PHYSIOLOGIA PLANTARUM 74: 531-536. Copenhagen 1988

Hexose kinases of avocado

Les Copeland and Gregory J. Tanner

Copeland, L. and Tanner, G. J. 1988. Hexose kinases of avocado. - Physiol. Plant.74: 531-536.

The subcellular location and properties of enzymes concerned with the phosphoryla-tion of glucose and fructose in avocado {Persea americana Mill. cv. Hass) have beenstudied. A substantial amount of glucose-phosphorylating activity was particulate andfractionation of extracts by sucrose density gradient centrifugation indicated thatmost of this activity was associated with the mitochondria. Three hexose-phosphory-lating enzymes were resolved by DEAE-cellulose chromatography of the cytosolicfraction. These were a hexokinase (EC 2.7.1.1), which had strong preference forglucose as substrate, and two specific fructokinases (EC 2.7.1.4). ATP was thepreferred phosphoryl donor for the hexose kinases of avocado.

Key words - Avocado, hexokinase, fructokinase, kinetic properties, Persea amer-icana, subcellular location.

L. Copeland {corresponding author), Dept of Agricultural Chemistry, Univ. of Syd-ney, N.S.W. 2006, Australia; and G. J. Tanner (present address), CSIRO, Division ofPlant Industry, Canberra, A.C.T. 2601, Australia.

Introduction

The first step in the metabolism of glucose and fructosein plant tissues is phosphorylation with ATP to from thecorresponding hexose 6-phosphate. Plant tissues appearto contain a number of hexose kinases which can cata-lyze this step. However, information on the propertiesof these enzymes is available from only a limited num-ber of plant sources. Four forms of hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1) oecur inwheat germ (Meunier et al. 1971) and spinach leaves(Baldus et al. 1981) and three isozymes have been foundin developing castor-bean seeds (Miernyk and Dennis1983). Pea seeds contain two hexokinases and, in addi-tion, two specific fructokinases (ATP:D-fructose 6-phos-photransferase; EC 2.7.1.4; Turner et al. 1977a, b, Co-peland et al. 1978, Turner and Copeland 1981). The peaseed hexokinases are thought to phosphorylate primar-ily glucose in vivo. Hexokinases which preferentiallyphosphorylate glucose, and specific fructokinases arealso present in pea stems (Tanner et al. 1983) and theplant cytosolic fraction of soybean nodules (Copelandand Morell 1985). in the present study we have in-vestigated the subeellular location and properties of theenzymes involved in the phosphorylation of glucose and

fructose in avocado. A substantial amount of glucose-phosphorylating activity in avocado extracts appearedto be assoeiated with the mitochondrial fraction. Thecytosolic fraction contained a hexokinase, which hadstrong preference for glucose as substrate, as well as twospecific fructokinases.

Abbreviations - AGATP, Agarose-hexane-adenosine-5'-tri-phosphate. Type 2 affinity resin; P^ inorganic phosphate; TES.2-{[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]-amino}ethanesul-fonic acid; Tris, tris(hydroxymethyl)aminomethane; V, maxi-mum velocity.

Materials and methods

Materials

All enzymes and chemicals were from Boehringer-Mannheim GmbH or Sigma Chemical Co. Mature avo-cados {Persea americana Mill. cv. Hass) were purchasedfrom a local market.

Preparation of extracts

All operations were carried out at 4°C. Avocado meso-carp (100 g) was cut into 1 cm cubes in 200 ml of 10 mM

Received 9 March. 1988; revised 10 May, 1988

Physiol. Plant. 74. 1988 531

TES (2- {[2-hydroxy-1,1 -bis(hydroxymethyl)ethyl]-aminojethanesulfonic acid)-KOH, pH 7.5, which con-tained 0.3 M mannitol, 1 mM EDTA and 15 mM re-duced glutathione (buffer A) and the suspension passedthrough a Braun juice extractor lined with 2 layers ofMiracloth. The extract was adjusted to pH 7.5 andeentrifuged at 1 000 g for 10 min. The pellet was washedby resuspending in 10 ml 0.3 M mannitol and een-trifuging at 1000 g for 10 min. The washed 1 000 g pelletwas resuspended in 5 ml 10 mM TES-KOH (pH 7.2).The 1 000 g supernatant and the wash supernatant wereeombined and centrifuged at 10000 g for 10 min. The10000 g pellet was washed in 10 ml 0.3 M mannitol andresuspended in 5 ml 10 mM TES-KOH (pH 7.2). The10000 g supernatant and the washings were combinedand centrifuged at 100000 g for 60 min. The pellet waswashed in 0.3 M mannitol and resuspended in 5 ml 10mM TES-KOH (pH 7.2).

