Alcohol Dehydrogenase of Apple

Journal of Experimental Botany, Vol. 31, No. 121, pp. 449-459, April 1980

Alcohol Dehydrogenase of Apple

IAN M. BARTLEY AND SHEILA J. HINDLEY1

East Mailing Research Station, East Mailing, Maids tone, Kent ME 19 6BJ

Received 3 July 1979

ABSTRACTThe alcohol dehydrogenase prepared from apple (Malus domestica Borkh.) possesses both NADH-and NADPH-linked activities, when assayed with acetaldehyde as substrate. The pyridinenucleotides bind to the same catalytic site on the enzyme. The alcohol dehydrogenase can alsocatalyse the reduction of C3-C6 aldehydes with either NADH or NADPH as cofactor.

I N T R O D U C T I O N

The enzyme, alcohol dehydrogenase (ADH) catalyses the oxidation of alcoholsand the reduction of aldehydes with NAD and NADH respectively as cofactors(Scandalios, 1977. Extracts prepared from a number of plant tissues includingmelon and apple fruits show NADH- and NADPH-linked ADH activities withacetaldehyde as substrate (Rhodes, 1973). In a preliminary study with ADH fromapple, Rhodes, Wooltorton, and Hulme (1969) were unable to separate theactivities using ion-exchange chromatography. NADPH phosphatase activity wasnot present in apple and the results suggested that the reduction of acetaldehydecould be catalysed by a single enzyme which accepts either cofactor. In furtherwork with melon ADH, Rhodes (1973) showed that the two activities could not beseparated using a number of chromatographic and electrophoretic techniques.Kinetic studies with the two pyridine nucleotides assayed singly and togethersuggested that the enzyme possesses a single catalytic site which will accept eitherNADH or NADPH.

The present paper describes a further study of the ADH prepared from applepeel which suggests that the enzyme from this tissue will also accept eitherpyridine nucleotide as cofactor and examines its possible role in the metabolism ofhigher alcohols which are precursors of the ester components of apple aroma(Nursten, 1970).

MATERIALS AND METHODSPyridine nucleotides (Sigma) were stored according to the suppliers instructions. Polyethyleneglycol-4000 (PEG-4000) was purchased from Sigma and pyrazole from Fluka. The polyvinyl-pyTolidones, PVP 44 000 (water-soluble) and Polyclar AT (highly cross-linked, insoluble form)were purchased from BDH.

1 Present address: Department of Biological Sciences, The Hatfield Polytechnic, Hatfield, Herts Al 10 9AB.

at University of A

berdeen on Novem

ber 16, 2014http://jxb.oxfordjournals.org/

Dow

nloaded from

http://jxb.oxfordjournals.org/

450 Bartley and Hindley—Alcohol Dehydrogenase of Apple

Source of apples

A bulk sample of apples (Malus domestica Borkh.) was obtained on 21 September, 1977 fromtrees of Cox's Orange Pippin and stored in 2% O2:98% N2 at 3-3 °C. Samples of 20 fruits wereremoved and held in a vessel supplied with a stream of 2% O2:98% N2 at 5 1 h~', prior to their use asthe source of ADH. The conditions used ensured a CO2 concentration of 0-3% or less in the storagejar.

Cox's Orange Pippin apples grown in New Zealand in the 1977/78 season and shipped to theU.K. in April, 1978 were stored in air at 3-5 °C. The fruit was used after 1-5 months in store.

Preparation of ADHThe apples were thoroughly washed with cold water and dried prior to peeling. ADH was

prepared from peel tissue at 2 °C. Method 1, below, was used routinely.

