Eur. J . Biochem. 83, 189-196 (1978)

Purification and Characterization of Aldehyde Dehydrogepase from Bovine Liver Wolfgang LEICHT, Fritz HEINZ, and Barbara FREIMULLER Institut fur Klinische Biochemie und Physiologische Chemie, Medizinische Hochschule Hannover (Received January 25/0ctober 25, 1977)

Aldehyde dehydrogenase from bovine liver has been purified to homogeneity. Amino acid composition showed a high content of cysteine of 32 mol/mol enzyme. The enzyme is composed of four identical subunits as judged by sodium dodecyl sulfate gel electrophoresis and end-group analysis. The molecular weight was determined to be 220 000 k 10000 by sedimentation equilibrium analysis in an analytical ultracentrifuge. The Michaelis constants for NAD +,glyceraldehyde and acetaldehyde were found to be 47 pM, 170 pM and 130 pM, respectively.

Detailed studies on kinetic properties of partially purified aldehyde dehydrogenase from different sources have been communicated during the last two decades. Due to its stability, aldehyde dehydrogenase from yeast seemed to be easier to purify and so Steinman and Jakoby [3] reported the isolation of a potassiumdependent aldehyde dehydrogenase. This enzyme is now already commercially available. Mammalian aldehyde dehydrogenases differ not only in their coenzyme specificity but also in their characteristic instability. Feldman and Weiner [4] first described a homogeneous enzyme from horse liver. Eckfeldt et al. [ 5 ] reported the isolation and characterization of two isoenzymes from horse liver. In the present work the purification and characterization of aldehyde dehydrogenase from bovine liver is described. MATERIALS AND METHODS Materials

Ammonium sulfate, mercaptoethanol, phenazine methosulfate, iodine nitrotetrazoliumchloride, dansylated amino acids, Visking dialysis tubing and charPreliminary results of this work were presented at the Joint Meeting of the Biochemical Societies of Belgium, Netherlands and the Federal Republic of Germany [l], Diisseldorf, 1974. This study was done in partial fulfillment of a diploma thesis [2]. Enzymes. Alcohol dehydrogenase (EC 1.1 . l . l ) ; formaldehyde dehydrogenase (EC 1.2.1.l); aldehyde dehydrogenase (EC 1.2.1.3); glyceraldehyde-3-phosphatedehydrogenase (EC I .2.1.12); catalase (EC 1.11.1.6); carboxypeptidase A (EC 3.4.12.2); carboxypeptidase B (EC 3.4.12.3); aldolase (EC 4.1.2.13).

coal (Norit A) were from Serva (Heidelberg). A11 chemicals not further specified were of analytical grade and as well as carboxypeptidase A and B were purchased from Merck (Darmstadt). Ellman's reagent [5,5'-dithio-bis(2-nitrobenzoic acid)] and reference proteins for sodium dodecyl gel electrophoresis were obtained from Boehringer (Mannheim). DE-52 and CM-52 cellulose were from Whatman (Maidstone) and Biogel P 150 from Biorad (California). Dithiothreito1 was from Seikagaku Kogyo Co. Ltd (Tokyo). DLGlyceraldehyde was obtained from Aldrich Europe (Dusseldorf). L-Glyceraldehyde was from Fluka, Buchs (Switzerland) and D-glyceraldehyde from Roth (Karlsruhe, West Germany). Ampholine was purchased from LKB (Uppsala, Sweden). Glass capillaries for electrophoresis were from Drummond Scientific Co. (U.S.A.). Water was double distilled prior to use. Bovine liver was obtained fresh from the slaughter house and stored frozen at - 20 "C if not immediately used for experiments. Enzymatic Activity Assay

The rate of formation of NADH was followed spectrophotometrically at 366 nm in a photometer 'Eppendorf at 25 "C. The enzymatic assay contained the following components in a total volume of 2.3 ml. : 0.87 M sodium pyrophosphate, p H 8.5; 0.65 mM NAD' ; 9.65 mM DL-glyceraldehyde; 0.5 - 2 enzyme units. The reaction was started by the addition of the glyceraldehyde. An enzyme unjt (U) was defined as the amount converting 1 pmol substrate in 1 min under the assay conditions.

