Purification of bacterial malate dehydrogenases by selective elution from a triazinyl dye affinity column

Biochimica etBiophysicaActa, 708 (1982) 17-25

17

Elsevier BiomedicalPress BBA31311

PURIFICATION OF BACTERIAL MALATE D E H Y D R O G E N A S E S BY SELECTIVE E L U T I O N F R O M A TRIAZINYL DYE AFFINITY C O L U M N K. SMITHa, T.K. SUNDARAM a,, M. KERNICK a and A.E. WILKINSON b '~ Department of Biochemistry, University of Manchester Insitute of Science and Technology, Manchester M60 1QD and b Deparment of Biochemistry, University of Manchester, Manchester M13 9PL (UK)

(Received May 21st, 1982)

Key words: Ternary complex," Affinity chromatography," Malate dehydrogenase

A technique of selective elution from affinity adsorbent columns has been devised for the purification of malate dehydrogenase (L-malate:NAD ÷ oxidoreductase, EC 1.1.1.37) from a number of mesophilic and thermophilic bacteria. It is dependent on the combined action of both NAD ÷ and L-malate, the two reactants in the malate dehydrogenase system. These two reactants individually or N A D H or oxaloacetate alone at its equilibrium concentration in the malate dehydrogenase system cannot effect elution of the enzyme. Evidence is presented suggesting that malate dehydrogenase is eluted as a ternary complex with NAD ÷ and L-malate. Using agarose-linked Procion Red HE3B as the affinity adsorbent, the purification method consists of a single step for some malate dehydrogenase species and two easy steps for the other species. It is rapid and efficient, producing malate dehydrogenase homogeneous by several criteria in good yield.

Introduction Affinity chromatography is one of the most potent techniques commonly used at present for the isolation of proteins. Our interest in malate dehydrogenase (EC 1.1.1.37) in thermophilic bacteria [1] led to the development of simple purification methods for this enzyme, the key step being affinity chromatography on 5'-AMP-Sepharose or NAD*-hexane-agarose involving elution of the enzyme with N A D H [2]. These affinity adsorbents are relatively expensive and the eluant, N A D H , is unlikely to exhibit a high degree of specificity for malate dehydrogenase because it is the coenzyme for a number of dehydrogenases. We have therefore inproved our earlier procedure by using a triazinyl dye linked to agarose [3], an affinity adsorbent appreciably less expensive and easily regenerated for repeated use, and by increas* To whom correspondenceshould be addressed 0167-4838/82/0000-0000/$02.75 © 1982 ElsevierBiomedicalPress

ing the specificity of elution of malate dehydrogenase from the affinity column by making the elution dependent on the presence of both the nucleotide coenzyme and L-malate, the two substrates in the malate dehydrogenase reaction. The principle of such specific elution has been invoked in a few systems. Elution of lactate dehydrogenase and alcohol dehydrogenase as ternary complexes has been achieved from affinity columns with N A D + and either pyruvate or hydroxylamine, respectively [4]. Mammalian malate dehydrogenase has been eluted by a similar principle with an adduct synthesized from N A D H and oxaloacetate [5]. To our knowledge elution of any malate dehydrogenase from affinity columns in a high state of purity with N A D + or N A D H and L-malate has not been reported before. The common practice in malate dehydrogenase purification by affinity chromatography has been to release the enzyme with relatively non-specific eluants such as N A D H or salt. We present here rapid, efficient purifica-

18 tion methods using specific elution by a mixture of N A D + and L-malate for a number of bacterial (mesophilic and thermophilic) malate dehydrogenases. Materials and Methods

