ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 196, No. 2, September, pp. 611-618, 1979

Purification

and Partial

Characterization from Wheat’

of Alcohol

Dehydrogenase

PAT J. LANGSTON,* GARY E. HART,3 AND C. NICK PACE Department of Biochemistry and Biophysics and Genetics Section of the Department of Plant Sciences, Texas A&M University and Texas Agricultural Experiment Station, College Station, Texas 77848

Received January 30, 1979; revised March 6, 1979 Further support for hypotheses proposed earlier for the genetic control and subunit composition of the alcohol dehydrogenase of T&cum has been obtained through the purification and partial characterization of the enzyme. The alcohol dehydrogenase of the wheat T. monococcum was purified 103-fold to a specific activity of 55,900 units/mg. Purification was achieved using streptomycin sulfate precipitation, gel filtration chromatography, DEAE-cellulose anion-exchange chromatography, and preparative isoelectric focusing. The native enzyme has a molecular weight of 116,000 and a dimeric subunit structure. The apparent Michaelis constants are 1.2 x lo-* M for ethanol and 1 X 10m4M for NAD. The substrate specificity of wheat alcohol dehydrogenase differs significantly from the substrate specificities of the enzymes of horse and yeast.

The current understanding of the genetic control and subunit composition of alcohol dehydrogenase (alcohol:NAD oxidoreductase, EC 1.1.1.1) of species in the grass tribe Triticeae has been derived largely from studies of polyploids. Three molecular forms of alcohol dehydrogenase, ADH-1, ADH-2, and ADH-3,4 have been detected in the mature grains of Triticum aestivum (hexaploid wheat, 2n = 42, genomes A, B, and D). Genetic analyses have provided evidence that these isozymes are composed of protomers, designated (Y, /3, and 6, which are encoded by three related (homologous) structural g&es, one located in each of the three genomes of the species in related chromosomes (1, 2). The results of these studies are consistent with the model that the protomers associate into dimeric en* The investigations reported were included in the dissertation submitted by P.J.L. to the Graduate College, Texas A t M University, in partial fulfdment of the requirements for the Ph.D. degree. * Present address: Biochemistry Department, University of Minnesota, St. Paul, Minn. 55108. J Send reprint requests to: G. E. Hart, Genetics Section, Texas A & M University, College Station, Tex. 77843. 4 Abbreviation used: ADH, Alcohol dehydrogenase.

zymes in all possible combinations to produce ADH-1 (aa dimers), ADH-2 (ap and as), and ADH-3 (pp, 66, and pa). The results of a genetic analysis of the tetraploid wheat T. turgidurn (2n = 28, genomes A and B) support the hypothesis that in this species ADH-1 is composed of aa dimers, ADH-2 of oq3 dimers, and ADH-3 of /3/3 dimers (3). An enzyme homologous to the tetraploid and hexaploid wheat isoiymes has been detected in each of the diploid species of Triticum that has been examined, including those which possess the A and D genomes (4). Further support for the proposal that the alcohol dehydrogenase isozymes of the Triticeae are composed of two polypeptide subunits was obtained by the production of hybrid enzymes in experiments in which genetically controlled electrophoretically variant isozymes of both T. turgidurn and T. tauschii (2n = 14, genome D) were dissociated into protomers and then reassociated into active molecules in crude tissue extracts (5). The availability of purified Triticeae alcohol dehydrogenase isozymes will provide a basis for further testing of the hypotheses of genetic control, subunit composition, and evolutionary relationships

611

0003-9861/79/100611-08$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

612

LANGSTON ET AL.

that have been advanced for them (l-5) and will also allow their characteristics to be compared with those of other plant and animal alcohol dehydrogenase. The purification and partial characterization of an alcohol dehydrogenase from the grain of T. r~zonococcum (2n = 14, genome A) is reported here. T. monococcum, which possessesone major alcohol dehydrogenase in its mature kernels, was chosen as the enzyme source for this initial study to avoid the difficulty of purification of the multiple forms of alcohol dehydrogenase which are present in the polyploid wheats. The principal findings reported are as follows: (i) The data provide the first direct evidence that the enzyme is composed of two polypeptide subunits and thus support the hypothesis of a dimeric structure; (ii) the molecular weight of the pure native enzyme, 116,000 + 2000, is the largest reported for any dimeric alcohol dehydrogenase; (iii) the substrate specificity of the enzyme differs significantly from that of other plant alcohol dehydrogenases and from the horse and yeast alcohol dehydrogenases. MATERIALS

AND METHODS

Wheat strain. Alcohol dehydrogenase was purified from whole kernels of strain G919 of the diploid species T. monococcum.