190

Aldehyde Dehydrogenase

Kinetic Measurements All Michaelis constants were determined in 0.1 M sodium pyrophosphate, pH 8.5 at 25 "C. For D,L and m-glyceraldehyde 0.04 U and for acetaldehyde 0.08 U/ml assay mixture was used. The concentrations of the aldehydes and of the coenzymes were determined enzymatically. The initial velocities were plotted double-reciprocally and the straight lines were fitted by eye. No correction for the hydration of the aldehyde substrates were made. Buffers Buffer A : 5 mM triethanolamine, 10 mM EDTA, 20 mM mercaptoethanol, pH 7.5. Buffer B: 3 mM triethanolamine, 200 mM mercaptoethanol, pH 8.0. Buffer C: 3 mM triethanolamine, 2 mM dithiothreitol, pH 8.0. Protein Determinution In the early stage of isolation the protein concentration was evaluated from the 260 nm and 280 nm absorption according to Layne [6]. The protein concentration of purified preparations was determined by using the absorption coefficient at 280 nm. Absorption C'o~ffirientDetermination A homogeneous enzyme solution (18 mgiml) was dialyzed exhaustively against 0.1 '%; ammonium carbonate, adjusted with carbon dioxide to pH 7. The absorbance of this solution was recorded and the absorption coefficient calculated from the dry weight of the enzyme. The latter was determined by drying 0.1 ml of the solution in vacuum at 110 "C over phosphorus pentoxide to constant weight and subtracting the dry weight of diffusate.

Gradient gel electrophoresis was performed in 5-pl capillaries as described by Neuhoff [8]. The run was terminated after about 30 min, when the current dropped from 50 - 60 pA to 10 - 20 pA/capillary at a constant voltage of 60 V. The gels were stained for protein using amido black [8]. Enzymatic activity in the gels was detected by exposure of the gels to a solution containing: 0.1 ml phenazine methosulfate (0.6 mgiml), 0.1 ml iodine nitrotetrazolium chloride (1.6 mgiml), 0.1 ml propionaldehyde (4 mg/ml) and 0.1 ml NAD' (10 mg/ml) in 3 mlO.1 M sodium pyrophosphate. Ultracentrijugation Sedimentation equilibrium and velocity runs were performed in a Beckman ultracentrifuge Model E equipped with an ultraviolet scanner. In sedimentation equilibrium runs were done at 6800, 8000. 9000, 10000 rev./min for 2 - 3 days at 4 "C with protein solutions at an initial concentration of 0.4 mglml. An equilibrium run was also performed at a higher concentration of protein (5.4 mgiml) at 8000 rev./min and using schlieren optics. All experiments were made in buffer C. A partial specific volume of 0.734 cm3/g, calculated from the amino acid composition according to Schachman [9], was used to calculate the molecular weight. Amino Acid Analysis Amino acid composition was determined according to Spackman et al. [lo], in an amino acid analyzer (Beckman). Samples were hydrolyzed at 110 ' C in toluene sulfonic acid in the presence of 0.2:~; tryptamine. Under these conditions tryptophan is protected [ll]. Cysteine was determined as cysteic acid after oxidation with performic acid [12]. End-Group Determination

Charcoal Treatment Charcoal was washed with 0.1 M EDTA solution, and then extensively washed with water and finally air-dried at 90 ' C before use. 500 mg of the charcoal was added to 3 - 4 ml of protein solution (7.5 mgiml). The solution was stirred for 5 min and centrifuged at 300 x g . The clear enzyme solution was withdrawn with a syringe, Gel ElectrophoreJis

Sodium dodecyl sulfate electrophoresis was carried out in tubes according to Weber and Osborn [7]. The relative mobility of the marker proteins and ofaldehyde dehydrogenase was determined in triplicate. The proteins were stained with Coomassie brilliant blue.

