REACTIVE OXYGEN SPECIES PRODUCTION BY POTATO TUBER

1

Running head: Hexokinase controls ROS formation in plant mitochondria

Dr. Antonio Galina from the Instituto de Bioquímica Médica, Programa de Biofísica e Bioquímica Celular

Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro 21941-590, Brazil, Av.

Brigadeiro Trompowsky, s/n, CCS, Bloco D, sub-solo sala D-013, Laboratório de Bioenergética e

Fisiologia Mitocondrial, Cidade Universitária, Rio de Janeiro, RJ, 21941-590. Tel: (55 21) 22609573

Brazil. E-mail: [email protected]

AREA: Bioenergetics and Photosynthesis

Plant Physiology Preview. Published on December 24, 2008, as DOI:10.1104/pp.108.129247

Copyright 2008 by the American Society of Plant Biologists

www.plantphysiol.orgon April 10, 2018 - Published by Downloaded from Copyright © 2008 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

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REACTIVE OXYGEN SPECIES PRODUCTION BY POTATO TUBER MITOCHONDRIA IS

MODULATED BY MITOCHONDRIALLY BOUND HEXOKINASE ACTIVITY.

Juliana Camacho-Pereira*, Laudiene Evangelista Meyer*, Lilia Bender Machado, Marcus Fernandes

Oliveira and Antonio Galina.

From Instituto de Bioquímica Médica, Programa de Biofísica e Bioquímica Celular and Programa de

Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Laboratório de

Bioenergética e Fisiologia Mitochondrial.

Av. Brigadeiro Trompowsky, s/n, CCS, Bloco D, sub-solo sala D-013, Cidade Universitária, , Rio de

Janeiro, RJ, 21941-590. Brazil.

* These authors contributed equally to the results of this work.



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FOOTNOTES:

This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico

(CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).

ABBREVIATIONS:

ANT: adenine nucleotide transporter. AOX: alternative oxidase. AUF: arbitrary units of fluorescence. EGTA:

ethylene glycol-bis (-aminoethyl ether)-N,N,N´,N´-tetraacetic acid. ETS: electron transport system. Faf-BSA:

fatty acid free bovine serum albumin. FCCP: carbonyl cyanide p-trifluoromethoxyphenylhydrazone. G6PDH:

glucose-6 phosphate dehydrogenase. LA: linolenic acid. MHP: mannoheptulose. mt-HK: mitochondrial-

associated hexokinase. NAG: N-acetylglucosamine. PEP: phosphoenolpyruvate. PMSF: phenyl methyl

sulfonyl fluoride. PUMP: plant uncoupling protein. ROS: reactive oxygen species. SOD: superoxide dismutase.

St-UCP: Solanum tuberosum uncoupling protein. UCP: uncoupling protein. VDAC: voltage-dependent anion

channel. ΔΨm: mitochondrial membrane potential.



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ABSTRACT

Potato tuber (Solanum tuberosum) mitochondria (PTM) have a mitochondrially bound hexokinase (mt-

HK) activity that exhibits a pronounced sensitivity to ADP inhibition. Here we investigated the role of

mitochondrial hexokinase activity in potato tuber mitochondria reactive oxygen species generation.

Mitochondrial hexokinase has a ten fold higher affinity for glucose than for fructose (KMGlc = 140 μM

versus KMFrc = 1375 μM). Activation of potato tuber mitochondria respiration by succinate led to an

increase in H2O2 release that was abrogated by mitochondrial hexokinase activation. Mitochondrial

hexokinase activity caused a decrease in the mitochondrial membrane potential (ΔΨm) and an increase

in oxygen consumption by potato tuber mitochondria. Inhibition of glucose phosphorylation by

mannoheptulose or N-acetylglucosamine induced a rapid increase in H2O2 release. The blockage of

H2O2 release sustained by glucose was reverted by oligomycin and atractyloside indicating that ADP re-

cycles through the adenine nucleotide translocator and FoF1ATP synthase is operative during the

mitochondrial hexokinase reaction. Inhibition of mitochondrial hexokinase activity by 60 to 70 % caused

an increase of 50 % in the maximal rate of H2O2 release. Inhibition in H2O2 release by mitochondrial

hexokinase activity was comparable to, or even more potent, than that observed for St-UCP activity. The

inhibition of H2O2 release in potato tuber mitochondria was two orders of magnitude more selective for

the ADP produced from the mitochondrial hexokinase reaction than for that derived from soluble yeast

hexokinase. Modulation of H2O2 release and oxygen consumption by glucose and mitochondrial

hexokinase inhibitors in potato tuber slices shows that hexoses and mitochondrial hexokinase may act

as a potent preventive antioxidant mechanism in potato tubers.