Method 1

Apples were peeled to give strips of tissue of approximately uniform thickness. The peel tissue (6g) was cut into small pieces, with scissors, and ground in a mortar, prechilled at 2 °C, with 24 mlpreparation medium (0-2 M Tris, pH 8-0, 0-25 M sucrose, 1 mM EDTA, 10 mM mercaptoethanol,and 3% (w/v) PVP 44 000) and 3 g acid-washed sand for approximately 5 min. Further volumes ofpreparation medium were added to give a final ratio of 10 ml medium per g peel tissue. Thehomogenate was centrifuged at 2000 g for 10 min at 5 °C (the temperature used throughout forcentrifugation) and the supernatant clarified by centrifugation at 20 000 g for 30 min. PEG-4000was added slowly to 40 ml of the 20 000 g supernatant, with stirring in an ice bath, to a finalconcentration of 0-4 g ml""1 supernatant (Ruffner and Kliewer, 1975). The homogenate wascentrifuged at 20 000 g for 15 min and the pellet resuspended in 10 mM Tris, pH 8-0, 5 mMmercaptoethanol (10 ml). The solution was clarified by centrifugation at 20 000 g for 15 min: thesupernatant was subdivided and stored at —20 °C. The recovery of ADH, following concentrationwith PEG-4000, was 88-93%.

Method 2Peel tissue (10 g) was cut into small pieces, with scissors, and homogenized for 1 min in 60 ml

preparation medium (0-2 M Tris, pH 8-0, 0-25 M sucrose, 1 mM EDTA, 10 mM mercaptoethanol,3% (w/v) Polyclar AT) using an Ultraturrax homogenizer at 40% of line voltage. The homogenatewas filtered through Miracloth, the homogenizer rinsed with a further 20 ml preparation medium,and the washings decanted through the same filter. The filtrate was clarified by centrifugation at2000 g for 10 min and 20 000 g for 30 min. (NHJjSO, was added slowly to the 20 000 gsupernatant (65 ml) with stirring in an ice bath, to 80% saturation. The suspension was centrifugedat 27 000 g for 20 min, the pellet resuspended in 10 mM Tris, pH 8-0, 5 mM mercaptoethanol (6-5ml) and the solution finally clarified by centrifugation at 27 000 g for 20 min.

Assay of ADHIn the forward direction. The oxidation of ethanol was followed spectrophotometrically at 25 °C

by measuring the change in absorbance at 340 nm of a reaction mixture consisting of 0-05 MK H j P O ^ a O H buffer, pH 9-5, 185 /M NAD or 161 /*M NADP, 50 mM ethanol, and enzyme.The total volume was 3-0 ml.

In the backward direction. The reduction of acetaldehyde was followed by measuring the changein absorbance at 340 nm of a reaction mixture consisting of 0-05 M KHjPO^/NaOH buffer, pH5-5, 106-7 )M NADH or 88-91 fiM NADPH, 16-7 mM acetaldehyde, and enzyme. The totalvolume was 3-0 ml. The reaction was initiated by the addition of acetaldehyde.

Modifications in the assay conditions are stated in the texLFreshly redistilled acetaldehyde was used to prepare a stock 200 mM solution which was used in

the assay. The stock solution was stored .at 2 °C in the dark and was used within 3—4 weeks ofpreparation (Tottmar, Pettersson, and Kiessfing, 1973).

The initial velocity was calculated from the tangent drawn to the reaction time course curve(Cossins, Kopala, Blawacky, and Spronk, 1968). The reduction of NAD with ethanol as substrateand the oxidation of NADH and NADPH with acetaldehyde as substrate were directlyproportional to the concentration of enzyme present.

The solubility of the Cj-C6 aldehydes in water decreases with increasing chain length (Eriksson,1968). Stock aqueous solutions of the aldehydes were prepared by the inclusion of the non-ionic

at University of A

berdeen on Novem


Dow

nloaded from


Bartley and Hindley—A Icohol Dehydrogenase of Apple 451

detergent Triton X-100 at a concentration of 0-4% (w/v). Aldehyde (5 /*1) and Triton X-100 werepipetted into a test tube and water added dropwise to a final volume of 5 ml, the tube being shakenon a vortex mixer during the addition of water. The presence of Triton X-100 in the reactionmixture did not affect the rates of oxidation of NADH or NADPH by ADH with acetaldehyde assubstrate.

pH optimaPotassium phosphate buffers pH 5—10 were used in the determination of the pH optima for

ADH.

Determination o/K,The Ki (inhibitor constant) was determined for the inhibition of ADH by pyrazole; the enzyme

was assayed in the forward direction with ethanol (10-50 mM) and NAD (107 fiM).