Materials The sources were as indicated: oxaloacetic acid from Calbiochem-Behring Corp.; N A D and N A D H from Boehringer Corporation (London) Ltd.; L- and D-malic acid and detergent Brij 58 from Sigma London Chemical Company Ltd.; M~trex Gel Red A, an affinity adsorbent in which the triazinyl dye, Procion Red HE3B, is linked to an agarose matrix, from Amicon Ltd.; acrylamide, N,N'-methylenebisacrylamide and N,N,N',N'-tetraethylenediamine from Bio-Rad Laboratories Ltd.; bovine serum albumin from Armour. Other chemicals were purchased from various commercial sources at the highest purity available. Growth of bacteria The bacteria used for enzyme purification were the following: the mesophiles, Escherichia co#, a K12 derivative requiring Lhistidine and nicotinic acid for growth [6], and Bacillus subtilis Marburg; the moderate thermophile, designated BI, which is a prototrophic Bacillus [2]; the extreme thermophiles, Bacillus caldotenax and Thermus aquaticus B. B. subtilis and BI were grown at 37°C and 55°C, respectively, in salts media [2] containing glucose or sucrose as carbon source and E. coli was grown at 37°C in salts medium [6] containing glucose, Lhistidine and nicotinic acid. B. caldotenax and T. aquaticus were grown at the Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K. and supplied to us as frozen cell pastes. Preparation of cell-free extracts. Cells were lysed by an adaptation of the method of Godson and Sinsheimer [7]. Cell suspensions in 40 mM Tris-HC1 buffer, pH 7.2 (pH 8 for E. coli) containing sucrose (25% w / v ) and 5 mM ethylenediaminetetraacetate (EDTA) were incubated with lysozyme (250 /~g/ml) at room temperature or at 30°C (B. caldotenax) for 10-15 min. An aqueous solution (5% w / v ) of the neutral detergent Brij 58 and MgSO 4 were added to final concentrations of 0.5% and 20 mM, respectively, and the mixture was

incubated for a further 10-15 min at room temperature or 30°C and then briefly homogenized with a tissue homogenizer. Centrifugation of this homogenate at 3 0 0 0 0 × g and 4°C for 30 min yielded a clear extract with most of the D N A in the sediment, which was discarded. Protein and malate dehydrogenase assays. For accurate determination of protein a microbiuret method [8] was used. When quick results were desired, e.g., in the monitoring of column fractions, the somewhat less specific Coomassie brilliant bleu G-250 binding assay [9] was preferred. Bovine serum albumin was the standard in both methods. Malate dehydrogenase was assayed at 30°C in a system which also contained 60 mM sodium/potassium phosphate (pH 7.5), 0.14 mM N A D H and 0.3 m M oxaloacetic acid; the rate of decrease in absorbance at 340 nm due to the addition of oxaloacetate was measured in a recording spectrophotometer. One unit of malate dehydrogenase activity is defined as catalyzing the oxidation of 1 /~mol N A D H / m i n at 30°C. Polyacrylamide gel electrophoresis. Native enzymes were electrophoresed at p H 9.2 or 7.5. The B. subtilis malate dehydrogenase was electrophoresed at p H 7.5 because of its instability at pH higher than 8 [10]. The system at p H 9.2 was a discontinuous one and based on that of Davis and Ornstein [11]. The system at p H 7.5 was a continuous one described by Garnak and Reeves [12]. The gels in both systems were cast in cylindrical tubes (0.5 × 8.5 cm). After electrophoresis at 2 m A / g e l the gels were stained for protein with Coomassie brilliant blue or for malate dehydrogenase activity [2]. Denatured proteins were electrophoresed in polyacrylamide gel containing 0.2% SDS, cast in slab form (0.15 × 35 × 150 cm), at 25 mA and the gel was stained for protein with Coomassie brilliant blue. Denatured proteins were prepared for electrophoresis as described previously [2]. Densitometric scanning of gels was done with a Beckman DU-8 computing spectrophotometer fitted with a gel scanning system. The instrument was programmed to determine the relative amounts of the proteins separated in a gel. Sedimentation equilibrium centrifugation. The centrifugation was carried out in a Beckman Model E ultracentrifuge essentially as described previously [1]. Photographs of the interference patterns

