Comparative studies on aldose reductase from bovine, rat and human lens

Biochimica etBiophysicaActa, 708 (1982) 348-357

348

Elsevier Biomedical Press BBA31354

COMPARATIVE STUDIES ON ALDOSE REDUCTASE FROM BOVINE, RAT AND HUMAN LENS SYLVIA M. CONRAD and C.C. DOUGHTY

Department of Biological Chemistry, Unwersity of Illinois at the Medical Center, Chicago, 835 South Wolcott Avenue, Chicago, 1L 60612

(U.S.A.) (Received May 10th, 1982)

Key words: Cooperativity," Aldose reductase; Affinity chromatography," (Lens)

A purification scheme for aldose reductase (aiditoi: NADP + 1-oxidoreductase, EC 1.1.1.21) developed using bovine lens tissue including an affinity chromatographic step is presented which is particulary suited for small quantities of lenses. Using the affinity chromatographic method as a key step also makes it possible to obtain preparations of rat lens aldose reductase which are homogeneous. The behavior of crude preparations of aldose reductase from human lens on both ion-exchange and affinity chromatography was similar to the chromatographic behavior of the enzyme from rat and bovine lens. Comparative studies of aldose reductase obtained from the lenses of the three species demonstrate the similarity of the enzymes. These comparisons were based on molecular weights, isoelectric points, chromatographic behavior and kinetic data. Homotropic cooperativity for both NADPH and glyceraldehyde, as evidenced by a downward curvature in the Lineweaver-Burk double-reciprocal plots, had been demonstrated for aldose reductase obtained from bovine lens (Sheaff, C.M. and Doughty, C.C. (1976) J. Biol. Chem. 251, 2696-2702). Similarly, cooperativity was observed with the enzyme from both rat and human lenses and the apparent K m values at both high and low concentrations of substrate are comparable for the lens aldose reductases from all three species for both substrates.

Introduction Aldose reductase (alditol:NADP + 1-oxidoreductase, EC 1.1.1.21) catalyzes the first reaction of the sorbitol pathway. It is an NADPH-dependent enzyme that reduces a broad spectrum of aldoses to the corresponding sugar alcohols [1,2], such as glucose to sorbitol. Since the enzyme was first characterized by Hers [1] it has been studied from numerous tissues by several investigators [3-6]. Clinical interest in the sorbitol pathway and in aldose reductase in particular derives from the role this metabolic pathway plays in the tissue damage

Abbreviation: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

sustained during the course of diabetes mellitus [7,81. It has been established that sorbitol accumulates in cells particularly in diabetes where the tissue glucose concentrations are abnormally high [9,10]. Elevated sorbitol levels are the cause of loss of osmotic integrity and subsequent cellular damage, particularly in the lens of the eye; this then is a factor in cataract formation [7,11-13]. In aldose reductase studies, bovine lens became a tissue of choice for the source of kilogram quantities of crude material from which to obtain enough highly purified enzyme for physical and chemical measurements [14]; in viva studies, however, such as experimental production of cataracts, necessitated the use of rats in the laboratory [12,15,16]. The question arose as to whether aldose

349

reductases from bovine and rat lens were homologous proteins and if they were similar enough to the human lens aldose reductase to serve as good experimental models. With this in mind the studies presented here compare physical, chemical and kinetic properties of lens aldose reductase from bovine, rat and human species.

of Illinois eye bank and stored at -60°C. Cell-free extracts from tissue from each of these three sources were similarly prepared. The lenses were homogenized by intermittent grinding and cooling cycles in 5 vol. 0.010M sodium phosphate, pH 7, at 0°C for 10-15 min. Following centrifugation at 18000 × g for 20 min, the cell-free extracts were obtained as the supernatant.

Methods

Affinity chromatography method Materials Chemicals of the highest purity were obtained from the following sources: DL-glyceraldehyde from K and K Laboratories; NADPH and paranitrobenzaldehyde from Sigma; enzyme grade ammonium sulfate from Schwarz-Mann; Sephadex G-100, DEAE-Sephacel, AH-Sepharose-4B, and molecular weight standards from Pharmacia; Hepes from Calbiochem; 2-mercaptoethanol from Eastman; NaBH3CN from Aldrich; ampholine was obtained from LKB Instruments. All other reagents were of the highest purity commercially available.