Sephadex G-100 chromatography

A 3 ml volume of 27 000 g supernatant (prepared from peel tissue by method 2) was applied to acolumn (39 cm x 2-5 cm) of Sephadex G-100 at 2 °C which had previously been equilibrated with10 mM Tris, pH 8-0, 5 mM mercaptoethanol, and eluted with this buffer at 10-8 ml h"1. 2-7 mlfractions were collected and 0-4 ml aliquots of each fraction were assayed for ADH activity asabove with 10 mM acetaldehyde. The assay mixture of buffer, NADH or NADPH (equimolarconcentrations), and H2O was made up in sufficient quantity for 30 assays and used within 1 h(Reeves and Fimognari, 1966).

R E S U L T S

Preparation of ADHThe level of ADH prepared from peel tissue of apple was influenced by the choiceof buffer and the form of polyvinylpyrrolidone used in the preparation medium(Table 1). A combination of Tris buffer, pH 8-0, and PVP 44 000 gave the highestactivity of ADH per gram of tissue. There was little difference in the activityprepared when using 3 or 4% PVP 44 000 in the preparation medium (Table 1) andtherefore the lower concentration of polymer was used routinely in the preparationof the enzyme.

pH optimaADH showed optimal activity at pH 5-5 with acetaldehyde and NADPH as

cofactor (Fig. 1) and the activity declined sharply above or below this pH. Incontrast, with NADH as cofactor, the enzyme displayed a much broader range ofactivity with the optimum at pH 5-5-6-0.

T A B L E 1. Preparation of ADH from peel tissue of apple

ADH was prepared from peel tissue using method 1. ADH was assayed at pH 7-0 using 20 000 gsupernatant as source of enzyme.

Preparation medium"

PVP

Polyclar AT, 3%Polydar AT, 3%PVP 44 000, 3%PVP 44 000,4%

Buffer

0-2 M phosphate, pH 7 00-2 M Tris, pH 8 00-2 M Tris, pH 8 00-2 M Tris, pH 8 0

ADH(jano\ NADH oxidized min ' g ' tissue)

0 1 00-461-381-42

1 Other components: 0-25 M sucrose, 1 mM EDTA, and 10 mM mercaptoethanol.

at University of A

berdeen on Novem


Dow

nloaded from



o.o10

Fio. 1. ADH activity as a function of pH. ADH was assayed with acetaldehyde with either NADH(A—A) or NADPH (•—A) as cofactor and with ethanol with NAD as cofactor (•—•).

ADH assayed with ethanol and NAD showed increasing activity in the pHrange 7-0-10-0 (Fig. 1). No activity was observed with NADP as cofactor.

StabilityADH was stable for at least 4 d when stored at — 20 °C. The enzyme was not

stable at 2 °C and lost approximately 25% of its activity within 4 d at thistemperature.

Inhibition of ADH by pyrazoleThe oxidation of ethanol by apple ADH was competitively inhibited by pyrazole,

an isomer of imidazole. The K, determined, 12 //M, was slightly lower than the valuesdetermined with ADH prepared from other plant tissues, 16-23 //M (Leblova andMancal, 1975). Pyrazole also inhibited the enzyme when it was assayed in thebackward direction with acetaldehyde as substrate (see below).

Evidence for a single ADH in apple using NADH and NADPH as cofactor(i) Comparison of NADH- and NADPH-linked activities during storage at

2 °C. The ratio of NADPH- to NADH-linked activities was determined in twoseparate experiments with ADH stored at 2 °C. The enzyme activity declined over50% during the period of each experiment (Table 2) whilst in contrast the ratio ofNADPH-to NADH-linked activities remained relatively constant.

(ii) Inhibition of NAD(P)H-linked activities by pyrazole. Pyrazole inhibited the

at University of A

berdeen on Novem


Dow

nloaded from



T A B L E 2. Ratio of NADPH- to NADH-linked activities ofADH during storageof the enzyme at 2 °C

The ratio of NADPH- to NADH-linked activities is presented as mean + s.d. The figures inparenthesis indicate the number of separate assays conducted.