Chemicals and reagents. The NAD (Grade III), blue dextran, streptomycin sulfate, and yeast (A3263) and horse (340-L2) alcohol dehydrogenases were obtained from Sigma Chemical Company (St. Louis, MO.). The DEAE-cellulose anion-exchange resin DE-52 was from Whatman, Inc. (Clifton, N. J.). Ultrogel AcA44 and Ampholine carrier ampholytes were supplied by LKB Instruments, Inc. (Bromma, Sweden). The molecular weight standards bovine serum albumin (A6003), ovalbumin (Grade VI), lactate dehydrogenase (Type VI), and fructose g-phosphate kinase (Type III), were from Sigma Chemical Company. The hexokinase (A Grade), was purchased from Calbiochem (La Jolla, Calif.). The a-chymotrypsin-A was from Schwarz/Mann (Orangeburg, N. Y.). All other chemicals used in this study were reagent grade. Proteivl determination. Protein concentrations were determined by the method of Lowry (6) using human serum albumin as a standard. All samples were dialyzed against 1 mM Tris buffer, pH 7.5, prior to the protein determination. Electrophoresis. Analytical disc gel electrophoresis was carried out using the procedure of Davis (‘7)with

115-mmlength gels. Following electrophoresis the gels were stained for enzyme activity by the method of Hart (3) or for protein with amido black. Sodium dodecyl sulfate electrophoresis was carried out using a modification of the procedure of Weber and Osborn (8). The procedure was changed to include incubation of samples with 2% sodium dodecyl sulfate and 5% 2-mercaptoethanol for 4 min in a boiling water bath. Enzyme assays. Alcohol dehydrogenase activity was measured as described by Bonnichsen and Brink (9) except than an NAD concentration of 0.8 InM and an alcohol concentration of 0.1 M were used for routine assays. In the substrate specificity studies the NAD concentration was 0.8 mM and the alcohol concentration was 0.03 M. Hexokinase activity was measured by the procedure of Beisenherz et al. (IO). One unit of activity is the amount of enzyme that catalyzes the reduction of 1 gmol of NAD* per minute. Specific activity is expressed as units of enzymic activity per milligram of protein. Molecular weight estimation by gel jiltration. The molecular weight of the native enzyme was determined by gel filtration on an Ultrogel AcA44 column (1.65 x 59.5 cm) using a flow rate of 8 ml per hour. The proteins used in calibrating the column are listed in the legend for Fig. 5. The protein standards and wheat alcohol dehydrogenase were chromatographed together and elution volumes were determined by scanning the absorbancy at 280 nm except for hexokinase and alcohol dehydrogenase where activity measurements were used. The value obtained is the result of three independent experiments. RESULTS

Enzyme Puri$cation

The steps used for the purification of alcohol dehydrogenase from wheat and some typical results are summarized in Table I. The purification was carried out at O-4%. All of the Tris buffers contain 0.5 mM dithiothreitol and 0.1 j.&M zinc sulfate. Extraction and streptomycin sulfate precipitation. Mature, whole kernels of T. monococcum wheat (30 g) were soaked

for 20 h in 60 ml of extraction buffer (0.075 M Tris-HCl, pH 7.3). The kernels were then ground in a Sorval Omni-mixer at 7500 rpm for 4 min followed by a lo-min soak and a second 4-min grinding. The homogenate was then centrifuged at 31,000g for 15 min. Streptomycin sulfate (180 mg/ml) in buffer was added to this supernatant to give a final concentration of 45 mg/ml. After standing

PURIFICATION

613

OF WHEAT ALCOHOL DEHYDROGENASE TABLE I

PURIFICATION PROCEDUREFORALCOHOLDEHYDROGENASEFROMT. monococcum

Protein b-g)

Total activity (units)

151.9

82,100

ND”

