Rat liver mitochondrial malate dehydrogenase: Purification, kinetic properties, and role in ethanol metabolism

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 290, No. 1, October, pp. 191-196, 1991

Rat Liver Mitochondrial Malate Dehydrogenase: Purification, Kinetic Properties, and Role in Ethanol Metabolism’ Mark

S. Wiseman,

Department

of

Duncan

McKay,

Chemistry and Biochemistry,

Received November

Kathryn

E. Crow, and Michael

Massey University,

Palmerston

J. Hardman’

North, New Zealand

14,1990, and in revised form June 3,1991

Malate dehydrogenase was purified from the mitochondrial fraction of rat liver by ion-exchange chromatography with affinity elution. The kinetic parameters for the enzyme were determined at pH 7.4 and 3’7”C, yielding the following values (PM): K,, 72; Ki,, 11; Kb, 110; K,, 1600; Kip, 7100; K,, 170; Kiqy 1100, where a = NADH, b = oxalacetate, p = malate, and q = NAD+. Kib velocwas estimated to be about 100 FM. The maximum ities for mitochondrial malate dehydrogenase in rat liver homogenates, at pH 7.4 and 37”C, were 380 ? 40 gmol/ min per gram of liver, wet weight, for oxalacetate reduction and 39 + 3 pmol/min per gram of liver, wet weight, for malate oxidation. Rates of the reaction catalyzed by mitochondrial malate dehydrogenase under conditions similar to those in viva were calculated using these kinetic parameters and were much lower than the maximum velocity of the enzyme. Since mitochondrial malate dehydrogenase is not saturated with malate at physiological concentrations, its kinetic parameters are probably important in the regulation of mitochondrial malate concentration during ethanol metabolism. For the mitochondrial enzyme to operate at a rate comparable to the flux through cytosolic malate dehydrogenase during ethanol metabolism (about 4 wmol min-’ per gram liver), the mitochondrial [malate] would need to be about 2 mM and the mitochondrial [oxalacetate] would need to be less than 1 MM. o 1091 Academic press. IIIC.

The oxidation of ethanol in rat liver is accompanied by reduction of cytosolic NADf to NADH by alcohol dehydrogenase (1). This leads to a decrease in the free cy’ Support for this work has been received from the Palmerston North Medical Research Foundation and the Massey University Research Fund. M.S.W. is supported by a Massey University Postdoctoral fellowship. * To whom correspondence should be addressed. 0003.9861,‘91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

tosolic [NAD’]/[NADH] ratio (2-4), due predominantly to an increase in the free NADH concentration (5). Ultimately NADH produced in the cytosol is reoxidized to NAD’ in the mitochondria by the electron transport chain. As the inner mitochondrial membrane is impermeable to NADH, hydrogen shuttle systems are required for the transport of the reducing equivalents of NADH across this membrane (6). The malate-aspartate shuttle has been shown to be the most important in the oxidation of cytosolic NADH produced during ethanol metabolism (6, 7). The cytosolic and mitochondrial forms of malate dehydrogenase are key enzymes in the malate-aspartate shuttle. The cytosolic form catalyzes the oxidation of cytosolic NADH to NAD+ with the concurrent reduction of oxalacetate to malate. Malate is transported by the dicarboxylate carrier mechanism (8) into the mitochondria, where it is oxidized back to oxalacetate by mitochondrial malate dehydrogenase with concurrent reduction of NAD+ to NADH. For the malate-aspartate shuttle to operate at a steady state, the rates of the mitochondrial and cytosolic dehydrogenases must be equal. We have previously determined the kinetic parameters for rat liver cytosolic malate dehydrogenase under conditions similar to those in viuo (9). We showed that the flux through this enzyme is sensitive to NADH concentration and may be important in determining the cytosolic NADH concentration during ethanol oxidation. During ethanol metabolism the flux through cytosolic malate dehydrogenase increases with increasing NADH concentration until a steady state is reached where the rate of NADH oxidation by cytosolic malate dehydrogenase matches the rate of production of NADH in the cytosol. We have now purified rat liver mitochondrial malate dehydrogenase, using a gentle affinity elution procedure, and have determined the kinetic parameters for the enzyme. We have used the data to calculate the rate of the reaction catalyzed by this enzyme at concentrations of 191

