Alcohol Dehydrogenase from Cucumber Seeds

Biochem. Physiol. Pflanzen 171, S.1-6 (1977)

Alcohol Dehydrogenase from Cucumber Seeds SYLVA LEBLOVA. and EVA PERGLEROVA. Department of Biochemistry, Natural Science Faculty, Charles University, Prague, Czechoslovakia

Key Term Index: alcohol dehydrogenase, substrate specificity, kinetic parameters; Cucumis sativus.

Summary Alcohol dehydrogenase (EC 1.1.1.1) was isolated from cucumber (Cucumis sativus L.) seeds after 40 h germination by means of precipitation of the sodium phosphate extract with ammonium sulphate to 40 % saturation, desalting the active sediment on Sephadex G-25 column and chromatography on DEAE-cellulose. Specific activity of the enzyme was increased by this procedure to 25,500 units per mg protein. NAD serves as co-enzyme for cucumber ADH. The Michaelis-Menten constant for the co-enzyme is in the order of 10-4 M. Besides ethanol mainly propanol and butanol serve as substrates of this alcohol dehydrogenase with the oxidation rate diminishing with the increasing number of carbon atoms in the molecule, but allyl alcohol has an initial rate twice as fast as ethanol. The Km value for both ethanol and allyl alcohol is in the order of 10-2 M. The enzyme catalyzes both ethanol oxidation and acetaldehyde reduction. The Km for acetaldehyde is 0.6 . 10-2 M. Oximes and amides inhibit the enzyme, acetoxime competitively, whereas acetamide, butyrylamide and cyclohexanone oxime inhibit non-competitively with respect to ethanol. Cucumber ADH has a mol. wt. of 57,000 ± 5,000 D and contains sulfhydryl groups. The intermediates of sugar metabolism inhibit ethanol oxidation: Malate and acetate being the strongest, succinate and lactate medium, and isocitrate and pyruvate weak inhibitors.

Introduction

Whereas in animal cells lactate is formed from pyruvate during sugar metabolism under anaerobic conditions by means of the transfer of reducing equivalents to pyruvate, plants have in addition a pyruvate decarboxylase which converts pyruvate into in to acetaldehyde: CHaCO COOH -> CHaCOH

+ CO

2

NADH is generated during the catabolism of glucose after the Embden-Meyerhof schema where 3-phospho-D-glyceraldehyde is oxidized to 1.3-diphospho-D-glycerate, and reoxidized when hydrogen is transferred to acetaldehyde: CHaCOH

+ NADH ~ CHaCH 0H + NAD 2

The reaction is catalyzed by alcohol dehydrogenase (EC-1.1.1.1). In this paperwe deal with some properties of alcohol dehydrogenase present in germinating cucumber seeds. 1 Biochem. PhYBiol. Pflanzen, Bd. 171

2

S. LEBLOVA and E. PERGLEROVA

Material and Methods Cucumber (Cucumis sativus L., cv. MlIlnicka) seeds were germinated in Petri-dishes (19 cm in diameter) on filter paper in deionized water. 20 g of dry seeds were placed in one Petri-dish together with 50 ml of water. The seeds were germinated in a thermostat at 22°C.

of ethanol in plant tissue 0.5 g of tissue homogenate was weighed into dishes fused to the stoppers of WID MARK vessels (WIDMARK 1922). 2 ml of K 2 Cr2 0 7 solution in concentrated sulphuric acid (2.5 ml of K 2 Cr2 0 7 in 100 ml of acid) were pippeted into the vessels. Stoppered WID MARK vessels were incubated for 150 min at 60°C. After cooling the content were diluted with destilled water to 25 ml. Absorbance was determinated at 366 nm in glass cuvettes using a Specol photometer equiepped with an amplifier. Determination