The N-terminal amino acids were determined by dansylation according to Woods and Wang [I 31, The dansylated amino acids were identified by twodimensional thin-layer chromatography on polyamide plates 3 x 3 cm (Schleicher and Schull). The plates were developed in the first dimension with formic acidlwater (3/250) and in the second dimension with toluene/acetic acid (9/l). To distinguish between dansylalanine and dansylamine a second run in the second dimension using the solvent system ethyl acetate/methanol/acetic acid (3/1/1) was necessary. In all cases, dansylated amino acids were used as reference substances. The carboxyterminal sequence was determined by enzymatic hydrolysis with carboxypeptidase A and B according to Sajgo and Devenyi [14]. The hydrolyzed

191

W. Leicht, F. Heinz, and B. Freimuller

amino acids were identified on ion-exchange thinlayer chromatography plates (Ionex 25, Macherey and Nagel) in 0.4 M citric buffer, pH 3.3, at 55 "C. Ninhydrin-positive spots were identified by comparison with reference amino acids.

ethanol was added. The temperature of the suspension was between -5 "C and - 8 "C. The mixture was centrifuged and the precipitate serially extracted three times with buffer B. The extracts were combined for further purification.

Determination of Sulfhydryl Groups with Ellman's Reagent

CM-Cellulose Treatment

Samples containing about 0.01 ' pmol sulfhydryl groups were assayed with 5,5'-dithio-bis(2-nitrobenzoic acid) according to Ellman [15]. Blanks consisting of diffusate alone were used. The change in absorbance at 412 nm was monitored in a differential spectrophotometer (Leitz Unicam SP 800). The absorption coefficient of the thiophenolate was determined with cysteine as reference. Isoelectric Focusing Isoelectric focusing in an Ampholine gradient (pH 3-10) was done in a 110-ml LKB apparatus according to the LKB instruction manual. Water was substituted by a 1% mercaptoethanol solution to suppress inactivation of the enzyme. Amounts of 10-20 mg protein (in buffer C) were used for the experiments. Isoelectric focusing was also performed in 5 % polyacrylamide gels in 5-pl capillaries according to the method of Neuhoff [8]. After 50 min the current dropped from 100 pA to less than 10 pA and the run was terminated. Protein staining was achieved using amido black after dialysis of the gel against water. Enzymatic activity was detected in the gels as described above. Purijication Procedure The entire purification procedure was carried out at 4 "C.

The extract was diluted by addition of 0.5 volume distilled water. To evaluate the optimal amount of CM-cellulose for the batch treatment the following procedure was used. An aliquot of the enzyme solution (50 ml) was adjusted with acetic acid to pH 5 and increasing amounts of CM-cellulose (equilibrated with 50 mM acetate buffer, pH 5 ) , starting from 1 g, were added. After stirring for 5 min the solution was filtered on a buchner funnel and the specific enzyme activity in the filtrate determined. The amount of CM-cellulose giving optimum yield and specific activity was scaled up for removing contaminating material from the bulk preparation. The material after this purification step was immediately adjusted to pH 8 with ammonium hydroxide solution. Fractionation by Ammonium Sulfate Precipitation Ammonium sulfate was added to the material after CM-cellulose treatment to 45 % saturation. The precipitate formed was removed by centrifugation (15OOOxg; 15 min) and the supernatant brought to 60 % saturation. The precipitate formed was collected anddissolved in buffer B and dialyzed overnight to remove contaminating ammonium sulfate. Ion-Exchange Chromatography on DEAE-Cellulose Enzyme solution was applied to a DEAE-cellulose column (2 x 32 cm), washed with buffer B and eluted by a sodium chloride concentration gradient in the presence of buffer B. The enzymatically active fractions were pooled and concentrated by ammonium sulfate precipitation (60% saturation).

Preparation of Cell-Free Extracl Bovine liver was homogenized in a Starmix blendor with three parts (v/w) of buffer A. After centrifugation (10 min; 15000 x g) the supernatant was filtered over quartz wool. Fractionation by Alcohol Precipitation To 1 volume of the supernatant, 0.7 volume of cold ethanol ( - 20 "C) was slowly added. The mixture was stirred and the temperature maintained during the reaction between 0 and -3 "C using a cold finger (Colora, Germany). After centrifugation (10 min ; 15000 x g) the supernatant was collected and another 0.4 volume (relative to the cell-free extract) of cold

Molecular Sieve Chromatography The precipitated protein was dialyzed overnight against buffer C. Molecular sieve chromatography was carried out with Biogel P 150 (54 x 3 cm). The enzymatically active fractions were combined and examined for homogeneity. RESULTS Purijication The elution pattern of the ion-exchange chromatography is seen in Fig. 1. After some experience with the described procedure, homogenous preparation with