192

WISEMAN

metabolites found in the mitochondria during ethanol oxidation in ho. We suggest a mechanism by which the rate of the mitochondrial enzyme may be increased to match the increased flux through cytosolic malate dehydrogenase which follows administration of ethanol. EXPERlMENTAL

PROCEDURES

Materials. All enzymes, substrates, and cofactors were purchased from Sigma. For kinetic studies, Grade III NADf and NADH and Grade I oxalacetate were used. Solutions of these compounds were prepared immediately prior to use. Carboxymethyl cellulose (CM-32) was obtained from Whatman. All other reagents were analytical grade products from standard sources. Enzyme assays. During enzyme purification, malate dehydrogenase was assayed at 25’C using 0.12 M L-malate and 2 mM NAD+ in 0.1 M glycine/NaOH buffer, pH 10.0. The increase in absorbance at 340 nm was monitored using a Cecil CE 292 spectrophotometer. Protein determination. Protein concentrations were determined by the Coomassie blue method (lo), using bovine serum albumin as standard. Protein concentration of eluates from chromatography columns was monitored using absorbance at 280 nm. Enzyme purification. Mitochondrial malate dehydrogenase was purified from the livers of 200. to 400-g male Sprague-Dawley rats. All procedures were carried out at 4°C except for column chromatography which was carried out at room temperature. All buffers contained 1 mM dithiothreitol and all buffers prior to column chromatography contained 1 mM phenylmethanesulfonyl fluoride as a protease inhibitor. Four to six livers (total wet weight 40-75 g) were homogenized in 5 mM sodium phosphate buffer, pH 7.3, containing 0.25 M sucrose, using an Ultra-Turrax homogenizer (3 X 30 s). Four milliliters of homogenizing buffer was used per gram wet weight of liver. The homogenate was centrifuged at 500g for 5 min and the resultant pellet discarded. The supernatant was centrifuged at 27,000g for 30 min and the resultant supernatant discarded. The remaining pellet, which contained the mitochondria, was resuspended in an equal volume of phosphate buffer, pH 6.0 (I = 0.02), using a ground glass homogenizer. This suspension was sonicated three times for 30 s at maximum power. The sample was kept on ice for 1 min between sonications. The disrupted mitochondria were centrifuged at 27,000g for 1 h after which the supernatant was saved and the pellet was resuspended, sonicated, and centrifuged as before. The final pellet was discarded and the two supernatants were combined. This supernatant, containing the released mitochondrial enzymes and some contaminating cytosolic enzymes, was further purified by ion-exchange chromatography with affinity elution (11). A column (3.2 X 16 cm) of CM-cellulose was equilibrated with phosphate buffer, pH 6.0 (I = 0.02). The combined supernatant was applied to the column and eluted with the pH 6.0 buffer until the absorbance at 280 nm was less than 0.01. Elution was then continued with phosphate buffer, pH 8.0 (I = 0.02), until the absorbance at 280 nm was again less than 0.01. The mitochondrial malate dehydrogenase was finally eluted by inclusion of 200 PM NADH in the pH 8.0 buffer. The fractions which were eluted by NADH and contained enzyme activity were combined and concentrated by ultrafiltration. The purified enzyme was stored in aliquots at -20°C. Steady-state kinetics. Initial velocities for malate dehydrogenase were determined in the presence of various concentrations of substrates and products at 37°C in 0.1 M phosphate buffer, pH 7.4, using an Aminco DW 2a uv/visible spectrophotometer. Prior to kinetic analysis, enzyme was dialyzed against this buffer, containing 1 mM dithiothreitol, to remove NADH. Kinetic constants were obtained by fitting all the data for an experiment to the equations for a sequential mechanism in the absence of products (12), or for linear competitive or noncompetitive inhibition in an ordered Bi-Bi system (13), using a nonlinear least squares curve-fitting program (14) with initial estimates of parameters obtained from reciprocal plots and replots.