5

INTRODUCTION

Production of reactive oxygen species (ROS) is an unavoidable consequence of aerobic respiration

(Chance et al., 1979). The mitochondrial electron transport system (ETS) is the major site of ROS

production in mammalian and non-photosynthesizing plant cells (Puntarulo et al., 1991; Halliwell and

Gutteridge, 1999). Depending on the mitochondrial respiratory states, a small portion of the consumable

oxygen is partially reduced to generate ROS (Skulachev, 1996; Liu, 1997; Turrens, 1997; Møller, 2001;

Considine et al., 2003; Smith et al., 2004). In plants, the monoelectronic reduction of oxygen by ETS

leads to the production of superoxide radicals (O2˙�) which can be dismutated by SOD, producing

hydrogen peroxide (H2O2), and further decomposed by catalase and/or ascorbate-glutathione peroxidase

cycles (Møller, 2001). An imbalance between the ROS production and antioxidant defenses can lead to

an oxidative stress condition. Increased levels of ROS may be a consequence of the action of plant

hormones, environmental stress, pathogens or high levels of sugars and fatty acids (Bolwell et al., 2002;

Couée et al., 2006; Gechev et al., 2006; Liu et al., 2007; Rhoads and Subbaiah, 2007). These conditions

may lead to storage deterioration or impairment of seedling growth decreasing on crop yield. To avoid

the harmful accumulation of ROS or to fine-tune the steady-state levels of ROS, various enzymatic

systems control the rate of ROS production in mitochondria (Schreck and Baeuerle, 1991; Møller, 2001).

Mitochondrial ROS production is highly dependent on the membrane potential (ΔΨm) generated

by the proton gradient formed across the inner mitochondrial membrane. High ΔΨm was shown to

stimulate ROS production when the ETS is predominantly in a reduced state (i.e. when NADH, FADH2

and O2 are present in abundance but ADP or Pi levels are low). This condition is reached in resting

metabolic states after a full oxidation of glucose or fatty acids. Stimulating electron flow by decreasing

ΔΨm either by the use of uncouplers or by coupling respiration to ATP synthesis, slows the ROS

generation rate (Boveris and Chance, 1973; Korshunov et al., 1997). It has been observed that in

isolated potato tuber mitochondria (PTM) the uncoupling protein (referred to as PUMP in plants, or UCP

in animals) causes a small decrease in ΔΨm when this proton carrier protein is activated by the presence

of anionic fatty acids, a condition that blocks ROS generation (Vercesi et al., 1995; Vercesi et al., 2006).

Nucleotides, such as ATP, antagonize this effect (Considine et al., 2003; Vercesi et al., 2006). On the

other hand, fluctuations in free hexose levels due to environmental or developmental conditions (Morrell

and ap Rees, 1986; Geigenberger and Stitt, 1993; Renz et al., 1993) lead to variations in the oxygen

consumption rate in heterotrophic tissues of plant (Brouquisse et al., 1991, Dieuaide et al., 1992). As a

result, ROS-producing pathways may be either stimulated or repressed (Couée et al., 2006). Unlike

PUMP activity, which is activated by an excess of free fatty acids, a specific mechanism for

mitochondrial ROS production caused by an excess of hexose remains elusive.

The metabolism of free hexoses begins by their phosphorylation in a reaction catalyzed by the

hexokinase:

Hexose + ATP → Hexose 6-P + ADP



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Hexokinase (HK) is an ubiquitous enzyme found in many organisms. In plants, the binding

mechanism of HK to the outer mitochondrial membrane is not fully established, but some reports indicate

that it may differ considerably from those properties described for mammal cells (Dry et al., 1983;

Miernyk and Dennis, 1983; Rezende et al., 2006). It has been shown that in several mature and

developing plant tissues, multiple HK isoforms are expressed with different kinetic properties and

subcellular localizations. The HK`s are found in cytosol, bound to the mitochondrial membrane or in

stroma of plastids in plant cells (Miernyk and Dennis, 1983; Galina et al., 1995; Damari-Weissler et al.,

2007). Beyond its obvious role in glycolysis regulation, HK activity may also function as a sugar sensor,

triggering a signal transduction pathway in plants (Rolland et al., 2006).