18,500

III Ultrogel chromatography

32.63

22,400

685

1.27

27

IV DEAE-cellulose chromatography

3.0

13,700

4,500

8.43

14.8

0.15

8,380

55,900

Fraction I Crude extract II Streptomycin sulfate supernatant

V Preparative isoelectric focusing

Specific activity (units/mg) 540 ND

Purification (-fold)

Yield (8)

1

100

ND

103

22.3

10.2

s Not determined.

in an ice bath for 40 min, the solution was centrifuged at 31,000g for 15 min to remove the precipitate. Gel jiltration and DEAE-cellulose chromatography. The streptomycin sulfate supernatant (3.0 ml) was applied to a gel filtration column of Ultrogel AcA44 (4.5 x 65 cm), equilibrated, and eluted with extraction buffer (Fig. 1). A flow rate of 45 ml per hour was used and Q-mlfractions were collected. The tubes containing the peak alcohol dehydrogenase activity (16-24)

20

were pooled and dialyzed for 5 h against 1 liter of 0.01 M Tris-HCI, pH 7.3 buffer. The enzyme was then applied to a DEAEcellulose column (1.3 x 8 cm) and the column then washed with a 0.01 M Tris-HCl, pH 7.3 buffer containing 0.3 M KC1 until the absorbancy at 215 nm was zero. A 500-ml linear KC1 concentration gradient of 0.03 to 0.12 M KC1 was used to elute the alcohol dehydrogenase (Fig. 2). The tubes containing peak alcohol dehydrogenase activity were pooled and dialyzed against 0.02 M

60

40 ERAWON

NUMBER

FIG. 1. Gel filtration chromatography of wheat alcohol dehydrogenase on an Ultrogel AcA44 column (4.5 x 65 cm). The column was equilibrated and eluted with a 0.075 M, pH 7.5 Tris buffer containing 0.5 mM dithiothreitol and 0.1 PM zinc sulfate. The flow rate was 45 ml/h and 9-ml fractions were collected. Fractions 16-24 were pooled.

614

LANGSTON ET AL.

4

.A 0’ O&d

hxh

-0

b0000000000000000

I 30

60 FRACTION

90

-0

120

NUMBER

FIG. 2. DEAE-cellulose column chromatography of wheat alcohol dehydrogenase. The column (1.3 x 8 cm) was equilibrated with a 0.01 M, pH 7.3 Tris buffer containing 0.03 M KCl, 0.5 mM dithiothreitol, and 0.1 pM zinc sulfate. For elution a 500-ml linear KC1 gradient (0.03 to 0.12 M KCl) was used with the same buffer. The flow rate was 20 ml/h and 4-ml fractions were collected. Fractions 17-32 were pooled.

Tris-HCl, pH 7.2 buffer to lower the ionic strength for isoelectric focusing. Preparative isoelectric focusing. Breparative isoelectric focusing was carried out with a 110 ml LKB electrofocusing column. A pH 5-7 Ampholine was used at a concentration of 2%. Sulfuric acid (1%) and ethylenediamine (1%) were used as electrolytes. Electrofocusing was carried out for 25 h at l-l.3 W. The alcohol dehydrogenase focused at approximately pH 5.5 in the dense portion of the sucrose gradient. The gradient was pumped from the column and collected in l-ml fractions. The fractions containing alcohol dehydrogenase activity were pooled and dialyzed for 36 h against three changes of 2 liters of 0.01 M Tris-HCl buffer, pH 7.3 to remove the Ampholines. Homogeneity Disc gel electrophoresis of a 40-pg sample of the purified enzyme revealed a single protein band (Fig. 3). A single band was also observed after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Enzyme Stability Alcohol dehydrogenase activity is completely lost in crude extracts after 10 days (Fig. 4). This inactivation can be decreased

by adding 0.01 M dithiothreitol or 0.1 M 2-mercaptoethanol. (The activity decrease in the presence of 2-mercaptoethanol results from competitive inhibition (11)). Not all sulfhydryl reagents are effective: L-cysteine has no effect on the inactivation of the enzyme, while glutathione inactivates the enzyme. Molecular Weight Determinations The molecular weight of the native enzyme was determined by gel filtration on a calibrated Ultrogel AcA44 column. Based on the data in Fig. 5, the molecular weight is 116,000 + 2000. The molecular weight of the subunits of alcohol dehydrogenase was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data shown in Fig. 5 indicate a subunit molecular weight of 58,000 k 2000. A least squares analysis of the data from three independent experiments was used to compute the line of best fit. A single band was observed which indicates identical subunits or subunits of very similar molecular weight. In addition to determining the molecular weight of homogeneous alcohol dehydrogenase, we also determined the molecular weight of alcohol dehydrogenase in crude extracts. The results were the same.