Enzyme quantitation Assay for enzyme activity were conducted at 37°C with a reaction mixture (1.0 ml) containing 0.4 M (NH4)2SO4, 0.1 M sodium-Hepes (pH 7.0), 5.6 mM DL-glyceraldehydeand 0.11 mM NADPH. The reaction was initiated by addition of the enzyme and activity was measured by recording the decrease in absorbance at 340 nm with a Gilford model 240 recording spectrophotometer. 1 unit of enzyme was defined as the amount of enzyme which catalyzed the oxidation of 1 nmol NADPH/min. Protein concentrations were determined by the absorbance ratio at 260/280 nm [17].

Purification of aldose reductase from the rat and human lenses employed an affinity chromatography step [18,19]. This method was originally developed using small pools of bovine lenses (5-10 lenses) [201, then applied in the purification scheme for rat and bovine lens aldose reductase. In preparation of the column beds for this type of chromatography, para-nitrobenzaldehyde, a highaffinity substrate, was attached covalently to beads of AH-Sepharose-4B (Pharmacia). This was done by means of the reaction between an aldehyde and an amine resulting in the formation of a Schiff base. The base was then reduced by NaBH3CN, a reducing agent selective for Schiff bases [21], to give a stable ligand (Fig. 1). 7 g of AH-Sepharose-4B lyophilized beads were initially swelled in 0.5 M NaC1 for 48 h: the swelled beads were transferred to a sintered glass filter, washed with 3 more liters 0.5 M sodium chloride followed by 1 liter triple-distilled water. After repeating this step, the beads were washed with 1 liter 0.5 M sodium phosphate, pH 6.0. This was

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preparation of cell-free extracts All buffers employed throughout all purification steps, beginning with the cell-free extracts, contained 0.005M 2-mercaptoethanol. Calf lenses were dissected at the abbatoir within 1 h after death, quickly frozen on solid CO 2 and stored at -60°C. Rat lenses were dissected immediately after the animals were sacrificed, also quickly frozen on solid CO 2 and stored at -60°C, Normal human lenses were obtained from the University

CH2

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Fig. 1. The structure of the ligand used for the affinity chromatography of aldose reductase. Sepharose bends are available from Pharmacia with spacer arms consisting of six methylene groups ending in an amino group. These amino groups were reacted with para-nitrobenzaldehyde and sodium cyanoborohydride to produce the ligand shown.

350 followed by the gradual addition of a solution containing equal volumes of methanol and 0.25 M sodium phosphate, p H 6, the solvent for the coupling reaction. The beads were then transferred in 200 ml of the above solution to a closed container. An amount of p-nitrobenzaldehyde in slight molar excess (300 #mol) dissolved in the same solution was added dropwise. The reductive amination was driven to completion by the addition of solid NaBH3CN (300 #mol). The reaction was allowed to continue for 18 h at 25°C with gentle shaking to allow for maximal coupling of the ligand. The beads were again washed by filtration on a sintered glass filter in 4 liters 1 M NaC1, followed by 1 liter 0.015 M sodium phosphate/0.015 M sodium chloride, p H 6.75. Between 7 and 7.5 g of dried beads resulted in 30 ml of prepared gel, which were poured in a 1.5 × 50 cm column; this gel contained 250-300 #mol of ligand. The enzyme Solution was dialylzed overnight against the appropriate molarity (depending on the source of tissue) of sodium chloride/sodium phosphate buffer, p H 6.75, to prepare it for loading on the affinity column, equilibrated in the same buffer. After the dialyzed enzyme preparation had been concentrated by means of an Amicon filtration device containing a pM-10 membrane to 10-20 m g / m l , it was loaded on the column at a flow rate of 3 m l / h . The column was washed at the same flow rate for 18 h or until the effluent fractions contained no more protein, as judged by monitoring of the effluent fractions at 280 nm. The aldose reductase was eluted with sequential gradients. First a 250 ml linear gradient of a substrate, glyceraldehyde, 0.0 to 0.10M, superimposed on the loading buffer, was applied. This was immediately followed by applying a 250 ml linear ionic strength gradient which varied from the ionic strength of the loading buffer up to 0.10M sodium chloride, 0 . t 0 M sodium phosphate, p H 6.75. During the course of the salt gradient, the enzyme eluted in a discrete peak (Fig. 2). This affinity chromatographic method, along with previously described DEAE-cellulose chromatography and G-100 molecular sieving [14], could be used in a three-step rapid method to p u r i f y bovine lens aldose reductase from small pools of lenses (10-20) (Table I).