192

Aldehyde Dehydrogenase

f\

' I

' I

I

I

Fraction Rumher

Fig. 1 . Elution profile of aldehyde dehydrogenasefrom DEAE-cellulose. A linear sodium chloride concentration gradient (0 - I M in the presence of buffer B. total volume 1.5 I) was used to eluate the enzyme. Transmission at 280 nm (---~). sodium chloride concentration (0--O), enzymntic n ~ t i ~ i t(A y --A)

Table 1 Purifitntron of aldehyde dehydrogenase from bovine liver

Molecular Weight

Fraction

The molecular weight of the oligomer was determined by sedimentation equilibrium in in an analytical ultracentrifuge. By plotting the logarithm of the concentration over the squared distances from the rotor center a linear function was observed as shown for a typical run in Fig. 4. From 7 different experiments, a molecular weight of 220 000 & 10000 was determined. A sedimentation equilibrium run with an initial concentration of 5.4 mg/ml using schlieren optics gave a value of 215000. This indicates that the apparent molecular weight seems to be independent from the protein concentration in the range from 0.4 - 5.4 mg/ml of enzyme. The subunit molecular weight was determined by comparing the relative mobilities of reference proteins with a homogeneous aldehyde dehydrogenase in sodium dodecyl sulfate electrophoresis. As shown in Fig. 5 the relative mobility of the enzyme corresponds to a subunit molecular weight of 55 000.

1

1, Crude extract

2. Fractionated ethanol precipitation 3. CM-cellulose batch 4. Fractionated (NH,),SO, precipitation 5. DEAF,-cellulose chromatography

Specific Protein Purifiactivity cation

Recovery

Ujmg 0.006 0.06

mg 72000 13000

-fold 1 I0

100 33

0.095 0.50

7370 640

16 83

14 12

150

190

10

1.14

specific activities between 1.O and 1.4 U/mg could be achieved after this step. This is shown in Table 1 for a preparation starting with 1.5 kg liver. In our initial experiments an additional purification on a molecular sieve was necessary to obtain a homogenous enzyme preparation. In this case a drop in specific activity was noted. We attribute this loss of specific enzyme activity to the characteristic instability of this protein (see below). Criteriajor Homogeneity

Electrophoresis as shown in Fig.2 in microgradient gels indicated a single protein band containing all the enzyme activity. Isoelectric focusing in gels also shows only one band which contains the enzymatic activity (not shown). The results of sodium dodecyl sulfate gel electrophoresis are shown in Fig.3 and indicate a single band. In sedimentation velocity runs in the analytical ultracentrifuge only one single symmetrical peak was observed.

Amino Acid Composition

The results of the amino acid analyses are shown in Table 2. The high value of cysteine was confirmed by titration of sulfhydryl groups with Ellman's reagent. In 8 M urea 32 - 34 mol-SH groups/mol enzyme (for samples with a specific activity of 0.5 U/mg) were found. Alanine was the only N-terminal amino acid found. The carboxyterminal sequence was found to be Glx-Ala. Stability

The effect of different media on the stability of the enzyme is shown in Fig. 6. It may be seen that while the

193

W. Leicht, F. Heinz, and B. Freimiiller

0 200

-0200

" -c -0600

-1000

-140L

490

485

49 5

i 500

r 2 m2)

Fig 4 Sedimentation equilibrium data for aldehyde dehydrogenase The initial protein concentration was 0 4 mg/ml and the speed 8000 rev /min

i

Fig. 2. Electrophoresis of aldehyde dehydrogenase on polyacrylamide gradient gels in 5-pl capillaries. The picture shows 2-mm-long parts of the gradient gels. (A) stained for proteins, (B) stained for enzymatic activity. The polyacrylamide gradient ranges from 0 to 40% according to Neuhoff [8]

i

25

'(I

'

0

1

I

I

I

I

01

02

03

04

05

06

Relative rnobllity

Fig. 5 . Subunit molecular weight determination of aldehyde dehydrogenase. The markers were: (1) bovine serum albumin (68000); (2) catalase (60 000) ; ( 3 ) ovalbumin (43 000) ; (4) aldolase (40 000) ; (5) glyceraldehyde-3-phosphatedehydrogenase (36 000). The relative mobility of aldehyde dehydrogenase is indicated by the arrow

rate of enzyme inactivation could be influenced by different media, none of them was successful in entirely stabilizing the enzyme. Isoelectric Focusing

Fig. 3. Sodium dodecyl surfate electrophoresis of aldehyde dehydrogenase. The polyacrylamide concentration was 10% in the gel

After focusing the enzyme in the ampholine gradient, enzymatic activity, the absorbance at 280 nm and the pH were measured and the compiled data of such an experiment are shown in Fig.7. A p I of 5 is found for the isoelectric point of the enzyme. Microheterogeneity, which was observed in an initial experiment, could not be reproduced with highly purified enzyme samples. Additionally, isoelectric focusing in microgels showed also a single band which contained all enzymatic activity.