In mammals, HK types I and II are associated with the mitochondrial outer membrane through

the voltage dependent anion channel (VDAC) and adenine nucleotide transporter (ANT). These

associations were found in tissues with a high energy demand, such as heart, brain and tumor cells

(Arora and Pedersen, 1988; BeltrandelRio and Wilson, 1992; Wilson, 2003). In addition, recent evidence

in mammalian cells has shown that binding of HK to VDAC located at the outer mitochondrial membrane

is somehow involved in the protection against pro-apoptotic stimuli (Nakashima et al., 1986; Pastorino et

al., 2002; Cesar and Wilson, 2004). Similar observations were reported for tobacco plant mitochondrial

HK (Kim et al., 2006). However, it has been shown that drugs such as the fungicide clotrimazole and the

anesthetic thiopental, which promptly disrupt the association between mt-HK and VDAC in mammalian

mitochondria, are unable to promote this effect in maize root mitochondria (Rezende et al., 2006). These

observations suggest a different type of association of mt-HK with plant mitochondria. The binding of mt-

HK with mitochondria in many plants involves a common N-terminal hydrophobic membrane anchor

domain of about 24 amino acids which is related to the membrane targeting, but the exact mechanism of

association is unknown (Damari-Weissler et al., 2007).

Recently, our group demonstrated that mt-HK activity plays a key preventive antioxidant role by

reducing mitochondrial ROS generation through a steady-state ADP re-cycling mechanism in rat brain

neurons. The mitochondrial ADP re-cycling leads to a decrease in the ΔΨm coupled to the synthesis of

ATP by oxidative phosphorylation (da-Silva et al., 2004; Meyer et al., 2006).

Although plant HK is recognized to fulfill a catalytic function, the role of mt-HK activity in the

regulation of both mitochondrial respiration and ROS production in plants is unknown. Recently, an

authentic HK activity was detected in PTM (Graham et al., 2007) and its involvement in potato tuber

glycolysis suggested, but its involvement in PTM ROS generation was not explored. We then raise the

hypothesis that HK bound to PTM would contribute to produce a steady-state ADP recycling that

regulates ROS formation. However, whether this association is capable of controlling the rate of ROS

generation in plant mitochondria is unknown. Here, we aim to investigate the role of mt-HK activity in

PTM physiology. The data indicate that mt-HK activity plays a key role as a regulator of ROS levels in

respiring plant tissues exposed to high hexose levels.



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RESULTS

An Authentic Hexokinase Activity is Associated with Potato Tuber Mitochondria.

Previous studies have shown that HK is bound to mitochondria in mammalian tissues, in different

plant species (Galina et al., 1995; da-Silva et al., 2001; Wilson, 2003; Meyer et al., 2006; Claeysen and

Rivoal, 2007), and even in PTM (Graham et al., 2007). To check whether a particulate HK activity from

potato tuber is also associated with this organelle, in Table I we determined the activities of HK,

oligomycin-sensitive FoF1ATPase, a mitochondrial enzyme, and glucose-6-phosphate dehydrogenase

(G6PDH), a cytosolic and plastid enzyme (Dennis and Green, 1975; Miernyk and Dennis, 1983) in

different fractions of potato tuber homogenates. Clearly, the majority of HK activity is concentrated in the

particulate fraction (P12) of potato tuber (Table I). As the FoF1ATPase and the G6PDH activities were

also substantial in P12 fraction, a further separation step was carried out on the P12 fraction by using a

self-generated Percoll gradient method, producing two main fractions. One was enriched in plastids

(fraction 1) and the other was enriched in mitochondria (fraction 2) (Neuburger, et al, 1982). Accordingly,

the FoF1ATPase activity was enriched 13 fold in fraction 2 of the Percoll gradient as compared to the

homogenate (Table I). The HK activity was present in both plastid and mitochondrial fractions from the

Percoll gradient, being enriched 8 fold in this fraction and only 2 fold in the plastid fraction (Table I). The

low G6PDH activity detected in the mitochondrial fraction indicates a very low degree of cross-

contamination between these fractions. Almost all particulate G6PDH activity was associated with the

plastid fraction (Table I). These data show that an authentic HK activity is associated with PTM.