ET AL. Maximum velocity of malate dehydrogenase. The maximum velocity of malate dehydrogenase in each direction was measured at 37°C in 0.1 M phosphate buffer, pH 7.4, in supernatant and mitochondrial fractions of individually homogenized rat livers. The separation of these fractions was essentially the same as for the enzyme purification procedure with the exception that the homogenization process was carried out for a shorter duration (2 X 30 s) and two extra wash stages were included. Following the 5OOgspin the pellet was resuspended in an equal volume of homogenizing buffer and centrifuged at 300g for 5 min. The two supernatants were combined. Following the first 27,000g spin the mitochondrial pellet was resuspended in homogenizing buffer and centrifuged at 27,000g for 30 min. The two supernatants were combined. The resuspended mitochondrial pellet and all other samples were sonicated three times at maximum power prior to assay but were not centrifuged following sonication. For oxalacetate reduction 1.2 mM oxalacetate and 0.2 mM NADH were used; for malate oxidation 30.0 mM malate and 5.0 mM NAD+ were used. V,,, values were calculated as micromoles per minute per gram of liver, wet weight. Glutamate dehydrogenase was assayed (15) at 37°C in 0.1 M phosphate buffer, pH 7.4, and used as a mitochondrial marker. Alcohol dehydrogenase was used as a cytosolic marker and its activity was assayed by monitoring the change in absorbance (340 nm) at 37°C in 66 mM sodium pyrophosphate/HCl buffer, pH 8.8, with 17.0 mM ethanol and 5.0 mM NAD+. Isoelectric focusing. Isoelectric focusing of mitochondrial malate dehydrogenase samples was carried out as previously described for the cytosolic enzyme (9).

RESULTS

AND

DISCUSSION

Enzyme Purification The isoelectric point of rat liver cytosolic malate dehydrogenase is pH 5.1 (9) and it is therefore eluted in the void volume from a CM-cellulose column at pH 6.0. The isoelectric point of the mitochondrial enzyme ranges from pH 8.9 to 9.6 (16) and it therefore remains bound to CMcellulose up to at least pH 8.0. Binding of NADH to the enzyme weakens the affinity of the enzyme for the resin, thus eluting it from the column. The combined effect of binding of mitochondrial malate dehydrogenase to the column at a relatively high pH followed by affinity elution from the column results in a very gentle purification procedure which yields enzyme of high purity. A similar procedure has previously been used for the purification of mitochondrial malate dehydrogenase from beef heart (11) . Typical results obtained during purification of the enzyme are shown in Table I. The 500- to 600-fold increase in specific activity seen during purification of the enzyme compares favorably with that reported for other purification schemes for rat liver mitochondrial malate dehydrogenase (17-19). Malate dehydrogenase activity was eluted from the column following addition of NADH, usually as two sharp but overlapping peaks. This may be an artifact of the procedure but the two peaks may also represent slightly modified forms of the enzyme. NO attempt was made to separate these two peaks further and all active fractions which were eluted with NADH were pooled. On isoelectric focusing of the purified enzyme, malate dehydrogenase activity appeared as a broad band, or possibly two bands, around pH 9.0. The pH range of the gel

RAT TABLE Purification

Fraction Total homogenate Cytosol (27000 X g spin; 30 min) Mitochondria, post sonication (27000 X g spin; 60 min, supernatant) CM-cellulose, NADH elution fraction

LIVER

MITOCHONDRIAL

I

of Malate Dehydrogenase Rat Liver Mitochondria

Total activity (units)