194

Aldehyde Dehydrogenase

Table 2 Amino crcicl anal! \ i of ~ allehvde dehydrogenare from bovine liver The recovery of protein was 94 2"/, for the mean value from two analyses per hydrolysis time

24 h 48 h 72 h Average hydrolysis hydrolysis hydrolysis

Amino acid

mol mol Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine Cysteine Ammonia

178.4 77.7 106.4 189.4 100.0 178.7 159.6 121.3 34.0 77.7 128.7 52.1 83.0 31.4 143.1 26.6 79.8

176.7 76.7 102.3 184.9 95.4 179.1 158.1 127.9 25.6 87.2 130.2 55.8 86.1 29.7 150.0 28.5 76.2

112.8

162.2

178.1 87.1 97.7 190.5 97.8 177.5 155.6 137.6 39.3 91 .O 138.2 57.3 92.1 32.0 148.9 27.5 75.3 -

~

177 79' 111' 188 98 178 157 137 39 91 138" 55" 87 31 149 27 77 32b

Fraction number

Fig. 7. Isoelectricfocusing of aldehyde drhydro~rtiosesin an cimpholine gradient. The focusing performed at 6 "C was terminated after 72 h at a constant voltage of 300 V and 2-ml fractions were collected. (0)Enzymatic activity in arbitrary units, ( x ) absorbance at 280 nm. (m)pH value

108.4

The highest \slues obtained were taken instead of the average. Determined as cysteic acid. Extrapolated to zero-time hydrolysis.

'

r

I

I

I

I

I

I

I

I

100

80

- 60

-

I

the proteins, was found to be 1.03. The ratio A,,,/A260 was 1.50 before charcoal treatment and 1.59 afterwards, indicating that the treatment removed some nucleotide material. This somewhat low ratio might be explained by a partial inactivation of homogeneous enzyme fractions, which could not be prevented by the method used.

x r >

-

Kinetic Constants

40

20

0 0

5

10

15

20

25

30

35

40

Time (days)

Fig. 6. Incrctirarion pirttrrn of uldehyde dehydrogenase ( I mglrnl) in diferenr hufcrs. All solutions were stored at 4 "C. ( m 4 ) Buffer B in presence of NAD' (10 mgiml); (0-0) buffer B with 304; glycerol: ( x x ) buffer B; (o--o) buffer B, 30%glycerol, 0.5 M sodium chloride. Enzyme solution which contained buffer B and 0.5 M sodium chloride lost its enzymatic activity within 3 h ~~~~

The Y, values for the substrates of aldehyde dehydrogenase were determined from a homogeneous enzyme preparation. For NAD' a value of 47 pM was found (Fig. 9) with both D- and i--glyceraldehyde (560 pM) as second substrates. The Michaelis constants for D-glyceraldehyde, L-glyceraldehyde and DL-glyceraldehyde showed identical values of 170 pM (Fig. 10). However, the maximum velocity of the mixed isomers is shown to be slightly faster. For acetaldehyde, a K, of 130 pM was found (Fig. 11). For the kinetic constants of the aldehyde a coenzyme concentration of 740 pM was kept constant.