PURIFICATION

615

OF WHEAT ALCOHOL DEHYDROGENASE

Kinetic Studies

-

The apparent Michaelis constants were determined to be 1 x lo4 M for NAD and 1.2 x lop2 M for ethanol from LineweaverBurk plots of initial velocity studies of the wheat alcohol dehydrogenase. The substrate specificity of the enzyme was investigated by measuring the rate of oxidation of a series of alcohols by wheat, horse, and yeast alcohol dehydrogenases. The initial rate was measured at the same concentration (0.03 M) for all of the alcohols in the presence of 0.8 InM NAD. The results are presented in Table II where the rate of oxidation of the alcohols is given as a percentage of the rate of oxidation of ethanol. DISCUSSION

This procedure produces purified alcohol dehydrogenase in reasonable yield from a workable quantity of material. Efforts to increase the yield with batch purification steps were not successful as ammonium sulfate, ethylene glycol, and ethanol precipitations either failed to increase the specific + FIG. 3. Disc gel electrophoresis of a crude wheat extract (I, 450 pg protein) and purified wheat alcohol dehydrogenase (II, 40 pg protein). The gels were stained for protein with amido black.

I II

I O2

4

4 TIME

8

IO

(Days)

FIG. 4. Stability of wheat alcohol dehydrogenase in crude extracts (0.01 M Tris, pH 7.5) at 25°C. Wheat alcohol dehydrogenase activity was measured as a function of time in the presence of 0.01 M dithiothreitol (m), 0.1 M 2-mercaptoethanol (Cl), 0.1 M glutathione (A), or no addition (01.

616

LANGSTON ET AL.

I

0

I

0.2 K av=-

I

0.4

0.6

OR RELATIVE

MOBILITY

0.8

1.0 @

FIG. 5. Native alcohol dehydrogenase molecular weight determination by gel filtration and subunit molecular weight determination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The logarithm of the molecular weight is plotted vs K,, = (V, - Vo)/(Vt - V,) where V, is the elution volume of the protein, V, is the void volume, and V, is the bed time, or relative mobility (=protein migration distance/dye migration distance). Purified alcohol dehydrogenase was chromatographed with five reference proteins (l-5) on an Ultrogel AcA44 column as described under Materials and Methods. Sodium dodecyl sulfate-electrophoresis was carried out as described under Materials and Methods using reference proteins 6-10. Alcohol dehydrogenase is represented by (0). The reference proteins (0) are bovine serum albumin dimer (1) and monomer (3); hexokinase (2 and 7); ovalbumin (4 and 8); cu-chymotrypsinogen (5 and 10); fructose 6-phosphate kinase (6); and lactate dehydrogenase (9).

activity or resulted in a large loss of activity. Purification of the enzyme was attempted by affinity chromatography on 5’-AMPSepharose 4B. Alcohol dehydrogenase was bound and specifically eluted from this adsorbant but a great loss of activity occurred; also nonspecifically bound protein was eluted with the enzyme activity. A major problem in developing this and other plant alcohol dehydrogenase purification procedures is enzyme instability. The procedure reported here produces a stable, purified enzyme. Although the recovery from the streptomycin sulfate precipitation is poor, the step is necessary to produce a stable enzyme preparation. When this step was omitted from the purification procedure 98% of the enzyme activity was lost within 48 h. Omission of only the gel filtration step also produced a very unstable purified enzyme preparation. A stable purified enzyme preparation was obtained only when both the streptomycin sulfate precipitation and gel filtration steps were used. It is possible that the use of these two steps could also increase the stability of the alcohol dehydrogenase of other plants. Dithiothreitol or mercaptoethanol was also required during purification for maintenance of enzyme activity. The requirement of a sulfhydryl reagent is characteristic of many (12-16)