Total protein (md

from

Specific activity (units/mg)

Recovery of activity (%I

1320

11,000

0.12

100

510

5,900

0.09

39

322

970

0.33

24

427

6.3

68

32

Note. Malate dehydrogenase was purified from rat liver mitochondria as described in the text. Protein concentrations and enzyme activity were determined as described under Experimental Procedures. The units of activity are kmol of NADH produced per minute.

used (pH 3.5-9.5) did not allow accurate determination of an isoelectric point. No protein bands were visible using Coomassie blue. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single protein band was seen with a molecular weight similar to that of a pig heart mitochondrial malate dehydrogenase standard. Jones and Vestling (16) have reported multiple bands of mitochondrial malate dehydrogenase activity with isoelectric points in the range pH 8.9-9.6 and have suggested that this is due to a varying degree of phospholipid binding by the enzyme, which may arise from association of the enzyme with the mitochondrial inner membrane.

Kinetics Typical results for the dependence of the rate of oxalacetate reduction on NADH and oxalacetate concen-

MALATE

trations in the absence of products are shown in Fig. 1. For experiments in the presence of product, the kinetic data showed that NAD+ and NADH are competitive inhibitors with respect to each other. Malate is a noncompetitive inhibitor with respect to oxalacetate but inhibition by oxalacetate appeared to be competitive with respect to malate. From these data it was not possible to determine a value for the inhibition constant for oxalacetate (Kib). A similar pattern of inhibition was also seen with rat liver (9) and human liver (20) cytosolic malate dehydrogenases, although in both these instances a finite value for Kib could be obtained and inhibition was taken to be noncompetitive. The inhibition pattern is consistent with an ordered Bi-Bi mechanism, as originally proposed for the pig heart enzyme (21). In the present study, we have estimated the value for Kib for the mitochondrial enzyme from data for inhibition by oxalacetate with respect to [NAD’] at different fixed malate concentrations (Ref. (22), p. 576). Kinetic constants calculated using the nonlinear curve fitting program are given in Table II. The values are similar to those previously determined for the cytosolic enzyme (9), but most are a little higher. In particular, the values of K, and Ki, are two- to three-fold higher for the mitochondrial enzyme. Some of the kinetic constants were determined in both the presence and the absence of product inhibitors and in general there was good agreement between the values, indicating that the observed differences between the mitochondrial and cytosolic enzymes are not due to experimental variation. However, for Ki(iq the three methods gave three different values, with the value determined in the absence of product being the lowest of the three. This difference, for which we have no explanation, was also seen with rat (9) and human (20) liver cytosolic malate dehydrogenases. The lowest of the three Ki(iqvalues is the most consistent with the Haldane relationship (Ref. (22), p. 574) and was used for the cal004

[oxalaceiate]

[NADH]

-‘()lM)

(KM)

193

DEHYDROGENASE

IO6

[NADHI(PM) I

7.5

~’

FIG. 1. Determination of the kinetic parameters of rat liver mitochondrial malate dehydrogenase. The figures show primary plots obtained in the absence of product inhibitors. The concentrations of the substrates are indicated on the appropriate figures. Experiments were performed as described under Experimental Procedures. The lines were calculated using kinetic parameters determined by nonlinear fitting of all the data for the experiment to the equation for a sequential mechanism in the absence of products (12).

194

WISEMAN TABLE

Kinetic Constant (PM) K, K, Kb K,b 4 K,, K, K1q

Constants

II

for Mitochondria]

Absence of inhibitor 72 t 12 f 86 f

8 (10) 2 (10) 11 (10)

1900 f 400 (6) 18Ok 30(6) 1100 f 100 (6)

Malate

Dehydrogenase

Presence of inhibitor 73 ? 61 150 + 1400 + 7100 + 140 ‘3100 + 1900 2

11” (3) 1 (3) 20"(5) 100b 100” (10) 800"(5) ZOO(3) 400'(3) 200d (4)