Kinetic Properties of Potato Tuber mt-HK and Dependence of Activity on Oxidative

Phosphorylation

The next step was to evaluate the activity of mt-HK after addition of ATP and glucose using

intact, Percoll-purified PTM (Figure 1). The mt-HK activity was detected immediately after addition of 1

mM ATP and 5 mM glucose (Fig. 1A, traces 1 and 2), but was progressively inhibited by more than 90 %

after 2 minutes. However, the mt-HK activity could be restored by the inclusion of succinate, an

oxidisable substrate that supports the conversion of ADP to ATP during oxidative phosphorylation,

indicating that inhibition observed for mt-HK was due to the ADP accumulated by the mt-HK reaction

(Fig. 1A, traces 1 and 2). The mt-HK sensitivity to ADP inhibition was confirmed when 0.3 mM ADP was

added to the reaction medium prior to starting the mt-HK reaction with ATP plus glucose (Fig. 1A, trace

3). The mt-HK reaction rate in this case was much lower than that observed in the first 2 minutes in the

absence of ADP (Fig. 1A, trace 1). The addition of succinate re-established the maximal rate of mt-HK

activity (Fig. 1A, trace 3). The authenticity of mt-HK activity was confirmed when its specific competitive

inhibitor, 50 mM mannoheptulose (MHP), was added to the reaction medium (Fig. 1A, trace 2). No

oxidative phosphorylation-supported mt-HK activity was observed in the presence of oligomycin (a

specific inhibitor of FoF1ATPase), atractyloside (a specific inhibitor of adenine-nucleotide translocator-



8

ANT) or in the absence of succinate (data not shown). In addition, these oxidative phosphorylation

inhibitors had no effect on PTM mt-HK activity (data not shown).

Figure 1B shows the ADP dependence for mt-HK inhibition using glucose or fructose as

substrate. ADP caused a half-inhibition of mt-HK activity in the micromolar range with glucose (IC50 ≅ 40

to 70 μM), but much more ADP was needed to cause a similar degree of inhibition (IC50 ≅ 400 to 700

μM) with fructose as substrate. This result suggests that the affinity of mt-HK for glucose and fructose is

different and the inhibitory activity of ADP depends on the substrate used. In fact, the affinity of mt-HK

from PTM was higher for glucose (KM 0.14 mM) than for fructose (KM 1.4 mM). The mt-HK activity

measured with saturating concentrations of hexoses was practically the same (Fig. 1C).

Potato Tuber mt-HK Activity Modulates Oxygen Consumption, ΔΨm and H2O2 Release

The experiments shown in Figure 1 indicated that PTM respiration is important to maintain the

mt-HK activity free of the ADP inhibition. In the next set of experiments, we evaluated the effect of the

mt-HK activity on several mitochondrial functional parameters (Figs. 2, 3 and 4). During PTM respiration,

the addition of small amounts of ADP (0.15 mM) accelerated the rate of O2 consumption (state 3) (Fig.

2A). When all the ADP was converted to ATP, the respiration rate decreased, returning to the resting

state (state 4), but a further addition of glucose permanently stimulated the O2 consumption (Fig. 2A).

Based on previous data from our group (da-Silva et al., 2004; Meyer et al., 2006), glucose-induced O2

consumption in PTM is a result of ADP generated by the mt-HK reaction being transported by the ANT to

the mitochondrial matrix and then being utilized as substrate by the FoF1ATP synthase, decreasing the

ΔΨm and leading to an acceleration of the electron flux in the respiratory electron chain. This can be

confirmed by ΔΨm measurements showing that sequential ADP additions transiently dissipated the ΔΨm

(Figs. 2B and 2C), reaching an estimated ATP concentration of 545 μM. Inclusion of glucose after all

ADP pulses promoted a small, but consistent decrease in ΔΨm (about 3 % of maximal value, Figs. 2B,

2C and 4A), which was reversed by the inclusion of 50 mM MHP (Figs. 2B and 2C). An increase in

glucose concentration to 10 mM further decreased the ΔΨm (Fig. 2C), and interruption of the ADP re-

cycling mechanism by ANT using atractyloside promoted an increase in the ΔΨm (Fig. 2C). These data

show that the mt-HK activity is able to modulate the proton motive force in PTM.