Days at 2 °C ADH Ratio NADPH- to0/mol NADH oxidized min"1 g"1 tissue) NADH-linked activities

Experiment A1 0-87 2-48 + 0-09 (3)5 0-40Experiment B1 0-90 2-43 + 0-28 (5)°7 0-42

" Ratio of rate of NADH oxidation (acetaldehyde) to NAD reduction (ethanol) for ADH inthis experiment was 4-86 ± 0-13 (5).

T A B L E 3. Inhibition ofNAD(P) H-linked activities of ADH by pyrazole

ADH was assayed with 10 mM acetaldehyde and 107 /iM NAD(P)H.

Pyrazole (mM)

00-11 05-0

ADH(% inhibition

NADH

0"23-470-691-3

by pyrazole)

NADPH

0*26-574-791-1

" 0-26 /imol NADH oxidized min"1 g"1 tissue.* 0-49 /anol NADPH oxidized min"1 g"1 tissue.

reduction of acetaldehyde by ADH with either cofactor (Table 3). The NADH-and NADPH-linked activities were inhibited to a similar degree at eachconcentration of pyrazole tested.

(iii) Sephadex G-100 chromatography. ADH was fractionated by gel filtrationusing Sephadex G-100. The NADH- and NADPH-linked activities were notresolved (Fig. 2).

(iv) Kinetics. The NADH- and NADPH-dependent activities followedMichaelis—Menten type kinetics. When ADH was assayed with a range of equimolarmixtures of NADH and NADPH, the rates of reaction observed were considerablylower than the sum of the rates with the two cofactors assayed separately (Fig. 3),indicating that there was mutual competition between the two cofactors for thesame active site on the enzyme (Lea and Thurman, 1972; Rhodes, 1973). The datawere also used in the theoretical treatment developed by Dixon and Webb (1964)to calculate the expected kinetics for an enzyme with a single catalytic site forwhich the two cofactors compete. The experimental data showed good agreementwith the predicted kinetics of the model (Fig. 3).

at University of A

berdeen on Novem


Dow

nloaded from



30 40Fraction number

Fio. 2. Chromatography of ADH on Sephadei G-100. NADPH-linked activity (•—A) andNADH-linked activity (A—A).

T A B L E 4. Kinetic properties of ADH from apple peel

The apparent Km and Vmtx were determined for apple ADH in the forward and backward direc-tions of assay at the pH optima established above. Vmtx is given as jmo\ pyridine nucleotideoxidized or reduced rain-1 g"1 tissue.

Variablesubstrate orcofactor

Constantcofactor orsubstrate

(M)

10-'10-'

A. Variable substrate, constant cofactorEthanol NAD (185 fM) 7-81 xAcetaldehyde NADH (107 fM) 0-61 xAcetaldehyde NADPH (107 fM) 1-18x10-'B. Variable cofactor, constant substrateNAD Ethanol (50 mM) 35-6x10-*NADH Acetaldehyde (10 mM) 12-8x10^NADPH Acetaldehyde (10 mM) 45-4x10-*

0-141-031-47

0-130-901-77

Km values of apple ADH

The apparent Km values determined for the ADH of peel tissue are listed inTable 4. The enzyme had a higher affinity (4-fold) for NADH than for NADPH inthe reduction of acetaldehyde, although the maximum velocity with NADPH wasgreater than that determined with NADH as cofactor. ADH also had a higheraffinity (2-fold) for acetaldehyde with NADH as cofactor than with NADPH.Comparison of the kinetic constants determined for ADH assayed in the forwardand backward directions, with NAD(H), showed that the enzyme had a 3-fold

at University of A

berdeen on Novem


Dow

nloaded from


Bar (ley and Hindley—A Icohol Dehydrogenase of Apple 455

higher affinity for NADH than NAD and a 13-fold higher affinity for acetaldehydethan ethanol.

Substrate specificityADH reduced C2-C6 aldehydes at a decreasing rate with increasing carbon

number with either NADH or NADPH as cofactor (Table 5). ADH also oxidizedprimary saturated alcohols (C4-C6) at a decreasing rate with increasing carbonnumber (Table 6). Although the enzyme oxidized 2-/rans-hexen-l-ol at asubstantially greater rate than hexan-1-ol it reduced 2-frans-hexenal and hexenal atsimilar rates.