19 were analyzed to assess the dispersity of the purified enzyme preparation. Determination of equilibrium constant for the malate dehydrogenase reaction and the equilibrium concentration of NADH. To a reactiom mixture containing 1 mM N A D + and 10 mM L-malate in 10 mM sodium phosphate potassium phosphate buffer, pH 7.2, purified BI malate dehydrogenase was added and the reaction allowed to reach equilibrium at various temperatures. The concentration of N A D H (and of oxaloacetate) at equilibrium was obtained from the increment in absorbance at 340 nm measured with a Beckman DU-8 computing spectrophotometer. From these data the equilibrium constants were calculated. A plot of log equilibrium constant against the reciprocal of absolute temperature was linear, as expected, in the temperature range 4-35°C. The concentration of N A D H at equilibrium attained at 4°C from initial concentrations of N A D + and L-malate of 0.15 mM and 10 mM, respectively, (representing conditions of elution of BI malate dehydrogenase from the Gel Red A column during the purification) was deduced from the equilibrium constant at 4°C. This method was adopted becasuse the absorbance at 340 nm due to the equilibrium concentration of N A D H under these conditions (1.35 /~M) is too low to be measured accurately. Purification of malate dehydrogenase. The general strategy was as follows. An extract of bacterial cells, prepared as described above and containing approx. 2 g protein, was applied to a column of M~trex Gel Red A (2 × 5 cm; 15 ml bed volume) equilibrated in the appropriate buffer. The column was exhaustively washed with a series of buffers until the protein in the effluent was reduced to zero or a constant, low level. The enzyme was then eluted with buffer mixture containing appropriate concentrations of NAD + and L-malate. All the malate dehydrogenase preparations, with the exception of the B. caldotenax and E. coli preparations, appeared to be virtually homogeneous at this stage. The B. caldotenax and E. coli preparations were fractionated on a second, small column of Gel Red A (1 × 2.5 cm; 2 ml bed volume); after being washed with suitable buffers, the enzyme was eluted with a buffer mixture containing NAD ÷ and L-malate. The flow rate through the larger column was approx. 150 m l / h and through the

smaller column approx. 70 ml/h. Further details for the purification of the individual malate dehydrogenases are given below. All buffers contained 1 mM EDTA, and the purification was carried out at about 4°C. B. subtilis malate dehydrogenase. The Gel Red A column was equilibrated with 10 mM sodium phosphate potassium phosphate buffer, pH 7.2. After the passage of the cell-free extract, the column was washed successively with (i) phosphate buffer containing 20 mM KC1 (about 3 litres), (ii) phosphate buffer containing 10 mM L-malate (150 ml), (iii) phosphate buffer containing 20 mM KC1 (100 ml) and (iv) phosphate buffer containing 20 mM KC1 and 0.15 mM N A D ÷ (400 ml). The malate dehydrogenase was then eluted with phosphate buffer containing 0.15 mM N A D ÷ and 10 mM L-malate. the active fractions (about 40 ml) were pooled and the protein was precipitated with ammonium sulphate added to 90% saturation. BI malate dehydrogenase. The details were essentially the same as for the B. subtilis enzyme. The volumes of the four buffers used to wash the columns were 3 litres, 200 ml, 50 ml and 300 ml, respectively. The malate dehydrogenase was eluted with phosphate buffer containing 0.15 mM N A D ÷ and 10 mM g-malate and concentrated by precipitation with ammonium sulphate. T. aquaticus malate dehydrogenase. The Gel Red A column was equilibrated with 10 mM phosphate buffer, pH 7.2, containing 1 mM 2-mercaptoethanol. The cell-free extract was diluted with 10 mM Tris-HC1 buffer, pH 7.2, containing 1 mM 2-mercaptoethanol, to a protein concentration of 6 m g / m l and applied to the Gel Red A column. The dilution of the extract facillitated the binding of all the malate dehydrogenase to the column; with a more concentrated extract an appreciable amount of the enzyme appeared in the effluent. The column was washed with phosphate buffer containing 20 mM KC1 (3 litres), phosphate buffer containing 10 mM L-malate (150 ml), phosphate buffer containing 20 mM KC1 (50 ml) and phosphate buffer containing 20 mM KCI and 0.2 mM NAD + (300 ml). The malate dehydrogenase was eluted with buffer containing 0.2 mM N A D + and 10 mM L-malate and concentrated by precipitation with ammonium sulphate. B. caldotenax malate dehydrogenase. Approxi-