For suerose density gradient centrifugation, extractswere prepared by squeezing avocado mesocarp (50 g in50 ml of buffer A) through a single layer of Miracloth.The extract was adjusted to pH 7.5, eentrifuged at 200 gfor 5 min and the supernatant centrifuged at 12 000 g for10 min. The pellet was carefully resuspended in bufferA which contained 0.1% (w/v) bovine serum albuminand layered onto a linear density gradient of 28 ml,prepared from 20 to 60% (w/v) sucrose solutions dis-solved in 1 mM TES-KOH (pH 7.5) and 1 mM EDTA.The preparation was centrifuged at 83 000 g (average)for 150 min in an SW-27 swing-out rotor and fractions of1.8 ml were pumped from the bottom of the gradient.Density was determined by measuring the refractiveindex of the fractions.

The cytosolic hexose kinases were isolated by passingavocado mesocarp (100 g) through a Braun juice extrac-tor with 100 ml mM TES-KOH (pH 7.2) which con-tained 1 mM EDTA (buffer B). The extract was ad-justed to pH 7.2 and centrifuged at 30000 g for 20 min.The supernatant was collected from underneath thefloating lipid layer and applied to a DEAE-cellulosecolumn (1.5 x 25 cm) which had been previously equih-brated in buffer B. The column was washed with bufferB until unbound protein was removed and eluted with agradient obtained by introducing 250 ml of buffer Bcontaining 0.5 M KCI into 250 ml of buffer B. Fractionsof 4.3 ml were collected at a flow rate of 0.5 ml min"'.Three sets of fractions were pooled, corresponding tohexokinase, fructokinase I and fructokinase II. Afteradding P, and MgClj to final concentrations of 10 and 5mM, respectively, the pooled hexokinase fractions werepassed through an agarose-hexane-adenosine-5'-tri-phosphate. Type 2 (AGATP) affinity column ( 1 x 5cm) which had previously been equilibrated in 10 mMTris-HCI (pH 8), 50 mM KCI, 5 mM MgClj and 10 mMPi (buffer C). Hexokinase passed through the columnwith the unbound material and was concentrated byultrafiltration (Amicon PM-10 membrane) to 10 ml anddialyzed against 10 mM Tris-HCI (pH 8), 50 mM KCI.

The fractions from the DEAE-cellulose columnwhich contained fructokinase I were made 10 mM withrespeet to P, and 5 mM with respect to MgCl2 andapplied to an AGATP affinity column ( 1 x 5 cm) inbuffer C. The eolumn was washed with buffer C untilunbound protein was removed and eluted with buffer Cwhich contained 5 mM ATP. Fractions with fructokinaseactivity were pooled and freed of ATP by passagethrough a Sephadex G-25 column. The pooled DEAE-cellulose column fractions which contained fructokinaseII were concentrated by ultrafiltration to 10 ml. Theenzyme preparations eould be stored in the presence of0.1% (w/v) bovine serum albumin at -15°C for 1 monthwithout loss of activity.

Enzyme assays

All assays were earried out at 30°C. Activity was mea-sured by eoupling the formation of product with thereduction of NADP+ and following the change in ab-sorbance at 340 nm.

Standard reaction mixtures for hexokinase contained,in a volume of 1 ml, 25 mM Tris-HCI (pH 8), 50 mMKCI, 2 mM D-glucose, 2 mM ATP, 3 mM MgClj, 0.33mM NADP^, 2 units glucose 6-phosphate dehydroge-nase (EC 1.1.1.49) and an appropriate volume of en-zyme.

Fructokinase aetivity was assayed in reaction mix-tures which contained, in a volume of 1 ml, 25 mMTris-HCI (pH 8), 50 mM KCI, 2 mM D-fruetose, 2 mMATP, 3 mM MgClz, 0.33 mM NADP% 2 units glucose6-phosphate dehydrogenase, 2 units phosphoglucoseisomerase (EC 5.3.1.9) and an appropriate volume ofenzyme. For assays of crude extraets and DEAE-cellu-lose column fractions 6-phosphogluconate dehydroge-nase (EC 1.1.1.43; 0.2 units) was added to the reactionmixtures.