Average 72+ llf 110 +

7 2 10

1600 f 200 170+

20

Note. Kinetic constants (PM) were determined as described under Experimental Procedures. The results are expressed as means f SEM for (n) separate determinations using at least two individual enzyme preparations. The subscripts a, b, p, and cl refer to NADH, oxalacetate, malate, and NAD+, respectively. a Since the nonvaried substrates were not saturating, the apparent values were corrected using the following equations (13), K. = K(1 t Kb/ B) - K,,Kb/B; Kb = K(1 + K,/A)/(l + K,,/A); Kip = K,J(l + K./A); Kp = K(l + K,/Q)/(l t K,,/Q); K, = K(1 + K,/P) - K,,K,/P, with values determined in the absence of inhibitors for the corrections. * An approximate value (see text). ‘K,, for linear competitive inhibition by NAD+. d Calculated from K,, for linear noncompetitive inhibition by malate, using K, and Kg obtained in the absence of inhibitors.

culation of rates of mitochondrial activity. Maximum

malate dehydrogenase

Velocity of Malate Dehydrogenase

To minimize mitochondrial breakage a gentle homogenization procedure was used. A significant proportion of the activity of the mitochondrial marker enzyme, glutamate dehydrogenase, and a lesser proportion of the cytosolic enzyme, alcohol dehydrogenase, was found in the precipitate of the initial centrifugation, which contains unbroken or partially broken cells. These losses in activity were taken into account in calculation of maximum velocities and the method gave reproducible results; therefore no attempt was made to optimize further the homogenization and separation procedure. The recovery of glutamate dehydrogenase was 102 2 4% (n = 10) and of alcohol dehydrogenase was 86 -t 3% (n = 10). In calculation of the maximum velocities a correction for the contamination of the mitochondrial fraction with the cytosol was made. A mean value of 11 * 1% of the total activity of the cytosolic marker enzyme, alcohol dehydrogenase, was found in the mitochondrial fraction. As only 5% of the total glutamate dehydrogenase was found in the cytosol, no correction was made for this. Maximum velocities for cytosolic and mitochondrial malate dehydrogenase are given in Table III. The ratio V,lV, for the mitochondrial enzyme was consistent with that found in the kinetic analysis. For the cytosolic en-

ET AL.

zyme the values for Vf and the ratio V,lV, are almost double the values we have previously published (9). This change is likely to be a result of using an outbred strain of rats. For the present data the sum of the maximum velocities for the mitochondrial and cytosolic dehydrogenases accounts for close to 100% of the total activity found in the liver. Calculation of Rate of Malate Oxidation by Malate Dehydrogenase in Mitochondria in Vivo The rate of production of NADH in the cytosol of rat liver during oxidation of ethanol is approximately 4 pmol/ min per gram of liver, wet weight (23). We have previously shown that the maximum velocity of cytosolic malate dehydrogenase for NADH oxidation greatly exceeds this rate and that the rate of the enzyme in viva is determined by the cytosolic [NADH] (9). The data in Table III show that the maximum velocity for NAD+ reduction by mitochondrial malate dehydrogenase under conditions similar to those in uivo is approximately lo-fold greater than the required flux through the enzyme. Rates of malate oxidation by mitochondrial malate dehydrogenase were calculated using the rate equation for an ordered Bi-Bi system (24). Values were calculated using the kinetic parameters shown in Table II, using the average values where appropriate. The effects of varying the concentrations of NADH and oxalacetate at different fixed malate concentrations are shown in Table IV. In the absence of alcohol the total [NAD+] in rat liver mitochondria is 4 to 6 mM (25) and only a small proportion of this is likely to be bound. Therefore we have used a fixed [NAD+] of 5.0 mM for calculation of the rate of mitochondrial malate dehydrogenase in uiuo. As the Michaelis constant for NAD+ (K,)