DISCUSSION

Apple ADH resembles melon ADH in using NAD as cofactor for the oxidation ofethanol and both NADH and NADPH as cofactors in the reduction ofacetaldehyde. The pH optima (Fig. 1) also compare closely, melon ADH having apH optimum of 9-6 with ethanol, NAD and pH 5-7 with acetaldehyde, NADH or

T A B L E 5. Reduction of various aldehydes by apple ADH

The substrate specificity of ADH was determined with the aldehydes at a concentration of lmMand NAD(P)H at 107 ftM.

Aldehyde

AcetaldehydeProp analButanal2-MethylpropanalPentanalHexanal2-fra/ts-Hexenal

Rate relative to acetaldehyde

NADH

100°30-137-2

4-331-1

5-77-2

NADPH

100*21-424-6

8-516-711-113-3

"0-21 fano\ NADH oxidized min"1 g-1 tissue.* 0-26 /anol NADPH oxidized min"1 g"1 tissue.

T A B L E 6. Oxidation of various alcohols by apple ADH

The substrate specificity of ADH was determined with the alcohols at a concentration of 10 mM.

Alcohol

MethanolEthanolPropan-1-olPropan-2-olButan-1-ol2-Methylpropan-1 -olPentan-1-olHexan-1-ol2-/ra/w-Hexen-l-ol

Rate relative to ethanol

0100°44-7

8-545-0

019118-3

121-3

10-03 fimo\ NAD reduced min"1 g"1 tissue.

at University of A

berdeen on Novem


Dow

nloaded from



NADPH (Rhodes, 1973). In contrast to the aliphatic ADH of melon and apple, thearomatic ADH prepared from potato and swede is an NADP(H)-specific enzyme(Davies, Ugochukwu, Patil, and Towers, 19736; Rhodes and Wooltorton, 1975).

The ratio of NADPH/NADH-linked activities of apple ADH, with acetaldehydeas substrate, remains relatively constant during storage of the enzyme at 2 °C(Table 2) and both activities are inhibited to similar degrees over a range ofpyrazole concentrations (Table 3). The NADH- and NADPH-linked activities arenot resolved by column chromatography with Sephadex G-100 (Fig. 2) or DEAE-cellulose (Rhodes et al., 1969). These observations suggest that there is a singleADH in apple peel which possesses both NADH- and NADPH-linked activitiesThe kinetic data obtained with the cofactors assayed separately and in equimolarconcentrations (Fig. 3) indicate that the enzyme has a single catalytic site whichwill accept either NADH or NADPH. The ADH of apple peel thus resembles theenzyme from melon fruit characterized recently by Rhodes (1973).

There are a number of dehydrogenases/reductases, in addition to alcoholdehydrogenase of apple and melon, which can use NADH and NADPH ascofactor including glyceraldehyde-3-phosphate dehydrogenase from pea and

50S,NAD(P)H(/iM)

100

FIG. 3. Assay of ADH with a range of concentrations of equimolar mixtures of NADH andNADPH: experimental (A—AX calculated (•—A) assuming a single catalytic site for whichNADH and NADPH compete. The continuous line represents the kinetics assuming NADH andNADPH bind at separate catalytic sites; the data for this plot was derived from the assay of ADH

with the two cofactors separately. The units of V were Alt0 min~' (Pupillo, 1972).