TABLE II SUBSTRATE SPECIFICITY OF ALCOHOL DEHYDROGENASE~

Source of alcohol dehydrogenase Alcohol

Wheat

Yeast

Horse

Pea*

Bean*

Lentil*

Ethanol n-Propanol n-Butanol n-Pen&no1 n-Hexanol Ally1 alcohol 2-Methylpropanol 3-Methylbutanol Cyclopentanol Cyclohexanol n-Glucitol

100 36 29 25 31 123 9 21 16 10 7

100

100

100

100

100

18

93

8 5 6 149 0 16 3 2 0

79 72 60 135 j 95 ’ 51 24 100 0

44 30 NDc 12 160 10 15 0 ND 0

55 30 ND 20 152 0 14 0 ND 0

46 32 ND 16 156 6 20 0 ND 0

a Values given are relative reaction velocities with the reaction rate for ethanol assigned the value of 100. * Data from Leblova and Mancal (13). c Not determined.

PURIFICATION OF WHEATALCOHOLDEHYDROGENASE but not all (17, 18) of the plant alcohol dehydrogenases which have been investigated. Curiously, glutathione, which is a sulfhydryl reagent sometimes used in plant enzyme purification, is a potent inactivator of the enzyme. Inactivation probably follows glutathione binding to the enzyme. Glutathione binds to glyceraldehyde 3-phosphate dehydrogenase by disulfide linkages (19) and the SH groups of the alcohol dehydrogenase are probably available for disulfide linkages with glutathione. The molecular weight of 116,000 reported for the wheat enzyme is the largest value reported for a plant alcohol dehydrogenase. Only the enzymes of peanut (M, 112,000 (17)) and tea (1M, 95,000 (20)) approach its size. The subunit molecular weight was measured using sodium dodecyl sulfateelectrophoresis and was found to be 58,000. This value, one-half of the molecular weight of the native enzyme, strongly indicates a dimeric structure for wheat alcohol dehydrogenase. This evidence, which is the first direct evidence for a dimeric structure, provides further support for the models of genetic control and dimeric subunit structure for wheat alcohol dehydrogenase proposed earlier on the basis of genetic studies (1, 3, 5). It is also consistent with the dimeric structure determined from physical studies of purified tea alcohol dehydrogenase (M, 95,000; subunit M, 47,000 (20)) and with the models of dimeric structure proposed on the basis of genetic studies of the enzyme from several other plant species (21-23). Alcohol dehydrogenase functions during the anaerobic portion of seed germination. The enzyme catalyzes the conversion of acetaldehyde and NADH to ethanol and NAD+. The NAD+ is utilized during the catabolism of the carbohydrate stores in the seed. The ethanol produced is accumulated in the seed until the end of anaerobosis when the enzyme catalyzes the oxidation of the ethanol to acetaldehyde which is then converted to pyruvate and further metabolized (24-26). The Michaelis constants for ethanol for the alcohol dehydrogenases from the seeds of eight different plant species all fall in the range 1 to 20 mM (13, 14,27-29). Thus,

617

the K, of 12 mM observed for wheat alcohol dehydrogenase is similar to that found for other plant enzymes, but is significantly greater than the K, for horse and yeast enzymes (30). The high K, of ethanol for plant alcohol dehydrogenase is not surprising since a low K, is not needed because germinating seeds contain high concentrations of ethanol readily available for oxidation. The K, of 0.08 mM for NAD for the wheat enzyme is also in the range from 0.05 to 0.16 mM commonly observed for plant alcohol dehydrogenases. The substrate specificity of the wheat alcohol dehydrogenase is similar to the specificity of the enzyme of pea, bean, and lentil except that only the wheat enzyme oxidizes cyclopentanol and D-glUCitO1 (13). The substrate specificity of wheat alcohol dehydrogenase differs significantly from that of the horse and yeast enzymes. The wheat enzyme exhibits the opposite preference for the three substrate pairs n-pentanolln-hexanol, 2-methylpropanol/3methylbutanol, and cyclopental/cyclohexanol. The wheat enzyme is the only alcohol dehydrogenase which oxidizes the sugar alcohol, D-glucitol. The substrate specificity of wheat alcohol dehydrogenase toward most of the alcohols studied lies between the very broad specificity of the horse enzyme and the very narrow specificity of the yeast enzyme. The wheat enzyme’s substrate specificity is clearly broader than is necessary to simply utilize ethanol and acetaldehyde as substrates. The unique substrate preferences and the broader substrate specificity suggest that the wheat alcohol dehydrogenase uses another, and possibly a larger, physiological substrate than ethanol. The data clearly reflect that the wheat alcohol dehydrogenase active site is different from the active sites of the horse or yeast enzymes. ACKNOWLEDGMENTS This paper is Technical Article No. 14546 of the Texas Agricultural Experiment Station. REFERENCES