20

mately half of the cell-free extract (11.5 mg protein/ml) was first applied to the Gel Red A column. The other half was diluted two-fold with 10 mM Tris-HC1, pH 7.2, before being passed through the column. This resulted in the retention on the column of all the enzyme in the extract. The column was washed sequentially with 10 mM phosphate buffer, pH 7.2, containing 20 mM KC1 (2-3 litres), buffer containing 10 mM L-malate (200 ml), buffer containing 20 mM KC1 (50 ml) and buffer containing 20 mM KC1 and 0.07 mM NAD + (400 ml). The malate dehydrogenase was eluted with buffer containing 0.07 mM NAD + and 10 mM L-malate. The active fractions were pooled and dialyzed against 10 mM sodium phosphate/potassium phosphate buffer, pH 7.2, overnight. The dialyzed sample was passed through a second column of Gel Red A (2 ml) equilibrated with the phosphate buffer. The column was washed successively with phosphate buffer containing 20 mM KCI (20 ml) and phosphate buffer containing 20 mM KCI and 0.07 mM NAD +. The malate dehydrogenase was eluted with buffer containing 0.07 mM NAD + and 10 mM L-malate and concentrated by precipitation with ammonium sulphate.

E. coli malate dehydrogenase. The cell-free extract (10.8 mg protein/ml) was adjusted to pH 7.2 with 1 M Tris base. Half of the extract was passed through the Gel Red A column equilibrated with 10 mM phosphate buffer, pH 7.2, containing 1 mM 2-mercaptoethanol. The other half was diluted two-fold with 10 mM Tris-HC1, pH 7.2, containing 1 mM 2-mercaptoethanol and then passed through the column. A small amount of malate dehydrogenase activity appeared in the effluent. The column was washed with phosphate buffer containing 20 mM KC1 (3 litres), buffer containing 10 mM L-malate (200 ml), buffer containing 20 mM KC1 (100 ml) and buffer containing 20 mM KC1 and 0.35 mM NAD + (350 ml). The dehydrogenase was eluted with buffer containing 0.35 mM NAD + and 10 mM r-malate. The active fractions were pooled and dialyzed against 10 mM phosphate buffer, pH 7.2, containing 1 mM 2-mercaptoethanol, overnight. The dialyzed preparation was passed through a second Gel Red A column equilibrated with 10 mM phosphate buffer, pH 7.2, containing 1 mM 2mercaptoethanol. The column was washed with buffer containing 20 mM KC1 (20 ml) and with buffer containing 20 mM KC1 and 0.35 mM

TABLE 1 P U R I F I C A T I O N OF BACTERIAL MALATE D E H Y D R O G E N A S E S Step

B. subtilis Cell-free extract Gel Red A affinity BI Cell-free extract Gel Red A affinity B. caldotenax Cell-free extract Gel Red A affinity Gel Red A affinity T. aquaticus Cell-free extract Gel Red A affinity E. coli Cell-free extract Gel Red A affinity Gel Red A affinity

Protein (rag)

Enzyme activity (units)

Spec. act. (units/mg protein)

Yield

(%)

chromatography

1 801 12.3

7 605 3 952

4.2 322

100 52

chromatography

2064 3.4

1385 640

0.7 187

100 46

chromatography I chromatography II

1 922 2.8 1.7

405 192 138

0.2 68.7 83

100 48 34

chromatography

1 979 6.5

1 148 650

0.6 100.5

100 57

chromatography I chromatography II

1 168 3.2 2.1

11070 5 442 4030

9.5 1 720 1 920

100 49 37

21 N A D + . The enzyme was eluted with buffer containing 0.35 mM N A D + and 10 mM L-malate and concentrated by precipitation with ammonium sulphate.