The phosphorylation of sugars other than glucose andfruetose was measured in reaction mixtures which con-tained, in a volume of 1 ml, 25 mM Tris-HCI (pH 8), 50mM KCI, 2 mM ATP, 3 mM MgCl2, 0.15 mM NADH,0.5 mM phosphoenolpyruvate, 4 units pyruvate kinase(EC 2.7.1.40), 4 units Iactate dehydrogenase (EC1.1.1.28), substrate as indicated, and an appropriatevolume of enzyme. Triose phosphate isomerase (EC5.3.1.1), cytochrome c oxidase (EC 1.9.3.1) and cata-lase (EC 1.11.1.6) were assayed as described by Tanneret al. (1983).

Kinetic constants and the SE of the values were calcu-lated by fitting the data to the Michaelis-Menten equa-tion as described by Duggleby (1981).

Protein eontent was determined by the method ofLowry et al. (1951) using bovine serum albumin as astandard.

Molecular weight was determined in a SephadexG-200 column according to the method of Andrews(1966) using bovine liver catalase (240 kDa), rabbitmuscle aldolase (158 kDa), bovine serum albumin (65

532 Physiol. Plant. 74. 1988

(ab. 1. Subcellular distribution of hexose phosphorylating activity in avocado extracts. Avocado mesocarp (100 g) was fraction-ited as described. The total activity in each fraction is shown. The data are from one of duplicate experiments.

Fraction

Crude homogenate1000 g Pellet10000 g Pellet100000 g Pellet100000 g Supernatant

Activity,

Glucosephosphorylating

4.440.470.960.292.02

(imol product formed

Fructosephosphorylating

3.100.210.190.282.05

min '

Cytochrome coxidase

5.921.244.920.030

Proteincontent,

mg

513.3

112.3

21

kDa) and horse heart cytochrome c (12.5 kDa) as cali-bration proteins.

Results

Crude extracts of avocado contained ca 1.4 times morephosphorylating activity with glucose as substrate than

S 1

0 5 10Fraction no.

Fig. 1. Distribution of enzymes in a 12000 g pellet from avo-cado following centrifugation in a sucrose density gradient.Cytochrome c oxidase, triose phosphate isomerase (TPI) and-^talase activities are in nmol product formed fraction"' min"'.Glucose and fructose phosphorylation are in nmol product'ormed fraction ' min ' and density is in g ml '.

with fructose (Tab. 1). Approximately 40% of the glu-cose-phosphorylating activity, together with 20% of thephosphorylating activity with fructose, was recovered inthe particulate fractions following differential centrifu-gation of crude extracts of avocado (Tab. 1). When aresuspended organelle pellet was centrifuged in a su-crose density gradient, most of the particulate hexosephosphorylating activity was located in a single fractionof density 1.17 g ml"' (Fig. 1). This corresponded to thefraction with maximum activity of the mitochondrialmarker, cytochrome c oxidase. Triose phosphate isom-erase, which occurs in plastids, as well as in the cy-toplasm (Quail 1979), had maximum activity in the gra-dient at slightly higher density of 1.18 g ml"'. The smallamount of triose phosphate isomerase at the top of thegradient indicated that breakage of the plastids was low.There was no hexose-phosphorylating aetivity in frac-tions of the gradient corresponding to the peroxisomal

100 200 300 400Elution vol 111)

500

Fig. 2. DEAE-cellulose chromatography of the soluble fractionof avocado extracts. Avocado mesocarp (100 g) was extractedas desribed and the soluble extract obtained by centrifuging at30000 g for 20 min. Activity is in nmol product formed frac-

'hysiol. Plant. 74. 1988 533

Tab. 2. Kinetic constants of the soluble hexose kinases of avocado with D-glucose and D-fructose. K^ values are in mM and V innmol product formed (mg protein)"' min"'. The kinetic constants and the SE were determined as described in at least 2 separateexperiments. The data are from representative experiments. The activities of fructokinases I and II with D-glucose were too low toenable constants to be determined for this substrate.

Enzyme

HexokinaseFructokinase IFructokinase II

D-Glucose

Km V

0.046±0.004 22.1±0.5

K^

0.12±0.030.14±0.010.10±0.02

D-Fructose

V

3.1±0.3100 ±391 ±5

marker, catalase. The hexose kinase associated with themitochondrial fraction in the sucrose density gradientshad a K^ for glucose of 0.10 ± 0.01 mM. The enzymehad little activity with fructose as substrate.