Preparation of alcohol dehydrogenase

80 g of germinating seedil were homogenized in chilled mortars with 150 ml 0.1 M sodium phosphate buffer, pH 8.5 containing 0.01 M mercaptoethanol. The homogenate was filtered through two layers of cheese-cloth and centrifuged at 0 °C for 20 min at 10,000. g. The supernatant was then precipitated with ammonium sulphate and the sediment was resuspended in a small amount of 0.01 M Tris-acetate buffer, pH 6.4 containing 0.01 M mercaptoethanol. The resuspended sediment was desalted on a column of Sephadex G-25 equilibrated with 0.01 M TRIS-acetate buffer pH, 6.4 containing 0.01 M mercaptoethanol. 20 ml-samples containing ca. 100 mg of protein were applied to a DEAE-cellulose DE 32 column. The column was eluted with 1000 ml of 0.01 M Tris-acetate buffer, pH 6.4, with linearly rising concentration of Tris from 0.01 to 0.6 M. The whole operation was performed in a refrigerator.

of alcohol dehydrogenase activity ADH activity was measured as the increment of absborbance at 366 nm in a medium of 0.1 M sodium phosphate buffer, pH 8.5, with 0.01 M mercaptoethanol, 100 nM ethanol and 860,um NAD. An enzyme unit arbitrarily defined as that amount of enzyme which increases absorbance of 0.001 per min. Determination

Determination of protein content

Protein content was determinated by the method of Lowry et al. (1951).

of molecular weight by means of gel filtration Mol. wt. was determinated on a Sephadex G-200 column, 1.6 X 18 cm equilibrated with. 0.01 M Tris-acetate buffer, pH 6.4. The following protein standards were used: Myoglobin(mol.wt. 17,800 D), ovalbumin (45,000), hemoglobin (64,500), albumin (67,000) and gamma globulin (157,000). The molecular weight of the enzyme was established by determination V.ofthe enzyme and substituting It into the diagramatic representation of the relation V.-Iog mol. wt. which for Sepha,dex G-200 is linear from 10,000 to 180,000 D. Determination

Determination of sulfhydryl groups

ELLMAN reagent (5,5' -dithio bis -2-nitrobenzoic acid-DTNN) forms a yellow reaction production product with sulfyhdryl groups (SEDLAK and LINDSAY 1968). The following solutions were used: 0.01 M DTNB in absolute ethanol, 0.2 M Tris-HCl buffer, pH 8.2, with 0.02 M EDTA, 0.5 % dodecyl.sulphate solution.

l

e g

o

l. M

i. n-

M D. 1

3

Alcohol Dehydrogenase from Cucumber Seeds

Results and Discussion

The ethanol content in germinating cucumber seeds was relatively low in comparison with other germinating seeds. It seems that a higher ethanol content occurs in bigger seeds (broad bean, pea, kidney bean) than in smaller ones (cucumber, soybean), and that the ethanol content is not influenced by the type of reserve substances. Under the same conditions of germination (amouth of water, seeds etc.) broad bean seeds contain 1.2 mg of ethanol/g- 1 fresh wt., whereas germinating lentil seeds contain only 0.7 mg (Fig. 1). Cucumber alcohol dehydrogenase was isolated from the seeds germinated for 4,0 h. The results of the purification operations are presented in Table 1. The decrease in activity of the individual fractions with time obtained during the purfication process is given in Table 2. The decrease in activity of the fraction obtained after DEAE-cellulose chromatography is slower than in crude extract. The Km values were determined according to Lineweaver and Burk (DAWES 1965). Reaction rates were measured with ethanol, allyl alcohol and acetaldehyde in concentration ranges from 5 . 10-2 M to 1 . 10-3 M and with NAD between 1 . 10-4 M and 1 . 10-5 M . Km for ethanol was found to be 1.1 . 10-2 M, for allyl alcohol 0.9 . 10-2 M and for NAD 1.1.10- 4 M, respectively. Substrate specificity is not limited to ethanol. The initial rate of the oxidation of n-propanol is 50% with respect to ethanol, of n-butanol 26%, and of n-hexanol 20%, respectively. Heptanol is only slowly oxidized by cucumber ADH. 2-propen-1-o1 is a better substrate for the cucumber enzyme than ethanol: The initial rate is by 70% higher rhan that of ethanol. Other unsaturated alcohols do not possess this affinity to the enzyme. With 2-hepten-1-o1 the enzyme shows only 45% of the activity obtained