It has been established that mitochondrial H2O2 formation is strongly dependent on high ΔΨm

values (Korshunov et al., 1997). In Figure 3 we observed substantial H2O2 release after induction of

respiration by succinate and ATP. The addition of 1 mM ADP strongly reduced H2O2 release, showing

that a small decrease in ΔΨm due to activation of state 3 respiration, is the main mechanism by which

mitochondrial H2O2 release is prevented (Fig. 3, left trace). A similar blockage in H2O2 release was

observed when ADP was replaced by 5 mM glucose (Fig. 3, right trace). This was reversed by either mt-

HK inhibitors such as MHP (Fig. 3) or NAG (Fig. 4C) by oxidative phosphorylation poisons such as

oligomycin or atractyloside, two inhibitors that promote an increase in ΔΨm in the presence of oxidizable

substrates (Figs. 2, 4A and 4B). These experiments demonstrate that mt-HK activity is able to modulate



9

the ΔΨm and H2O2 release in PTM by an ADP recycling mechanism through ANT/ FoF1ATP synthase

activities. The regulation of H2O2 release from PTM by mt-HK is attained regardless the source of ATP

for the mt-HK is external (Fig. 4B) or from oxidative phosphorylation (Fig. 4A).

Glucose Dependence for mt-HK Activity is Inversely Related to the Rate of H2O2 Release by PTM

The mt-HK activity and the rate of H2O2 release by PTM were measured simultaneously with

increasing amounts of glucose (Fig. 5A). We observed an inverse relation between mt-HK activity and

mitochondrial H2O2 release. The IC50 to inhibit H2O2 release was approximately 20 μM glucose (Fig. 5A).

The dependence on mt-HK activity to abrogate the H2O2 release was confirmed in Figure 5B, when 25

mM NAG, a competitive inhibitor of mt-HK was included in the assay medium. Under this condition, the

IC50 for glucose to inhibit H2O2 release rose to about 800 μM. Importantly, a half maximum inhibition of

the mt-HK activity was able to maintain the rate of H2O2 release at about 15 % of its maximal value (Fig.

5C).

Comparison of the Preventive Antioxidant Role of mt-HK and PUMP Activities in PTM

The rate of H2O2 release, oxygen consumption and ΔΨm levels were titrated with increasing

amounts of a proton ionophore, FCCP, or with the PUMP activator linolenic acid (LA) (Vercesi et al.,

2006) (Fig. 6). In the titration with FCCP (Fig. 6A), it became apparent that a small decrease in the

maximal ΔΨm (about 3 % of AUF of safranine O) by 700 nM FCCP (Fig. 6A, open circles) promoted a

two-fold stimulation of oxygen consumption (Fig. 6A, open stars) and a complete inhibition of H2O2

release (Fig. 6A, open triangles). The effect of maximal mt-HK activity compared with these parameters

was determined and the set point gives a 3 % decrease in ΔΨm, expressed as AUF of safranine O

(closed circle), an increase of 1.8 fold in oxygen consumption (closed stars) and a reduction of at least

95 % in H2O2 release (closed triangle) (see Fig. 6A, vertical dotted line for set point).

In an attempt to compare the impact of mt-HK activity with, that of PUMP activation on the

bioenergetic parameters evaluated above, we next measured the effect of increasing amounts of LA,

either with (Fig. 6B) or without faf-BSA (a protein that sequesters LA) (Fig. 6C). The IC50 for LA to

inhibit H2O2 release was about 30 μM in the presence of faf-BSA (Fig. 6B, open triangles). Under this

specific condition mt-HK activation exerted an even more powerful effect on H2O2 release than did

PUMP activation (Fig. 6C, closed triangle). The IC50 for LA to inhibit H2O2 release when faf-BSA was

omitted from the reaction medium (Fig. 6C) was about 4 μM, confirming the ability of this fatty acid to

activate PUMP. In this condition, 30 μM LA almost completely blocked H2O2 release, similar to mt-HK

activation (Fig. 6C, open and closed triangles).