1. HART, G. E. (1970) Proc. Nut. Acad. Sci. USA 66, 1136-141.

618

LANGSTON

2. HART, G. E., AND LANGSTON, P. J. (1977) Heredity 39, 263-277. 3. HART, G. E. (1969) Biochem. Genet. 3, 617-625. 4. HART, G. E. (1971) Zsozyme Bull. 4, 15. 5. HART, G. E. (1971) Mol. Gen. Genet. 111,61-65. 6. LOWRY, 0. H., ROSENBOUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1961) J. Biol. Chem. 193, 265-275. 7. DAVIS, B. (1964) Ann. N. Y. Acad. Sci. 121, 404-427. 8. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 9. BONNICHSEN, R. K., AND BRINK, N. G. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 495-500, Academic Press, New York. 10. BEISENHERZ, G., BULTZE, H. J., BUCHER, T., CZOK, R., GARBADE, K. M., MEYER-ARENDT, E., AND PFEIDERER, G. (1953) Z. Naturforsch. 86, 555-569. 11. GEREN, C. R., OLOMUN, C. M., JONES, T. T., AND EBNER, K. E. (1977) Arch. Biochem. Biophys. 179, 415-419. 12. FELDER, M. R., SCANDALIOS, J. G., AND LIU, E. H. (1973) Biochim. Biophys. Acta 318, 149-159. 13. LEBLOVA, S., AND MANCAL, P. (1975) Physiol. Plant. 34, 246-249. 14. LEBLOVA, S., AND STIBORVA, M. (1976) Physiol. Plant. 38, 176-180. 15. MANSELL, R. L., GROSS, G. G., STOCKIGHT, J., FROCHE, H., AND ZENK, M. A. (1974) Phytochemistry 13, 2427-2435.

ET AL. 16. DAVIES, D. D., PATIL, K. D., UGOCHUKWU, E. H., AND TOWERS, G. H. N. (1973) Phytochemistry 12, 523-530. 17. SWASGOOD, H. E., AND PATTEE, H. E. (1968) J. Food Sci. 33, 400-405. 18. BRUEMMER, J. H., AND ROE, B. (1971) J. Agr. Food Chem. 19, 266-268. 19. RACKER, E., AND KRIMSKY, I. (1954) Fed. Proc. 13, 246-258. 20. HATANAKA, A., KAJIWARA, T., TOMOHIO, S., AND YAMASHITA, H. (1974) Agr. Biol. Chem. 38, 1835-1844. 21. SCANDALIOS, J. G. (1969) Biochem. Genet. 3, 37-79. 22. TORRES, A. M. (1974) Biochem. Genet. 11,17-24. 23. GO’ITLIEB, L. D. (1974) Proc. Nut. Acad. Sci. USA 28, 610-614. E. A., ANDTURNER, E. R. (1963)J. Exp. 24. COSSINS, Bot. 14, 290-298. 25. EFFER, W. R., AND RANSON, S. L. (1967) Plant Physiol. 42, 1053-1058. 26. COSSINS, E. A., KAPALA, L. C., BLAWACKY, B., AND SPRONK, A. M. (1968) Phytochemistry 7, 1125-1134. 27. LEBLOVA, S., AND PERGLEROVA, E. (1976) Phytochemistry 15, 813-815. 28. LEBLOVA, S., AND PERGLEROVA, E. (1977) Biochem. Physiol. Pflanzen 171, 1-6. 29. LEBLOVA, S., AND PERGLEROVA, E. (1976) Collect. Czech. Chem. Commun. 41,3482-3488. 30. SUND, H., AND THEORELL, H. (1963) in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., eds.), 2nd ed., Vol. 7, pp. 25-83, Academic Press, New York.

Purification and partial characterization of alcohol dehydrogenase from wheat.

ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 196, No. 2, September, pp. 611-618, 1979 Purification and Partial Characterization from Wheat’ of Alco...
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