The particulate hexose-phosphorylating activity ofavocado was not readily solubilized by metabolites orsalts. When a 10000 g pellet from a crude extract wasresuspended and incubated for 10 min at 4°C in 0.3 Mmannitol in 10 mM TES-KOH (pH 7.5) and then cen-trifuged at 10000 g, ca 25% of the phosphorylatingactivity (with glucose as substrate) was released into thesupernatant. The addition of glucose 6-phosphate (finalconcentration 2 mM), MgATP (1 mM) or KCI (0.5 M)to the incubation mixture did not increase the amountof glucose-phosphorylating activity that was solubilized(results not shown).

Three peaks of hexose-phosphorylating activity wereseparated from the cytosolic fraction of avocado ex-tracts by DEAE-cellulose chromatography. The firstpeak eluted from the DEAE-cellulose column by theKCI gradient contained an enzyme which phosphory-lated mainly glucose (Fig. 2). The two enzymes thatwere eluted subsequently phosphorylated fructose, buthad little or no activity with glucose. The enzymes weretermed, in order of elution, hexokinase, fructokinase Iand fruetokinase II. This nomenclature is based on thatadopted for the hexose kinases of other plant tissues(Turner and Copeland 1981, Copeland and Morell1985). The elution profile shown in Fig. 2 was notaltered by the inclusion of 0.3 M mannitol in the extrae-tion medium for the cytosolic hexose kinases. Hexoki-nase and fructokinase I were further purified by chro-matography using an AGATP affinity column. Fructo-

kinase I bound to the affinity column whereashexokinase did not, and this enabled the two enzymesto be completely separated. Fructokinase II was studiedwithout further purification. The size of all three solublehexose kinases was determined by gel filtration to be 85± 3 kDa.

The three cytosolic hexose kinases had optimum ac-tivity at pH 8.2. The enzymes all displayed typical Mi-chaelis-Menten kineties with respect to the hexose andnucleoside triphosphate substrates. Hexokinase had alower K^ and higher maximum velocity (V) with glu-cose as substrate than with fructose (Tab. 2). The en-zyme also phosphorylated 2-deoxy-D-glucose but theactivity with this substrate (final eoneentration 5 mM)was 30% of the V with glueose. Hexokinase had noactivity with D-mannose, D-galactose or D-xylose (finalconcentration 5 mM). The kinetie eonstants of fructoki-nases I and II with fructose were eomparable (Tab. 2).Fructokinase I had no activity with glucose, whereas theactivity of fructokinase II with 5 mM glucose was 8% ofthe V with fructose. Neither fructokinase I nor fructoki-nase II phosphorylated D-mannose, D-galaetose, D-xy-lose or D-arabinose (final concentration 5 mM).

The soluble hexose kinases required Mg + and hadmaximum activity when the concentration of Mg + andATP were equivalent. None of the enzymes was inhib-ited by an exeess of 2 mM Mg-+ over ATP. The cytosoUchexokinase had a K^ for MgATP (determined in thepresence of a fixed excess of 1 mM Mg-+) of 0.14 ± 0.02mM. The activity of hexokinase with MgGTP. MgUTPand MgCTP (final concentration 2 mM with 1 mMexcess Mg^+) was 7, 10 and 14%, respectively, of theactivity with 2 mM MgATP. Fructokinase I had a similar V

Tab. 3. Kinetic constants of fructokinases I and II of avocado with MgATP, MgGTP, MgUTP and MgCTP. K^ values are in mMand V in nmol product formed (mg protein)"' min '. The kinetic constants and the SE were determined as described in at least 2separate experiments. The data are from representative experiments.

Substrate

MgATPMgGTPMgUTPMgCTP

534

Fructokinase I

0.037±0.0O40.64 ±0.151.7 ±0.70.36 ±0.05

V

117± 3159±18105±23137± 6

V/K,

316024862

381

Fructokinase 11

K„,

0.106±0.0060.91 ±0.065.2 ±1.00.64 ±0.12

V

83± 177± 2

122±1852± 4

Physiol.

V/K,

780852381

Platit. 74, 1988

when MgATP, MgGTP, MgUTP or MgCTP acted as thephosphoryl donor. However, the enzyme had a muchlower K^ for MgATP than for the other nucleosidetriphosphates (Tab. 3). Fructokinase II was most aetivewith MgUTP, but had highest affinity for MgATP. Theaffinity of fructokinase I for MgATP was almost 3 timeshigher than that of fruetokinase II (Tab. 3).