TABLE Maximum

Velocities

III

for Rat Liver

Malate

Dehydrogenase in Viuo

Cytosolic enzyme Maximum velocity for oxalacetate reduction ( V,) Maximum velocity for malate oxidation (V,) VflV,

1130

Mitochondrial enzyme

f 60"

57 f 19.8 f

2 0.8

380

k40'

39 f 9.8 f

3 0.6

Recovery (% of total liver activity)

98 + 2

101 + 3

Note. Maximum velocities are in units of Fmol/min per g liver, wet wt, and were determined as described in the text. Data represent the mean f SEM for 10 individual rat livers. a Corrected for lack of saturation with NADH using the equation (Ref. using the value for CW, P. 567) V,,,., = V,,,,,apparent(l + KJ[NADH]), K, in the absence of product (Table II).

RAT TABLE

LIVER

MITOCHONDRIAL

IV

Oxalacetate, and Malate Concentrations on the Rate of Operation of Mitochondrial Malate Dehydrogenase

Effect

of NADH,

Malate (mM)

NADH (PM)

0.01

0.1

0.5

1.0

2.0

0.5 0.5 0.5 0.5 0.5

200 400 600 800 1000

2.1 1.2 0.83 0.64 0.51

1.88 0.95 0.57 0.36 0.24

0.91 ~0.16 -0.60 -0.83 -0.98

-0.27 -1.52 -2.04 -2.32 -2.49

-2.58 -4.19 -4.86 -5.22 -5.45

1.0 1.0 1.0 1.0 1.0

200 400 600 800 1000

3.88 2.26 1.59 1.23 1.00

3.67 2.03 1.35 0.97 0.73

2.77 0.99 0.24 -0.16 -0.42

1.66 -0.30 -1.11 -1.56 -1.85

-0.49 -2.81 ~3.78 -4.32 -4.65

2.0 2.0 2.0 2.0 2.0

200 400 600 800 1000

6.67 4.02 2.87 2.23 1.82

6.49 3.81 2.65 2.00 1.59

5.69 2.88 1.66 0.97 0.54

4.71 1.73 0.43 -0.29 -0.76

2.81 -0.51 -1.96 -2.78 -3.30

4.0 4.0 4.0 4.0 4.0

200 400 600 800 1000

10.41 6.53 4.75 3.73 3.07

10.26 6.35 4.56 3.54 2.87

9.61 5.58 3.73 2.67 1.99

8.81 4.63 2.71 1.61 0.90

7.24 2.77 0.71 -0.48 -1.25

Oxalacetate(PM)

Note. Rates were calculated using the rate equation for an ordered Bi-Bi system (24). The kinetic constants used in the calculations were the values in Table II; average values were used where appropriate. The value for K,, obtained in the absence of products was used. The concentration of NAD+ was constant (5 mM). Rates are in units of amol NADH produced per minute per gram liver, wet wt.

of the enzyme is less than 0.2 mM (Table II) the enzyme is unlikely to be very sensitive to small changes in [NAD+] within the physiological range given above. The total [NADH] in the mitochondria is about 600 PM (25). Ethanol oxidation is accompanied by a decrease in the mitochondrial free [NAD+]/[NADH] ratio from about 15 to 8.5 (2). The free NADH is therefore likely to be in the range 300-600 PM and the NADH concentrations used in Table IV cover the likely physiological range. The data in Table IV show that, in order for the mitochondrial enzyme to operate during ethanol oxidation at a rate sufficient to balance the flux through the cytosolic enzyme (approximately 4 pmol NADH produced per minute per gram liver), the free mitochondrial [oxalacetate] must be very low (600 PM) reasonable rates of malate oxidation by the mitochondrial enzyme are achieved