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Purification of malate dehydrogenase The typical course of purification of malate dehydrogenase from five bacteria is presented in Table I. Although in the majority of organisms the enzyme is a dimeric molecule, in a significant number of bacteria it is tetrameric [1]. The five bacteria selected in this study represent both the dimeric (T. aquaticus and E. coli) and the tetrameric (Bacilli) types as well as the mesophilic, moderately thermophilic and extremly thermophilic classes of bacteria. The key step in the purification schedule is the selective elution of malate dehydrogenase by buffer containing both substrates in the catalysed reaction, NAD + and L-malate. Although the malate concentration (10 mM) was relatively high, it did not elute any enzyme by itself. The N A D + concentration in the eluting buffer was carefully adjusted for each malate dehydrogenase species so that the coenzyme on its own did not release any enzyme from the affinity column but together with L-malate (10 mM) eluted the dehydrogenase efficiently. The purity of the final enzyme preparations was monitored by electrophoresis of the proteins in the native and denatured forms in polyacrylamide. In each case the preparation was apparently homogenous, exhibiting a single major protein band in the native and denatured states (Fig. 1). The native protein band stained for malate dehydrogenase activity. Some of the gels were also scanned densitometrically; the result of one such scan is shown in Fig. 2. With the T. aquaticus native malate dehydrogenase preparation two minor bands, both possessing activity, were also seen as reported previously [2]. Two of the preparations, those form T. aquaticus and B. caldotenax, were also tested by the criterion of sedimentation equilibrium centrifugation. The results, presented in Fig. 3, are a further reflection of their high state purity. For the B. caldotenax and E. coli malate dehydrogenases a second (small) affinity column step was required to remove all detectable contaminants (TableI).

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The final yields were relatively high for all the enzyme species, ranging from 34 to 57% (Table I).

Elution behaviour of malate dehydrogenase The efficient elution of enzyme from the affinity column achieved in this investigation was dependent on the presence of both NAD + and L-

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Fig. 2. Densitometric scan of polyacrylamide gel after electrophoresis. Purified BI malate dehydrogenase was electrophoresed in the native state (see Fig. 1). The destained gel was scanned at 580 nm at slit width of 0.1 mm, scanning speed of 2 cm/min and chart speed of 2 cm/min. The peak corresponds to the malate dehydrogenaseprotein band in the gel. The purity of the enzyme was estimated to be at least 97% by the scanning spectrophotometer, which was programmed to determine the relative amounts of protein components separated in the gel.

malate. This elution step might therefore be expected to be specific for malate dehydrogenase. It is possible, however, that N A D H a n d oxaloacetate might be produced in this step from the N A D + a n d malate by the action of the malate dehydrogenase present at relatively high c o n c e n t r a t i o n on the column. It is not k n o w n for certain whether this happens, but if it did the elution might simply be caused by N A D H or oxaloacetate on its own a n d therefore not be as highly specific for malate dehydrogenase as initially expected. This p o i n t further explored in a study of the elution of BI malate dehydrogenase from a Red A column. BI dehydrogenase from a cell-free extract was adsorbed to a small c o l u m n of Gel Red A (1 × 2.5 cm). The equilibrium c o n c e n t r a t i o n of N A D H (and of oxaloacetate) u n d e r the conditions of the elution from the Red A c o l u m n during the purifi-

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r 2 (cm 2) Fig. 3. Sedimentation equilibrium centrifugation of malate dehydrogenases. Purified malate dehydrogenases from B. caldotenax MDH (protein concentration 1.8 mg/ml) (A) and Z aquaticus MDH (protein concentration 3.4 mg/ml) (B) in 0.1 M sodium phosphate potassium phosphate buffer, pH 7, were centrifuged at 8.6°C and a rotor speed of 8766 rpm. AQJ is the fringe rise across a constant interval Q, and r is the distance from the centre of rotation. The plots are the best-fit lines determined by the method of least-squares. The linearity of the plots indicates the monodisperse nature and hence the high state of purity of the malate dehydrogenase preparations.

cation ( N A D + a n d L-malate c o n c e n t r a t i o n s of 0.15 m M a n d 10 m M a n d 4°C) was calculated to be 1.35 ~ M from the equilibrium constant. It was observed that no enzyme was eluted from the Red A c o l u m n with 10 m M phosphate buffer (pH 7.2) c o n t a i n i n g 20 m M KCI (to simulate the electrolyte c o n c e n t r a t i o n due to the 10 m M L-malate in the elution buffer) a n d 1.4/tM N A D H , a n d even with 5 /LM N A D H there was only negligible elution.