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Fig. 2. The elution profile of bovine lens aldose reductase from the affinity column. Initially a 250-ml linear gradient of glyceraldehyde (in 0.015 M sodium chloride/0.015 M sodium phosphate, pH 6.75) from 0.0 to 0.10 M was applied to the column. This was followed by a second linear gradient consisting of 250 ml total volume with a concentration range from 0.015 M to 0.100 M in both sodium chloride and sodium phosphate (pH 6.75). 3-ml fractions were collected and selected fractions were monitored for total protein (e e) by A26o/A2ao and units of aldose reductase. The elution of the enzyme by this gradient is shown. Activity, O -- -- -- O; spec. act., A - - - - - & .

Purification method." rat lens tissue Crude precipitation steps. In purifying rat lens aldose reductase certain precipitation steps were performed on the cell-free extract before any chromatographic steps were begun. The cell-free extract of 100-200 rat lenses, maintained at 4°C, was titrated with 0.1 N H2SO 4 to p H 5.35 with continuous monitoring. The precipitate which formed was removed following centrifugation (18000 × g, 20 min) and the supernatant was immediately adjusted to p H 7.0 with 0.5 M N a O H . Solid (NH4)2SO 4 was added to a total concentration of 22.6 g / 1 0 0 ml and after a 15 min equilibration the precipitate removed following centrifugation. The pellet was discarded and additional (NH4)2SO 4 added to the supernatant in the amount of 8.9 g / 1 0 0 ml. Again the centrifuged precipitate was discarded, the aldose reductase remaining in the supernatant. The enzyme was dialyzed into 0.005 M sodium phosphate, p H 7, to prepare for loading on a D E A E ion-exchange column. (The conductivity of the dialysate was used as an indicator that the loading sample of enzyme was at the correct molarity). DEAE-Sephacel chromatography. In purifying

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Fig. 3. The elution profile of rat lens aldose reductase from DEAE-Sephacel. After loading the sample in 0.005 M sodium phosphate, pH 7, and washing with this buffer until the concentration of protein in the effluent, as monitored by 280 nm absorbance ratios, was zero, the enzyme was eluted by applying a linear gradient of (NH4)2SO4 which was 250 ml total volume. The concentration of the gradient ranged from 0.0 to 0.040 M in (NH4)zSO4 (superimposed on the loading buffer). 3-ml fractions were collected and monitored for total protein (0 0), enzyme activity (0; spec. act., A) and conductivity ([]).

aldose reductase from rat lens, DEAE-Sephacel (Pharmacia-granular) columns with a 10-ml bed volume were used, equilibrated with 0.005 M sodium phosphate, p H 7. These columns were maintained at 4 ° C with a 10 m l / h flow rate. Rat lens aldose reductase dialyzed into this buffer b o u n d to the DEAE-Sephacel. After loading the sample and washing the column overnight with this buffer (or until the 280 n m absorbance readings indicated no more protein was eluting), the aldose reductase was eluted by applying a 250 ml linear gradient of ( N H 4 ) 2 S O 4 from 0.00 to 0.04 M superimposed on the loading buffer, (Fig. 3). The fractions containing the activity peak were pooled, concentrated with an A m i c o n filtration device and dialyzed into 0.005 M sodium chloride/0.005 M sodium phosphate, p H 6.75, to prepare for loading o n the affinity column. Affinity chromatography. A 10-ml or 25-ml bed volume column prepared as previously described and maintained at 4 ° C was equilibrated in 0.005 M sodium chloride/0.005 M sodium phosphate, p H 6.75. The enzyme solution (concentrated to 1 0 - 2 0 m g / m l ) was loaded at 3 m l / h and the column washed overnight or until protein, as monitored b y the absorbance of 280 nm, no longer