MALATE

DEHYDROGENASE

195

only at high concentrations of malate. In the absence of alcohol the mitochondrial [malate] in rat liver is within the range 0.5 to 3.7 mM (26). Following administration of alcohol to rats there has been shown to be a nearly twofold increase in the mitochondrial [malate] (27). This suggests that during alcohol oxidation the [malate] could be as high as 4 mM. Our data show that mitochondrial malate dehydrogenase is sensitive to changes in [malate] within the physiological concentration range. The mitochondrial [malate] is likely to influence the activity of the enzyme in a manner similar to that previously postulated for the effect of the free cytosolic [NADH] on the cytosolic malate dehydrogenase. As the mitochondrial [malate] increases, as a result of an increase in the rate of production in the cytosol and transport across the mitochondrial membrane, the activity of mitochondrial malate dehydrogenase will increase. When the rate of malate oxidation in the mitochondria equals the rate of malate transport into the mitochondria a higher steady-state [malate] will be reached. If the rate of transport of malate into the mitochondria is equal to the rate of malate production in the cytosol, then this mechanism allows the rates of the cytosolic and mitochondrial malate dehydrogenases to be closely linked. Both the enzymes will respond to the increased requirement for reoxidation of NADH during alcohol oxidation. Ultimately NADH must be reoxidized by the mitochondrial electron transport chain. The first enzyme of this system, NADH dehydrogenase, may respond to changes in the concentration of mitochondrial free [NADH]. As [NADH] increases, due to an increase in the production of NADH in the mitochondria by enzymes such as mitochondrial malate dehydrogenase, the activity of NADH dehydrogenase may also increase until the rate of removal of NADH equals the rate of production of NADH. The steady-state mitochondrial [NADH] may thus be dependent on the balance between the activities of NADH dehydrogenase and the NADH-producing dehydrogenases. This would explain the increased steadystate [NADH] seen in the mitochondria during alcohol oxidation. The reoxidation of cytosolic NADH, produced during ethanol oxidation, by the malate-aspartate shuttle and ultimately the electron transport chain, has been suggested to be the rate-limiting step for the overall metabolism of alcohol (28). We do not believe that this system plays the only rate-determining role in alcohol metabolism because the first enzyme in the pathway, alcohol dehydrogenase, is a major rate-determining enzyme (1). However, NADH reoxidation may share control with other enzymes in the overall pathway. The degree of control exerted by individual reactions of the malate-aspartate shuttle and the electron transport chain remains to be determined. The cytosolic and mitochondrial malate dehydrogenases are unlikely to have a major role in regu-

196

WISEMAN

lation of flux, since their maximal activities are much greater than the flux through the pathway. They are, however, important in regulation of the concentrations of NADH (in the cytosol) and malate (in the mitochondria) during ethanol metabolism. REFERENCES 1. Crow, K. E., and Hardman, M. J. (1989) in Human Metabolism of Alcohol (Crow, K. E., and Batt, R. D., Eds.), Vol. 2, pp. 3-16, CRC Press, Boca Raton, Florida. 2. Veech, R. L., Guynn, R., and Veloso, D. (1972) Biochem. J. 127, 387-397. 3. Fellenius, E., Bjorkroth, U., and Kiessling, K.-H. (1973) Actu Chem. Scand. 27,2361-2366. 4. Posti, A. R., and Forsander, 0. A. (1976) Acta Chem. &and. Ser. B. 30,801-806. 5. Crow, K. E., Braggins, T. J., Batt, R. D., and Hardman, M. J. (1983) Pharmacol. Biochem. Behau. 18(Suppl. l), 233-236. 6. Dawson, A. G. (1979) Trends Biochem. Sci. 4, 171-176. 7. Nordmann, R., Petit, M.-A., and Nordmann, J. (1975) Biochem. Pharmacol. 24, 139-143. 8. Palmieri, F., Prezioso, G., Quagliariello, E., and Klingenberg, M. (1971) Eur. J. Biochem. 22,66-74. 9. Crow, K. E., Braggins, T. J., Batt, R. D., and Hardman, J. Biol. Chem. 257, 14,217-14,225. 10. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254.

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