23

Nor was any malate dehydrogenase eluted with buffer-KCl mixture containing 1.4 laM N A D H + 0.15 mM N A D +, 2 /~M oxaloacetate, 10 /~M oxaloacetate, 0.15 mM NAD + + 2 /~M oxaloacetare or 2/~M N A D H + 2/~M oxaloacetate. Phosphate buffer containing 1.4/~M N A D H + 10 mM D-malate effected a significant elution; when the concentration of N A D H was increased, the elution became extremely efficient. Efficient elution could also be achieved with a concentration of N A D H significantly higher than 1.4 /~M+ 10 mM Lmalate. Buffer containing 0.15 mM NAD + + 1 0 mM D-malate did not cause any elution. These observations suggested that, for the elution of BI malate dehydrogenase from Gel Red A in the purification method developed by us, N A D + or N A D H and L-malate are essential. In a further experiment the eluting capacity of 0.15 mM

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Fig. 4. Elution of BI malate dehydrogenase form M~trex Gel Red A with N A D + + L-malate and with N A D H + L-malate. BI malate hydrogenase (86 units) was adsorbed from a cell-free extract to each of two Gel Red A columns and the columns were washed with buffer as described in the text. One column was eluted with buffer containing 0.15 mM N A D + and 10 mM L-malate ( I ) and the other column was eluted with buffer containing 1.4 tLM N A D H and 10 mM L-malate (0). The experiment was carried out at about 4°C. Fractions (1 ml) were collected and assayed for malate dehydrogenase activity.

N A D + + 10 mM L-malate was compared with that of 1.4/~M N A D H (equilibrium concentration) + 10 mM L-malate. Two identical Red A columns were set up with malate dehydrogenase adsorbed from a cell-free extract of BI cells, the columns were washed as described for the purification of BI enzyme. One column was then eluted with phosphate buffer (pH 7.2) containing 0.15 mM NAD + + 10 mM L-malate and the other column was eluted with buffer containing 1.4/~M N A D H + 10 mM L-malate. The elution profiles observed are shown in Fig. 4. Clearly buffer containing N A D + + L-malate is a considerably more efficient eluant than that containing N A D H (1.4/~M) + L-malate.

Discussion The most likely explanation of the elution of malate dehydrogenase from the affinity column in our present study is that the enzyme forms a ternary complex with L-malate and NAD + . This would clearly be the case if during the elution the M D H did not catalyze the formation of any N A D H and oxaloacetate from NAD + and Lmalate. If, however, the enzyme did catalyze malate dehydrogenation on the column, other possibilities must be considered and the maximum concentrations of N A D H and oxaloacetate present in the system would be the equilibrium concentrations. These compounds individually were not able to elute the enzyme even at concentrations appreciably higher than the equilibrium concentration. In the mammalian malate dehydrogenase system the formation of the productive complexes, enzymeNAD+-L-malate and enzyme-NADH-oxaloacetate, and of the abortive complex, enzyme-NADHL-malate, has been postulated and supported by some experimental evidence [13]. If this situation might be extrapolated to the bacterial system, the enzyme could be eluted as any one of the three complexes. However, the results presented in Fig. 4 suggest strongly that the complex with N A D + and L-malate is involved in the elution rather than that with N A D H and L-malate. The observation that buffer containing N A D H (2/tM) and oxaloacetate (2/~M) did not elute the BI malate dehydrogenase cannot by itself rule out a function for the complex of the enzyme with these compounds in the elution because it might be argued that the nature

24 of the equilibrium in the malate dehydrogenase reaction would favour their extensive conversion to N A D ÷ and L-malate in the absence of any added N A D + or L-malate but that, when the elution is carried out with N A D ÷ and L-malate, N A D H and oxaloacetate would probably exist in the system at their equilibrium concentrations. However, in the light of the finding that N A D H (1.4 #M) in conjunction with 10 m M L-malate was not an efficient eluant (Fig. 4), it appears unlikely that N A D H (1.4 /~M) would release the enzyme efficiently aided by oxaloacetate (1.4 /zM). An involvement of the complex, enzyme-NAD +oxaloacetate, in elution may be discounted not only because there is no evidence for its formation in the mammalian system but also because of our observation that enzyme was not released with buffer containing 0.15 m M N A D ÷ and 2 ~M oxaloacetate. It is noteworthy too that no elution occurred with buffer containing 1.4 /~M N A D H and 0.15 m M N A D + ; this suggests that the second substrate (L-malate) is required for elution. Thus, as mentioned above, the overwhelming probability is that the enzyme is eluted as a complex with N A D + and L-malate in our purification protocol. The inability of L-malate to elute the enzyme on its own at the relatively high concentration of 10 m M is explainable by the observation in the mammalian system that this substrate binds to the enzyme only if the nucleotide cofactor is already bound to it [13]. It has been demonstrated with the malate dehydrogenase from Pseudomonas testosteroni [14] that D-malate acts as a noncompetitive inhibitor with respect to L-malate and as a competitive inhibitor with respect to oxaloacetate. Our finding that D-malate forms an effective eluant with N A D H but not with N A D + is consistent with this idea and confirms that elution of malate dehydrogenase entails the formation of a ternary complex in which the substrates or their analogues occupy the active sites. In recent years agarose-linked triazinyl dyes have come to the fore in enzyme isolation and they have proved superior to other affinity adsorbents such as agarose-linked 5'-AMP and agarose-linked N A D ÷ because, besides being less expensive, they can be regenerated for repeated use with strong reagents such as urea-NaOH, and any lipids con-