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Fig. 4. The elution profile of rat lens aldose reductase from the affinity column. Initially a 250-ml linear gradient of glyceraldehyde (in 0.005 M sodium chloride/0.005 M sodium phosphate, pH 6.75) from 0.0 to 0.10 M was applied to the column. This was followed by a second linear gradient consisting of 250 ml total volume with a concentration range from 0.05 M to 0.100 M in both sodium chloride and sodium phosphate. 3-ml fractions were collected and selected fractions were monitored for total protein (0) and units of aldose reductase (activity, O; spec. act., A). The elution of the enzyme by this gradient is shown. (In this figure the first 70 ml, eluted after the salt gradient was initiated at zero elution volume, are not shown.)

appeared in the effluent. The enzyme was eluted b y the sequential gradients previously described. Fig. 4 illustrates the elution of the rat aldose reductase from the affinity column of a loading sample treated only by the precipitation steps. In the final purification scheme, however, D E A E c h r o m a t o g r a p h y also preceded the affinity step (see Table II).

Purification method: human lens tissue Crude precipitation step. In purifying the enzyme from the small a m o u n t of h u m a n material ( 2 0 - 4 0 lenses) available only one precipitation step was done. Solid ( N H 4 ) 2 S O 4 was added to the cell-free extract, maintained at 4 ° C to a final concentration of 22.6 g / 1 0 0 ml; after a 15 rain equilibration the precipitate was removed following centrifugation. The aldose reductase, which remained in the supernatant fluid, was then dialyzed into 0.005 M sodium phosphate, p H 7, to prepare for loading on the DEAE-Sephacel column. Chromatographic steps. The methods used for b o t h D E A E - c h r o m a t o g r a p h y and affinity chrom a t o g r a p h y for the h u m a n lens material were exactly the same as in the rat lens purification scheme.

352

Molecular sieving Sephadex G-100 column chromatography was also used for molecular weight estimation. A downward flow column (1.5 X 50 cm) was employed at a flow rate of 6 m l / h at 4°C in 0.1 M sodium chloride/0.05 M sodium phosphate/0.005 M 2-mercaptoethanol, pH 7. Marker proteins used were: ribonuclease A ( M r 13700), chymotrypsinogen A (M r 25000), ovalbumin ( M r 43000), bovine serum albumin (M r 67000). The void volume (110) was determined using Blue Dextran 2000.

Isoelectric focusing A l l0°ml vertical column (LKB) was used in the isofocusing experiments to determine isoelectric points for the enzymes, according to the method described by Dons and Doughty [5]. The pH gradient of the ampholines was 4-6. After electrofocusing was completed, the column was drained by use of a peristaltic pump at a flow rate of 1 ml/min. The pH and aldose reductase activity of the individual fractions were then determined.

Kinetic studies In performing the assays for the kinetics studies the assay procedure initially described was used, with the exception that the substrate concentrations were varied by the addition of exact amounts of standard substrate solutions into the cuvette, keeping the final volume to 1 ml. Care was taken to standardize the time allowed for temperature equilibration of the reaction mixture in the cuvette (5 min) before the addition of the enzyme and the assays were run for 3 min to determine the change in absorbance at 340 nm per min [22]. In the studies described here, the lines on the kinetic graphs were determined by a linear regression analysis SD-03A program of a Hewlett-Packard 97.

Polyacrylamide gel electrophoresis Assessment of enzyme purity was made using the vertical slab gel electrophoretic technique of O'Farrell [23]. The running gel consisted of 10% acrylamide, (36.5:1; acrylamide to N,N'-methylene bisacrylamide), 0.1% SDS, 0.375 M Tris-HC1 buffer, pH 8.8. Polymerization was initiated with ammonium persulfate and TEMED (N,N,N;N'-

tetramethylethylenediamine). The stacking gel consisted of 3% acrylamide, (36.5 : 1; acrylamide to N,N'-methylene bis acrylamide), 0.1% SDS, 2 M urea, 0.125 M Tris-HC1, pH 6.8. The gels were stained overnight in Coomassie brilliant blue solution. They were destained in an ethanol/acetic acid/water mixture (3:1:6) and preserved in an ethanol/acetic acid/glycerol/ water mixture (3 : 1 : 1 : 5). 0.5 #g protein was easily detectable by this method. Results