at University of A

berdeen on Novem


Dow

nloaded from



mustard chloroplasts (McGowan and Gibbs, 1974; Cerff, 1978), glutamatedehydrogenase from chloroplasts and mitochondria of lettuce (Lea and Thurman,1972), homoserine dehydrogenase from maize (Bryan, 1969), and nitrate reductasefrom the alga Dunaliella parva (Heimer, 1976). The enzymes localized in thechloroplast, i.e. glyceraldehyde-3-phosphate dehydrogenase will use NADPH ascofactor whilst enzymes localized in mitochondria, i.e. glutamate dehydrogenase,use NADH. With alcohol dehydrogenase which is localized in the cytoplasm(Ragland and Hacket, 1964; Scandalios, 1977) the enzyme may use either NADHor NADPH or both pyridine nucleotides as cofactor in vivo. Although the pH ofthe cytoplasm is not known with any certainty, the acid-lability of NADH andparticularly NADPH suggests that it is above 7 (Davies, 1973). At pH 7, theNADH-linked activity of ADH is approximately 4-fold greater than the NADPH -linked activity (Fig. 1.). The ratio of NADPH to NADP in the peel tissue of applesduring ripening of the fruit in air at 12 °C has a mean value of 5-85 (Rhodes andWooltorton, 1968) whilst the ratio of NADH to NAD has a mean value of 0-15.The ratios of the pyridine nucleotides (reduced to oxidized state) remain relativelyconstant during ripening. If these ratios reflect the position in the cytoplasm of peeltissue cells then ADH might be expected to use NADPH as cofactor in vivo in thereduction of aldehyde. Heber and Santarius (1965) have noted that in the cytoplasmof leaf cells most of the NADP(H) occurs in the reduced state whilst most of theNAD(H) occurs in the oxidized state.

Comparison of the kinetic properties of apple and melon ADH (Table 7) showsthat apple ADH has a higher affinity for NAD(P)H and acetaldehyde than themelon enzyme. The enzymes also have a number of properties in commonincluding a higher affinity for NADH than NADPH, with acetaldehyde assubstrate, a higher affinity for acetaldehyde with NADH as cofactor than with

T A B L E 7. Comparison of kinetic properties of ADH of apple peeP and melonfruit*

VmMX is given as //mol NAD(P)H oxidized min"1 g"' tissue.

ADH Variablesubstrate orcofactor

Constantcofactor orsubstrate

A. Variable substrate, constant cofactorApple

Melon

Acetaldehyde

Acetaldehyde

NADHNADPHNADHNADPH

B. Variable cofactor, constant substrateApple

Melon

NADHNADPHNADHNADPH

Acetaldehyde

Acetaldehyde

(M)

0-611-181-3 x8-8 x

12-8 x45-4 x

2-4 x3-2 x

x 10-3

x lO"3

io-3

io-3

io-*io-*io-3

io-<

rnu

1-031-47

0-901-775-2

14-9

° Data, Table 4 this paper.6 Rhodes (1973).

at University of A

berdeen on Novem


Dow

nloaded from



T A B L E 8. Comparison of substrate specificity of various alcohol dehydrogenases

All values have been calculated as a percentage of the rate with ethanol as substrate.

Alcohol

MethanolEthanolPropan-1-olPropan-2-olButan-1-ol2-Methylpropan- l-olPentan-1-olHexan-1-ol2-/ra/ts-Hexan-1 -ol

ADH

Pea"

1-510044

309-8

12-3

Bean"

010046-5

326-2

16

Until"

010055-5

300

20

Citrus*

10072

84264942

168

Apple'

010044-7

8-545-0

019-118-3

121-3

" Leblova and Macal (1975).b Bruemmer and Roe (1971).c Data, Table 6 this paper.

NADPH, and higher Vmtx values with acetaldehyde, NADPH as substrate andcofactor respectively, than with acetaldehyde, NADH.

Apple ADH reduces C2-C6 aldehyde at a decreasing rate with increasing carbonnumber (Table 5) and thus resembles ADH of pea (Ericksson, 1968) and potato(Davies, Patil, Ugochukwu, and Towers, 1973a) rather than citrus ADH whichreduces C3—C6 aldehydes at 80% of the rate determined with acetaldehyde assubstrate (Bruemmer and Roe, 1971). The results with butanal and 2-methyl-propanal (Table 5) suggest that the enzyme, like potato ADH (Davies et ah,19736), reduces branched-chain aldehydes at a lower rate than their straight-chainisomers. The similarity in the rates of reduction of 2-/rans-hexenal and hexanal(Table 5) suggests that the presence of a double bond in the substrate does notaffect the reaction rate (Bruemer and Roe, 1971).

