indicating the presence of still other forms of the enzyme. Thus
10 366 Jedziniak and Kinoshita Investigative Ophthalmology May 1971 more work is needed to establish how these various forms are related and what are the factors that determine the state of equilibrium between these forms. The action of /?-metoh also needs to be better understood. In activating the enzyme, /?- metoh appears to associate smaller units of inactive protein to a larger active enzyme. It is a little difficult to see how simply maintaining the SH group in a reduced state could lead to a larger protein. An explanation for this appears forthcoming from more extensive kinetic studies now in progress. They suggest that aldose reductase exhibits the property of allosterism and that /?-metoh serves as a modifier in this process. The authors would like to thank Mrs. Ellen M. Yates for her expert technical assistance. This investigation was supported by contract AT (30-1)1368 from the United States Atomic Energy Commission, and by grants EY 17082, EY and EY from the National Eye Institute of the National Institutes of Health. REFERENCES 1. Kuck, J. F. R., Jr.: Cataract formation, in Graymore, C. N., editor: Biochemistry of the eye, New York, 1970, Academic Press, Inc., Chap. 5, pp van Heyningen, R.: The lens: Metabolism and cataract, in Davson, H., editor: The eye Volume 1. London, 1969, Academic Press, Inc., Chap. 6, pp Gabbay, K. H., and O'Sullivan, J. B.: The sorbitol pathway in diabetes and galactosemia, Diabetes 17: 300, Gabbay, K. H., and O'Sullivan, J. B.: The sorbitol pathway: Enzyme localization and content in normal and diabetic nerve and cord, Diabetes 17:239, Warburg, O., and Christian, W.: in Colowick, S. P., and Kaplan, N. O., editors: Methods of enzymology, New York, 1957, Academic Press. Inc., Vol. Ill, p Lowry, O. H., Rosebrough, N. J., Fair, A. L., and Randall, R. J.: Protein measurement with folin reagent, J. Biol. Chem. 193: 265, Hayman, S., and Kinoshita, J. H.: Isolation and properties of the lens aldose reductase, J. Biol. Chem. 240: 877, Davis, B. J.: Disc electrophoresis. II. Method and application to human serum protein, Ann. N. Y. Acad. Sci. 121: 404, Weber, K., and Osborn, M.: The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis, J. Biol. Chem. 244: 4406, Kinoshita, J. H., Dvornik, D., Kraml, M., and Gabbay, K. H.: The effect of aldose reductase inhibitor on the galactose-exposed lens, Biochim. Biophys. Acta 158: 472, Sippel, T.: Rat lens pyridine nucleotide, Exp. Eye Res. 1: 368, Dvornik, D.: Gordon Research Conference proceedings, conference on medicinal chemistry, p. 61, 1970.
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