An affinity chromatographic method for purification of aldose reductase was devised for this enzyme obtained from lens tissue. This was done to facilitate obtaining enzyme from small samples of tissue, since existing methods were designed for kilogram quantities of starting material. Bovine lens aldose reductase was used to develop this method because of the limiting supply of both rat and normal human lens tissue. This method then became a key step of a simplified purification scheme for aldose reductase adaptable to small amounts of material. The three-step method for purifying enzyme from 10-20 bovine lens is shown in Table I. It employes DEAE-cellulose chromatography and G-100 molecular sieving as already described for bovine lens purification by Sheaff and Doughty [14] in addition to the affinity

TABLE I A SIMPLIFIED PURIFICATION PROCEDURE FOR BOVINE LENS ALDOSE REDUCTASE WITH l0 g CRUDE MATERIAL (10 OR FEWER LENSES) Step

Cell-free extract DEAE-cellulose ion-exchange chromatography Affinity chromatography (3-100 Sephadex chromatography

Spec. act. (units/mg)

0.16

Overall yield (%)

Fold purification

100

1

35

50

219

300

45

1 875

947

45

5 919

353 TABLE II THE PURIFICATION PROCEDURE FOR RAT LENS ALDOSE REDUCTASE Step

Spec. act. (units/mg)

Overall yield (%)

Fold purification

Cell-free extract

6

100

1

Acid precipitation

7

90

1

(NH4)2SO4 fractionation

12

75

2

DEAE-Sephacel ion-exchange chromatography

1 200

37

200

Affinity chromatography

2100

10

350

chromatographic step. In purifying rat lens aldose reductase beginning with 3-6 g lens tissue the affinity chromatographic step was the final step employed. (Table II). The DEAE ion-exchange chromatography, employed by Sheaff and Doughty [14] as one step of many in the purification of bovine lens aldose reductase from kilogram quantities of lenses, also proved to be an effective step in purifying aldose reductase from rat lenses following the (NH4)2SO 4 precipitation step. The final purification for the rat lens aldose reductase was based on the scheme shown in Table II. Pooling all the active fractions from the affinity

Fig. 5. Densitometer tracing at 633 nm of a vertical slab SDS-polyacrylamide gel stained with Coomassie blue. Sample from the pool of active fractions of rat lens aldose reductase from the final affinity column step of the purification scheme described in Table II was subjected t o electrophoresis as described in Methods. The specific activity of this preparation was 2100 and the major band visible is the aldose reductase.

chromatography step resulted in a preparation which showed two minor contaminants in addition to the aldose reductase bands (Fig. 5); however, a homogeneous one-band preparation was observed if the latter halves of the active fractions were pooled separately. With 20-40 normal human lenses from a limited supply furnished by the University of Illinois eye bank, a partial purification of human lens aldose reductase was attempted. An initial (NH4)2SO 4 fractionation as described in Methods was done on the cell-free extract. The human enzyme contained in the dialyzed supernatant from the salt precipitation bound to both DEAE-cellulose ionexchange and affinity columns. Although it was eluted by the same procedures used for the rat lens aldose reductase, the specific activities of the peaks eluted from either column were low. The purification was approx. 30-fold. Vertical slab polyacrylamide gels of the pooled fractions containing peaks of activity from the columns showed a preponderance of bands in the low molecular weight region from 18000 to 28000. Some of these bands may represent fragments of proteins which could be generated by the lens proteases [24,25] during the time lag before the human lenses were frozen at the eye bank. In comparing properties of lens aldose reductase from three different species (bovine, rat and human) the molecular weights were compared by means of gel chromatrography in consecutive determinations on the same calibrated G-100 column under identical conditions. The results of this study (using ribonuclease A, chymotrypsinogen A, ovalbumin and bovine serum albumin as markers) demonstrate the similar elution volume values for the three proteins, indicating that the molecular weights are similar. Since the isoelectric point of bovine lens aldose reductase had already been determined [14] as 4.85, this property of rat aldose reductase was found to be 4.75. Although only limited purification was obtained with the human lens enzyme, these experiments did demonstrate that human lens aldose reductase behaved chromatographically very similarly to both bovine lens and rat lens aldose reductases. The conditions for binding the rat and the human enzyme to DEAE ion-exchange col-

354 TABLE III APPARENT MICHAELIS-MENTEN CONSTANTS FOR ALDOSE REDUCTASE OBTAINED FROM BOVINE, RAT A N D H U M A N LENSES Source