- - - pea

,..., ~ .c III

1.5

.,~

M D), he ng is

~

Cl

- - - - - maize _._._- soybean _ .. _ .. -

kidney bean

_ ... _ ...- lentil

oS

-_. __ .- broad bean

I- 1.0

............ cucumber

Z w

I-

Z

0

()

on d: yl-

8

16

24

32

40

48

56

64

72

TIME [hJ

Fig. 1. Ethanol content in germinating seeds or various plants during ike first 72 h of germination. 1*

4

S. LEBLOVA and E. PERGLEROVA

Table 1. Isolation steps for cucumber alcohol dehydrogenase. Fraction

Activity units

Protein mg

Spec. act. units mg-1

Multiple of spec. act.

Phosphate extract Ammonium sulphate fraction Desalted am. sulph. fraction Fraction after DEAE-cellulose

315,000 112,500 103,000 65,000

373 35 25.7 2.6

850 3,210 4,005 25,500

1 4 5 30

with ethanol, and with penten-1-o1 only 8 %. Cinnamyl alcohol and phenyl alcohol are oxidized only to a minute extent, whereas methanol, n-heptanol, isopropanol, isoamyl alcohol, isooctanol, lo3-butanediol, lo4-butanediol, cyclohexanol, glycerol, benzyl alcohol, 2-mercaptoethanol, 2-aminoethanol and terpenic alcohols are not oxidized at all. As far as substrate specificity is concerned, cucumber alcohol dehydrogenase resembles other plant alcohol dehydrogenases, i. e. those of pea, broad bean, lentil, kidney bean (ERIKSSON 1967, 1968, LEBLOVA and MANCAL 1975). T1~ble 2. Loss of activity of alcohol dehydrogenase isolated from cucumber seeds during storage at 0 to 4 °0 for 3 days. Rem~inder

of initial activity (%)

Fraction

Initial activity

1st day

2nd day

3rd day

N aPi extract Sulphate fraction Desalted sulphate fraction Fraction after DEAE-cellulose

100 100 100 100

95 55 85 100

70 12 61 95

34 0 20 80

The reaction of NADH is considered to be decisive for the velocity of ethanol oxidation by alcohol dehydrogenase. For this reason different cell substrates were assayed as possible hydrogen acceptors of the binary complex enzyme-coenzyme. Whereas with rat liver ADH some substrates such as succinate enhanced the rate of oxidation (ARSLANIAN et al. 1971) others, such as malate inhibited it completely, and some as lactate and acetate were without influence. In our experiments with cucumber ADH all compounds listed in Table 3 proved to be inhibitory for the reaction. If alone added these compounds were neither oxidized nor reduced by the enzyme. Amides and oximes which were considered as potential anti-alcoholic drugs in the treatment of alcoholism are efficient inhibitors of animal alcohol dehydrogenase (THEORELL 1965, LESTER and BENSON 1970, NACHMAN et al.1970). Plant alcohol dehydrogellase is also inhibited by the derivates of these compounds. Oximes are stronger inhibitors than amides (Table 4) and their effect on acetaldehyde reduction is stronger than their effect on ethanol oxidation. Acetamide, butyrylamide and cyclohexanone oxime are noncompetitive inhibitors whereas acetoxime is a competitive one. Susceptibility of plant alcohol dehydrogenase is much smaller than that of liver ADH. Also the Km for co-

Alcohol Dehydrogenase from Cucumber Seeds

5

l'able 3. Relative .velocity of the oxidation of 0.1 M etllanol by cucumber alcohol dellydrogenase in tile presence of substrates at 0.1 M concentration.

e yl yl at mey

to

xiyed ith

Substrate

Oxidation velocity

Ethanol Eth. + lactate Eth. + pyruvate Eth. + acetate Eth. + malate Eth. + succinate Eth. + isocitrate