taminating the adsorbent can be removed with a c h l o r o f o r m / m e t h a n o l (1 : 1) mixture. This will be a great advantage in the large-scale isolation of malate dehydrogenase and other enzymes. One drawback inherent in the use of the triazinyl dye adsorbents is their ability to retain a wide range of enzymes which catalyze reactions involving nucleotides as substrates or cofactors and in some instances even other proteins. This can be largely overcome, however, by a selective elution procedure of the kind we have adopted for the purification of bacterial malate dehydrogenase. In this procedure elution of the enzyme is rapid with N A D + and L-malate both present at appropriate concentrations. These components individually or N A D H or oxaloacetate alone at its equilibrium concentration cannot cause a significant rate of elution. The purification schedule based on this principle is rapid and efficient, producing enzyme homogenous by several criteria in good yield and consisting of a single step for some species and of two easy steps for the other species (Table I). The whole purification operation takes only 36-48 h. This elution technique can also be used with other affinity adsorbents such as 5'-AMP-Sepharose. In earlier experiments we tested agarose-linked Cibacron Blue 3GA (marketed under the name of M~trex Gel Blue A by Amicon Corporation). For malate dehydrogenase purification Gel Red A, with its higher capacity for, and tighter binding of, the enzyme, is superior to Gel Blue A.

Acknowledgements We are grateful to the Royal Society, Great Britain for support through equipment grants. Cells of Bacillus caldotenax and Thermus aquaticus B were supplied to us by the Centre for Applied Microbiology and Research, Porton Down under a block grant scheme financed by the Science and Engineering Research Council and the Medical Research Council.

References 1Sundaram. T.K., Wright, I.P. and Wilkinson, A.E. (1980) Biochemistry 19, 2017-2022 2 Wright, I.P. and Sundararn, T.K. (1979) Biochem. J. 177, 441-448

25 3 Dye-Ligand Chromatography (1980) Amicon Corporation, Lexington, MA 4 0 h l s s o n , R., Brodelius, P. and Mosbach, K. (1972) FEBS Lett. 25, 234-241 5 Kaplan, N.O., Everse, J., Dixon, J.E., Stolzenbach, F.E., Lee, C.-Y., Lee, C.-L., Taylor, S.S. and Mosbach, K. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3450-3454 6 Dickinson, E.S. and Sundaram, T.K. (1970) J. Bacteriol. 101, 1090-1091 7 Godson, G.N and Sinsheimer, R.L. (1967) Biochim. Biophys. Acta 149, 476-488 8 Munkres, K.D. and Richards, R.M. (1965) Arch. Biochem. Biophys. 109, 466-479

9 Bio-Rad Protein Assay (1979) Bulletin 1069 EG. Bio-Rad Laboratories, Richmond, CA 10 Yoshida, A. (1965)J. Biol. Chem. 240, 1118-1124 11 Davis, B.J. and Ornstein, L. (1961) Disc Electrophoresis, Distillation Products Industries, Rochester, New York 12 Garnak, M. and Reeves, H.C. (1979) J. Biol. Chem. 254, 7915-7920 13 Banaszak, L.J. and Bradshaw, R.A. (1975) Enzymes, 3rd Edn. 11,369-396 14 You, K. and Kaplan, N.O. (1975) J. Bacteriol. 123, 704-716