Spectral Proprtit>,r

DISCUSSION

The enzyme solution shows a maximum of absorbance at 280 nm as shown in Fig. 8 and no absorbance in the visible spectral range. The absorption coefficient at 280 nm, determined by the dry weight of

The purification method described results in a homogeneous preparation of aldehyde dehydrogenase with a yield of 100 mg from 1 kg bovine liver. Different specific activities ranging from 0.7 to 1.4 Uimg for

W. Leicht, F. Heinz, and B. Freimuller

195

240 250 260 270 280 290 300 310 Wavelength (nm)

i

L

0 320 330 340

-10'

0

10

20

30

40

50

60

70

I/[S] (mM-')

Fig. 8. Ultraviolet spectrum of aldehyde dehydrogenase. The spectrum was recorded in a Cary double-beam spectrophotometer. The enzyme solution was dialyzed against 0.01 M ammonium carbonate buffer at pH 7

Fig. 10. Initial velocities of aldehyde-dehydrogenase-catalyzed oxidation of glyceraldehydes at high concentrations of NAD'. The initial velocities at different substrate concentrations [S] are plotted double-reciprocally. L-glyceraldehyde (0), D-glyceraldehyde (O), DL-glyceraldehyde (A)

i

i

A 70

80 11 [5]p r v ; ~ ' )

l/[S] (mM-') Fig. 9. Initial velocities of aldehyde-dehydrogenase-catalyzedreduction of NAD' at high concentrations ofglyceraldehydes. Initial velocities at different coenzyme concentrations [S] are plotted double-reciprocally. L-Glyceraldehyde (0)and D-glyceraldehyde).( served as substrates (560 pM)

Fig. 1 1. Initial velocities o f aldehyde-dehydrogenase-catalyzed oxidation of acetaldehyde at high concentrations of NAD' . Initial velocities at different substrate concentrations [S] are plotted doublereciprocally

preparations from different batches were obtained. However, the criteria for homogeneity were equally fulfilled for all preparations. Attempts to maintain enzyme activity were not successful. Preparations with different activities did not show different degrees of dissociation or conformational changes, as judged by the methods employed. Preliminary experiments, however, suggest that the loss of activity may be correlated with the loss of titratable sulfhydryl groups PI. The molecular weight of aldehyde dehydrogenase from bovine liver was found to be 220000+ 10000. This may be compared with the figure of other aldehyde dehydrogenases. Steiman and Jacoby [3] found a value

of 200000 D for the well-characterized enzyme from yeast. Aldehyde dehydrogenases from mammalian sources have also been investigated. Feldman and Weiner [4] found for the homogeneous enzyme from horse liver values between 220000 and 260000 depending on the methods used. With an improved preparation procedure Eckfeldt et al. [ S ] have isolated two isoenzymes from horse liver, with molecular weights of 230000 and 240000. For human aldehyde dehydrogenase Greenfield and Pietruszko [I 61 found two isoenzymes with molecular weights of 245 000 and 225 000. The aldehyde dehydrogenase was found to be composed of four identical subunits. This is based on

196

W. Leicht. F. Heinz, and B. Freimuller: Aldehyde Dehydrogenase

the observations that in dodecyl sulfate gel electrophoresis only one band is seen corresponding to a molecular weight of 55 000 and one N-terminal amino acid was found. A tetrameric structure has also been shown for other eucaryotic aldehyde dehydrogenases investigated [3 - 5,161. Our preparations did not indicate the presence of any isoenzymes. This is consistent with an earlier finding by Robbins [18]. Since Lamprecht and Heinz [I91 find no indication for isoenzymes in both the mitochondrial and cytoplasmic fractions, it would appear that the same enzyme is present in the different subcellular compartments. However, isoenzymes found from other mammalian sources have been reported. Marjanen 1201 found different aldehyde dehydrogenases in the cytoplasmic and mitochondrial fractions derived from rat liver In human liver two enzymes also have been described [21,16] although Kraemer and Deitrich [22] reported only one. Two enzymes from horse liver have also been isolated [S]. The kinetic properties of the isolated bovine liver enzyme are similar to the reported values for the horse liver enzyme isolated by Feldman and Weiner [4], which according to Eckfeldt and Yonetani [17] is mainly located in mitochondrial fractions. The only remarkable difference between this horse liver enzyme and that isolated from bovine by our method seems to be its higher Michaelis constant for acetaldehyde. The same discrepancy is seen by a comparison of one of the very recently characterized isoenzymes from human liver [16] This enzyme seems to be also strikingly similar to our enzyme preparation in respect to both subunit and oligomer molecular weight (54 200 and 225 000 as compared to SS 000 and 220000), 280 nm/260 nm ratios of absorbance (1.67 as compared to 1.59)and absorption coefficient (1 .OO as compared to 1.03) The amino acid analysis for the aldehyde dehydrogenase from bovine liver unexpectedly resembled that for the yeast enzyme [3] rather than either of the two horse liver isoenzymes [S]. However, all preparations show a high content of cysteine. In our study, we find 32 mol cysteine/mol enzyme in agreement with the figure calculated from the data given by Eckfeld et d. [5]. However. only four of these are involved in the catalytic sites. Also another enzyme, alcohol dehydrogenase, in the same pathway is characterized by a high cysteine content and also its enzyme activity is correlated with the number of free sulfhydryl groups [23,24].