High substrate (,aM)

Bovine K m (glyceraldehyde) g m (NADPH) Rat K m (glylceraldehyde) K m (NADPH) Human K m (glyceraldehyde) K m (NADPH)

Low substrate (,aM)

100 a, 250 b 17 a, 23 b

10 a 2a

70-95 19-21

~- 2-28 ~2-7

90-1 l0 17-23

~ 10-30 ~2-5

a Data from Thrash [26]. b Data from Sheaff and Doughty [14].

umns were identical and were similar to the bovine lens binding conditions. The conditions for elution were also similar for the enzyme from all three species (see Methods Ref. 14). In the affinity chro-

matographic step the conditions for binding the rat and human enzyme to the matrix were identical and similar to those for the bovine enzyme, and conditions for eluting all three enzymes were the same (see Methods). The kinetic behaviors of the rat and the human enzymes were studied. The bovine lens enzyme had been studied by Thrash [26] with partially purified enzyme, and non-linear Lineweaver-Burk plots had been observed in that both the K m and Vm,x values increased above a certain level of substrate concentration. This was confirmed with homogeneous bovine lens enzyme by Sheaff and Doughty [14]. The values for the two different Michaelis-Menten constants for both substrates, NADPH and glyceraldehyde, reported by these investigators are shown in Table III. Although they did not assign numerical values to the Michaelis-Menten constants at the lower levels of substrate concentration they clearly showed the non-linearity in kinetic plots for both substrates [14] using homogeneous bovine lens aldose reductase. 400-

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Fig. 6. The apparent K m values for N A D P H for rat lens aldose reductase. The enzyme preparation for these experiments was a partially purified fraction obtained by DEAE ion-exchange chromatography of a cell-free extract; the reciprocal of the initial velocity is plotted against the reciprocal of the N A D P H concentration. The fixed level of glyceraldehyde concentration used for each line is as follows: A , 0.05 mM; A , 0.25 raM; Q, 1.0 raM. The values for the intercepts were determined by linear regression of the points between 0.004 and 0.083 1/[NADPH] (,aM) for the apparent K m for the high substrate levels. The values for the intercepts for the apparent K m for the low substrate levels were determined by linear regression of the points between 0.133 and 0.267 I/[NADPH] (,aM).

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Fig. 7. The apparent K m values for glyceraldehyde for rat lens aldose reductase. The enzyme preparation for these experiments was a partially purified fraction obtained by DEAE ion-exchange chromatography of a cell-free extract; the reciprocal of the initial velocity is plotted against the reciprocal of the glyceraldehyde concentration. The fixed level of N A D P H concentration used for each line is as follows: A, l0 #M; A , 20/aM; O, 40 ,aM; 0 , 120 ,aM. The values for the intercepts were determined by linear regression of the points between 0.001 and 0.008 1/[glyceraldehyde] (,aM) for the apparent K m for the high substrate levels. The values for the intercepts for the apparent K m for the low substrate levels were determined by linear regression of the points between 0.016 and 0.040 1/[glyceraldehyde] (,aM).

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Fig. 8. Lineweaver-Burk plot of reciprocal initial velocity vs. reciprocal NADPH concentration for highly purified rat lens aldose reductase (spec. act. 2000). The plot shows a deviation from linearity in the kinetics of the purified enzyme. [], 0.25 raM, and A, 1 mM glyceraldehyde.

Figs. 6 a n d 7 show Lineweaver-Burk plots of initial velocity studies of rat lens aldose reductase partially purified by D E A E ion-exchange chrom a t o g r a p h y a n d the n o n - l i n e a r i t y of the plots is evident. T o o b t a i n the a p p a r e n t K m values from these figures the linear regression of each set of p o i n t s representing a particular c o n c e n t r a t i o n of the second substrate is used to o b t a i n the intercept o n the abscissa which is the negative reciprocal of the a p p a r e n t Kr, [27]. The m e a n values of these intercepts with the s t a n d a r d deviation calculated e n a b l e one to express the K m as a c o n c e n t r a t i o n range. At levels of N A D P H above 20 btM the K m is 19-21 /~M, while at lower levels it approaches 2 - 7 /~M. F o r glyceraldehyde at c o n c e n t r a t i o n s greater t h a n 5 0 / ~ M the K m is 7 0 - 9 5 v M , while at lower levels it approaches 2 - 2 8 / ~ M . I/V 500