Apple ADH, in common with the enzyme prepared from other plant tissues,oxidizes a number of primary alcohols (Table 8). The introduction of a methylbranch in the substrate decreases the reaction rate whilst the presence of a doublebond increases the rate of oxidation (Eriksson, 1968). Secondary alcohols are poorsubstrates for ADH (Davies et al, 19736) and this also appears to be the casewith the enzyme from apple.

Apples develop a characteristic aroma during ripening arising from the synthesisof a mixture of volatile esters. The volatiles are principally compounds with evencarbon numbers, i.e. butyl acetate and hexyl acetate (Grevers and Doesburg, 1965)which suggests that their metabolism may be connected with that of fatty acids(Paillard, 1975). Incubation of peel and cortical tissue discs of apple withpropionic, butyric, or hexanoic acids leads to the formation of the correspondingalcohol and this suggests that the reductive pathway acid -» aldehyde -• alcoholfunctions in apple. Alcohol dehydrogenase is present in peel and cortical tissue butthe enzyme(s) catalysing reduction of acid to aldehyde have still to becharacterized.

at University of A

berdeen on Novem


Dow

nloaded from


Bartley and Hindley—Alcohol Dehydrogenase of Apple 459

LITERATURE CITEDBRUEMMER, J. H., and ROE, B., 1971. / . Agric. Fd Chem. 19,266-8.BRYAN, J. K., 1969. Blochim. biophys. Acta, 171,205-16.CERFF, R., 1978. Eur. J. Biochem. 82,45-53.COSSINS, E. A., KOPALA, L. C, BLAWACKY, B., and SPRONK, A. M., 1968. Phytochemistry, 7,

1125-34.DAVIES, D. D., 1973. In Biosynthesis and its control in plants. Ed. B. V. Milborrow. Academic

Press, London. Pp. 1-20.PATIL, K. D., UGOCHUKWU, E. N., and TOWERS, G. H. N., 1973a. Phytochemistry, 12,523-30.UGOCHUKWU, E. N., PATH, K. D., and TOWERS, G. H. N., 19736. Ibid. 12,531-6.

DKON, M., and WEBB, E. C, 1964. Enzymes. Longmans, London.ERIKSSON, C. E., 1968. /. Fd Sci. 33,525-32.GREVERS, G., and DOESBURO, J. J., 1965. Ibid. 30,412-15.HEBER, U. W., and SANTARIUS, K. A., 1965. Biochim. biophys. Acta, 109,390-408.HEIMER, Y. M., 1976. PL PhysioL, Lancaster, 58, 57-9.LEA, P. J., and THURMAN, D. A., 1972. / . exp. Bot. 23,440-9.LEBLOVA, S., and MANCAL, P., 1975. Physiologia PL 34,246-9.MCGOWAN, R. E., and GIBBS, M., 1974. PL PhysioL, Lancaster, 54, 312-9.NURSTEN, H. E., 1970. In The biochemistry of fruits and their products. Ed. A. C. Hulme. Academic

Press, London. Pp. 239-68.PAILLARD, N., 1975. Colloques natn. Rech. Scient., Paris, 321-6.PUPHXO, P., 1972. Phytochemistry, 11,153-61.RAGLAND, T. E., and HACKETT, D. P., 1964. Archs Biochem. Biophys. 108,479-89.REEVES, JR., W. J., and FIMOGNARI, G. M., 1966. Meth. Enzym. 9, 288-94.RHODES, M. J. C, 1973. Phytochemistry, 12,307-12.

and WOOLTORTON, L. S. C, 1968. Ibid. 7,337-53.1975, Ibid. 14,1235-40.and HULME, A. C, 1969. Qualitas PL Mater, veg. 19,167-83.

RUFFNER, H. P., and KLIEWER, W. M., 1975. PL PhysioL, Lancaster, 56, 67-71.SCANDAUOS, J. G., 1977. In Regulation of enzyme synthesis and activity in higher plants. Ed. H.

Smith. Academic Press, London. Pp. 129-53.TOTTMAR, S. O. C, PETTERSSON, H., and KIESSLING, K.-H., 1973. Biochem. J. 135,577-86.

at University of A

berdeen on Novem


Dow

nloaded from


Documents

Alcohol Dehydrogenase of Apple