The functional role for the large number of noncatalytic free thiol groups is not clear. It is possible that they have a regulatory role or are involved in multienzyme complex formation. There is an indication [25] that in situ the enzyme aldehyde dehydrogenase exists as a complex together with formaldehyde dehydrogenase and alcohol dehydrogenase. However, further studies are necessary to establish a functional role of free thiol groups in complex formations or regulatory properties. We thank Mrs Heide and Dr Riesner for performing the experiments on the analytical ultracentrifuge, Mrs Sylvia Reckel for the carefully performed amino acid analysis and Mr M. Vogel for determining the subunit molecular weight of the enzyme in sodium dodecyl sulfate electrophoresis.

REFERENCES 1. Leicht, W., Heinz, F., Freimuller, B. & Lamprecht, W. (1974) Hoppe-Seyler’s Z. Physiol. Chem. 355. (55) - (56). 2. Leicht, W. (1974), Diplomarbeit, Techn. Universitiit Hannover, Germany. 3. Steinman, G . R. & Jacoby, W. B. (1967) J . Biol. Chrm. 243, 730 - 734. 4. Feldman, R. I. & Weiner, H. (1972) J . Biol. Chem. 247, 260266. 5. Eckfeldt, J., Mope, L., Takio, K. & Yonetani, T. (1970) J . Biol. Chem. 251, 236-240. 6. Layne, E. (1957) Methods Enzymol. 3, 447-454. 7. Weber, K. &Osborn, M. (1969) J . Biol. Chrm. 244,4406-4412. 8, Neuhoff, V. (1973) Mol. Biol. Biochem. Biophys. 14. 1-83, 9. Schachman, K. (1957) Methods Enzymol. 4. 65-71. 10. Spackman, D. H., Stein, W. H. & Moore, S . (1958) Anal. Chem. 30, 1190 - 1206. 11. Lu, T. (1957) Methods Enzymol. 25, 44 - 55. 12. Moore, S. (1963) J . Biol. Chem. 238, 235-237. 13. Woods, K. R. & Wang, K . T. (1967) Biocl7im. Biophys. Acta, 133, 369 - 377. 14. Sajgo, M. & Devenyi, T. (1972) Acta Biochem. Biophys. Acad. Sci. Hung. 7, 233-236. 15. Ellman, C . (1959) Arch. Biochem. Biophys. 82, 70-81. 16. Greenfield, N. J. & Pietruszko, R. (1977) Bzochim. Biophys. Acta, 483, 35 -45. 17. Eckfeldt, J. & Yonetani, T. (1976) Arch, Biochem. Biophys. 175, 717-722. 18. Robbins, J. H. (1966) Arch. Biochrni. Biophvs. 214. 585 - 592. 19. Lamprecht, W. & Heinz, F. (1958) Z . Nrzturforsch. 13h. 464. 20, Marjanen, L. A. (1973) Biochim. Biophys. Actn. 327, 238-246. 21. Blair, A. €I. & Bodley, F. H. (1969) Cun. J . Bioc,hrnz.47. 265.272. 22. Kraemer, K. J. & Deitrich, R , A. (1968) J . B i d . Chem. 243, 6402 - 6408. 23. Jornvall, H. (1970) Eur. J . Biochrm. 16, 25-40. 24. Buhner, M. & Sund, H. (1969) Eur. J , Biochem. 11. 73 -79. 25. Goodman, J. I. & Tephly, T. R. (1971) Biochim. Biophys. Acta, 252,489 - 505.

W. Leicht, Polymer Department, Weizmann Institute of Science, P. 0. Box 26, Rehovot, Israel F. Heinz and B. Freimiiller, Institut fur Klinische Biochemie und Physiologische Chemie, Medizinische Hochschule Hannover, Karl-Wiechert-Allee 9, D-3000 Hannover-Kleefeld, Federal Republic of Germany