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Fig. 10. The apparent /C m values for glyceraldehyde from human lens aldose reductase. The enzyme preparation for these experiments was a cell-free extract which had been subjected to o n e ( N H 4 ) S O 4 precipitation; the reciprocal of the initial velocity is plotted against the reciprocal of the glyceraldehydeconcentration. The fixed level of NADPH used for each line is as follows: A, 10/xM; O, 20/xM; e, 120 vM. The values for the intercepts were determined by linear regression of the points between 0.001 and 0.008 1/[glyceraldehyde] (#M) for the apparent Km for the high substrate levels. The values for the intercepts for the apparent Km for the low substrate levels were determined by linear regression of the point between 0.016 and 0.040 1/[glyceraldehyde](t~M).

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Fig. 9. Lineweaver-Burk plot of reciprocal initial velocity vs. reciprocal glyceraldehyde concentration for highly purified rat lens aldose reductase (spec. act. 2000). The plot demonstrates a deviation from linearity in the kinetics of the purified enzyme. A, 40 ~aM, and O, 120 tM NADPH.

Fig. 11. The apparent Krn values for NADPH for human lens aldose reductase. The enzyme preparation for these experiments was a cell-free extract which had been subjected to one (NH4)2SO4 precipitation. The reciprocal of the initial velocity is plotted against the reciprocal of the NADPH concentration. The fixed level of glyceraldehyde used for each line is as follows: A, 0.05 raM; O, 0.25 raM; e , 1 raM. The values for the intercepts were determined by linear regression of the points between 0.004 and 0.067 I/[NADPH] (/~M) for the apparent K m for the high substrate levels. The values for the intercepts for the apparent K m for the low substrate levels were determined by linear regression of the points between 0.133 and 0.267 I/[NADPH] (vM).

356 Figs. 8 and 9 show the same kind of initial velocity experiments repeated with highly purified rat enzyme. These graphs illustrate that the discontinuities in the lines are still apparent and that classical Michaelis-Menten kinetics do not apply. Similar experiments were done using cell-free extracts from h u m a n lens subjected to one ( N H 4 ) 2 S O 4 precipitation. Results are shown in Figs. 10 and 11. The K m ranges obtained from these graphs are as follows: glyceraldehyde, above 50 # M levels, 9 0 - 1 1 0 # M ; at lower substrate levels, 10-30 # M ; N A D P H , above 20 # M levels, 17-23 # M ; at lower substrate levels, 2 - 5 #M. These experimentally determined K m values for the lens enzyme obtained from the rat and h u m a n sources are summarized in Table III. The values for the bovine lens aldose reductases are from the literature and are included in Table III to allow comparison of the values for all three species for both Michaelis-Menten constants.

Discussion These comparative studies on aldose reductases derived from bovine, rat and h u m a n lens tissue demonstrate their similarities. Elution volumes from a calibrated gel filtration column on identical runs indicate the close proximity of their molecular weights. These are acidic proteins with similar isoelectric points. In developing purification procedures it became clear that h u m a n lens aldose reductase became b o u n d and eluted under conditions very similar to those for bovine lens enzyme and under the same conditions as rat lens enzyme from both the affinity column described in this paper and the D E A E ion-exchange column. This is further evidence for the similarities of these enzymes. The comparative kinetic studies are particularly significant, since they demonstrate that the aldose reductase enzymes from rat lens and h u m a n lens deviate from classical Michaelis-Menten kinetics just as had been shown for bovine lens aldose reductase [14,26] and recently for aldehyde reductase from erythrocytes [28]. All three lens aldose reductase exhibit a concave d o w n w a r d curvature in the Lineweaver-Burk double reciprocal plots at higher substrate concentrations. Also, as shown in Table III, the corresponding high and low sub-

strate values for the Michaelis-Menten constants for N A D P H all fall in the same range, and the values for the glyceraldehyde substrate are also all in close agreement.

Acknowledgements This work was supported by National Institutes of Health grants R01 EY 03025 and R01 EY 00449 and by a grant from the Juvenile Diabetes Foundation, 81 R 126.

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