Discussion

Avocado mesocarp contains several enzymes eapable ofcatalyzing the phosphorylation of hexoses. A substan-tial amount of the glucose-phosphorylating activity wasparticulate (Tab. 1). Attempts to separate intact orga-nelles from crude extracts were unsueeessful and eoose-quently the subcellular location of the particulate glu-cose-phosphorylating activity was investigated by su-crose density gradient centrifugation of a resuspendedorganelle pellet. Most of the hexose-phosphoryiatingactivity in the sucrose density gradient appeared to beassociated with the mitochoncirial fraction, although thepossibility that a small amount of activity was associatedwith the plastids could not be excluded (Fig. 1). Thefractions in the suerose density gradient that had maxi-mum activity of cytochrome c oxidase and triose phos-phate isomerase occurred at densities of 1.17 and 1.18 gml"', respectively, which were lower than densities gen-rally observed for mitocbondrial and plastid markers inother plant tissues (Quail 1979). The lower apparentdensities of the mitochondria and plastids in extracts ofavocado, and the difficulty in clearly separating theseorganelles, were presumably due to the high lipid con-tent of the extracts. Hexose-phosphorytating activityhas been shown to be associated with the mitochondriaof other plant tissues. In pea stems all of the glucose-phosphorylating activity is associated with the mito-chondria, whereas essentially all of the fructose-phos-phoryiating activity is soluble (Tanner et al. 1983).Hcxokinase activity is associated with mitochondria ofCttscuta reflexa (Baijal and Sanwal 1977) and pea leaves(Dry et al. 1983) and with mitochondria and plastids ofdeveloping castor bean endosperm (Miernyk and Den-nis 1983). Hexokinase activity has been reported inplastids of developing cauliflower buds (Journet andDouce 1985). The particulate hexose kinase activity inavocado extracts was not readily solubilized by metabo-lites. This is similar to the situation in pea stems (Tanneret al. 1983) and pea leaves (Dry et al. 1983), but incontrast to the mitochondrial hexokinase of castorbeans, which is solubilized by low concentrations ofhexose monophosphates and nucleoside triphosphates(Miernyk and Dennis 1983).

The cytosolic fraction from avocado contained ahexokinase and two fructokinases (Fig. 2). Ail threeenzymes had a native size of 85 kDa. This is comparableto the molecular weight of fructokinase I of pea seeds(Copeland et al. 1984) and the fructokinase from theplant cytosol of soybean nodules (Copeland and Moreil

1985). Pea seed fructokinase 1 is a monomeric proteinwith a subunit size of 79 kDa. In contrast, a value of 38kDa has been reported for the size of the hexokinases ofcastor-bean endosperm (Miernyk and Dennis 1983).

The eytosolic hexokinase of avocado had strong pref-erence for glucose as substrate compared with fructose.Fructokinases I and II had a high degree of specificityfor fructose (Tab. 2). This indicates that the phosphory-lation of glucose and fructose in avocado may be cata-lyzed by different enzymes which have metabolic speci-ficity for the respective hexoses. Thus, glucose maymainly be phosphorylated by the soluble hexokinaseand by the kinase associated with the mitochondria,whereas the specific fructokinases are likely to be in-volved in the phosphorylation of fructose. Highly spe-cific fruetokinases have now been isolated from a num-ber of different plant tissues, suggesting that the occur-rence of these enzymes may be widespread in plants.Fructokinases I and II of avocado were not inhibited byhigh concentrations of fructose and in this respect dif-fered from the fructokinases of pea seeds (Turner et al.1977b, Copeland et al. 1978), and soybean nodules (Co-peland and Moreil 1985). The fructokinases from thesetissues are inhibited by concentrations of fructose grea-ter than 0.2-0.5 mM.

Fructokinases I and II of avocado were able to utilizeMgATP. MgGTP, MgUTP and MgCTP as phosphoryldonors. However, both enzymes had much higher affin-ity for MgATP tban for the other nucleoside triphos-phates (Tab. 3). Furthermore, the values of the param-eter V/Kn,, which represents the first-order rate constantfor the combination of an enzyme with its substrate attow concentrations of the substrate, indicate that nucie-oside triphosphates other tban MgATP would be poorsubstrates for the fructokinases of avocado. It has beenproposed that in some plant tissues UTP, formed frompyrophosphate and UDP-glucose in the reaction cata-lyzed by UDP-glucose pyrophosphorylase, may act asphosphoryl donor for the phosphorylation of fructose,thereby regenerating UDP for the breakdown of su-crose by sucrose synthase (Huber and Akazawa 1986,Black et al. 1987). Our findings indicate that this isunlikely to occur in avocado. The kinetic properties ofthe fructokinases would not favour UTP acting as aphosphoryl donor in preference to ATP.

Acknowledgement - The authors wish to thank Ms L. C. You-nie for technical assistance.

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Edited by I. M. Miller

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