100 51 89 35 25 47 81

enzyme and substrate are much higher in the case of plant ADH than in the case of animal ADH. The mol. wt. of cucumber ADH is 57,000 ± 5,000, its molecule has according to preliminary calculations 7.4 sulfhydryl groups. A mol. wt. of 60,000 was reported for pea alcohol dehydrogenase by COSSINS et al. (1968) and this value was also confirmed in our experiments (LEBLOVA and MANCAL 1971). On the contrary, tea alcohol dehydrogenase (HATANAKA 1972, HATANAKA and HARADA 1972) shows a mol. wt. nearly twice as high. Table 4. Influence of amides and oximes on etllanol oxidation and acetaldehyde reduction by cucumber alcohol dellydrogenase. The numbers express % of inhibition. Inhibitor 1,10-2 M

Oxidation of 1,10-2 M ethanol

Reduction of 5 '10- 2 M acetaldehyde

Acetamide Butyrylamide Acetoxime Cyclohexanone oxime

4.5 4.5 28.4 15.0

13.7 31.0 67.5 71.5

RS-

ate mese

he Eoase han ect onant co-

As far as we know alcohol dehydrogenase from cucumber has not yet been isolated and characterized. We obtained a fairly active preparation of ADH which was considerably labile in vitro and which showed a broad specificity towards primary saturated and unsaturated alcohols. We determinated its mol. wt., its Km for ethanol, allylalcohol and acetaldehyde, its Km for its co-enzyme, and the influence of some amides, oximes and intermediates of sugar metabolism. References ARSLANIAN, M. J., PASCOE, E., and REIHOLD, J. G., Rat liver alcohol dehydroegnase. Biochem. J. 125,1039-1047 (1971). COSSINS, E. A., KOPALA, L. C., BLAWACKY, B., and SPRONK, A. M., Some properties of higher plant alcohol dehydrogenase. Phytochemistry 7, 1125-11R4 (1968).

6

S. LEBLOVA and E. PERGLEROVA, Alcohol Dehydrogenase from Cucumber Seeds

DAWES, E. A., Quantitative Problems in Biochemistry, p.165. In Czech. Pubi. House Czechosl. Acad. Sci, Praha 1965. ERIKSSON, C. E., Alcohol: NAD oxidoreductase from peas (Pisum sativum). Acta chern. scand. 21, 304 (1967). ERIKSSON, C. E., Alcohol: NAD oxidoreductase (E.C. 1.1.1.1.) from peas. J. Food Sci. 33, 525-532 (1968). HATANAKA, A., Leaf alcohol: NAD oxidoreductase from tea seeds. Bull. lnst. Chern. Res., Kyoto Univ. 60, 135-1411972). HATANAKA, A., and HARADA, T., Purification and properties of alcohol dehydrogenase from tea seeds. Agr. bioI. Chern. 36, 2033-2035 (1972). LEBLOVA, S., and MANCAL, P., Characterization of pant alcohol dehydrogenase. Physiol. Plantar. 34, 246-249 (1975). LESTER, D., and BENSON, G. D., Alcohol oxidation in rats inhibited by pyrazol, oximes and amides. Science 169, 282-284 (1970). LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J., Protein measurement with the Folin phenol reagent. J. BioI. Chem. 193, 265-275 (1970). NACHMAN, M., LESTER, D., and LEMAGNEN, J., Alcohol aversion in the rat: behavioral assessment of noxious drug effects. Science 168, 1244-1246 (1970). SEDLAK, J., and LINDSAY, R. H., Estimation of total, protain-bound and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal. Biochem. 26, 192-205 (1968). THEORELL, H., Die Alkoholdehydrogenase ihre Wirkungsweisen und Komplexverbindungen. Experientia 21, 553-561 (1965). WIDMARK, E. M. P., Eine Mikromethode zur Bestimmung von Athylalkoholin Blut. Biochem. Z. 131, 473-484 (1922). Received August 4, 1976. Authors' address: SYLVA LEBLOVA and EVA PERGLEROVA, Department of Biochemistry, Nature Science Faculty, Charles University, Prague, Czechoslovakia.