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Prevention of H2O2 Release by PTM mt-HK Requires Specific Enzyme Association to

Mitochondria

In order to evaluate whether a specific location of mt-HK on mitochondria is required to prevent

H2O2 release we next measured the rate of H2O2 release in PTM using two alternative hexose

substrates, glucose or fructose at 0.5 mM (Fig. 7A). Mt-HK activity using fructose as substrate is much

lower when compared with glucose, as expected from the apparent affinities of these two substrates

(Fig. 1C). In fact, when 0.5 mM fructose was added to the reaction mixture, a negligible effect was

observed on the rate of H2O2 release, contrasting with the strong reduction in the rate of H2O2 release

when 0.5 mM glucose was used as substrate (Fig. 7A). Using fructose as a substrate, we only observed

a decrease in the rate of H2O2 release when the hexose concentration was increased to 10 mM (Fig.

7A). Regardless of the substrate used, oligomycin promptly increased the H2O2 release by impairing the

ADP recycling activity mediated by mt-HK.

In contrast to PTM mt-HK activity, soluble yeast HK exhibits a high affinity for fructose with an

apparent Km of 0.7 mM (Avigad and Englard, 1968; Bernard 1975). Taking advantage of this difference

in kinetic properties between the two enzymes, we next compared the effect of soluble yeast HK

activation, with that of activating the endogenous PTM mt-HK by using fructose as substrate and

measuring the rate of H2O2 release. The rationale was that at 0.5 mM fructose, PTM mt-HK would not be

active, thus not producing ADP, but addition of soluble yeast HK, which has a higher affinity for fructose,

would promptly catalyze this reaction. Thus, under this condition, the ADP produced from the HK

reaction comes only from the exogenous HK activity. Clearly, increasing amounts of the soluble yeast-

HK decreased the rate of H2O2 release in PTM (Fig. 7A). On comparing at the levels of soluble yeast HK

activity required to impair H2O2 release in PTM with the activity of endogenous mt-HK, we observed a

difference of at least two orders of magnitude (Fig. 7B). This result led us to conclude that specific

location of mt-HK at the outer PTM membrane is absolutely essential to improve ADP delivery for the

oxidative phosphorylation, decreasing PTM H2O2 release.

Glucose Impairs H2O2 Release in Potato Tuber Slices

Potato tuber slices incubated with glucose showed a rate of oxygen consumption 1.6-fold higher

than those without glucose or with sucrose (Fig. 8A). The potato tuber slices treated with FCCP showed

oxygen consumption 2.2-fold higher than that observed for the control (Fig. 8A). To ascertain whether

these increased rates in O2 consumption were related to the rate of H2O2 release, we measured the

accumulation of H2O2 in potato tuber slices medium. The response was the opposite to that observed for

the rate of respiration by potato tuber slices in each condition (Figs. 8B and C). The rate H2O2 release in

disk slices was three-fold lower when the slices were incubated with 5 mM glucose compared to the rate

measured in the absence or in the presence of sucrose, a non-phosphorylatable sugar for mt-HK (Fig.

8B, closed circle and open triangle against open circle). When H2O2 release by potato tuber slices was

measured in the presence of glucose and 50 mM NAG, an inhibitor of mt-HK, the rate of H2O2 release

tended to be higher than that measured only in the presence of glucose (Fig. 8B, closed triangles). This



11

result is in accordance with those shown in Figures 3, 4 and 5 using isolated PTM. The H2O2 release

measured in the presence of FCCP and without glucose was as low as that recorded for the potato

slices incubated with 5 mM glucose without FCCP (Fig. 8B, open square). These results indicate that the

preventive effects of glucose on H2O2 release mediated by the mt-HK activity observed in isolated PTM

are working in a similar manner in the potato tuber slices and are associated with increased O2

consumption.

DISCUSSION

Changes in free hexose levels by carbohydrate starvation and reverse feeding lead to variations

in the respiration rate in heterotrophic plant tissues (Brouquisse et al., 1991; Dieuaide et al., 1992).

These free hexose fluctuations may correspond to certain environmental or developmental conditions

where sugar availability may be limiting (Morrell and ap Rees, 1986; Geigenberger and Stitt, 1993; Renz

et al., 1993; Couée et al., 2006) and are associated with ROS generation.

High glucose levels are toxic in several models because of increased release of ROS by glucose

auto-oxidation and metabolism (Couée et al., 2006). The leakage of ROS from mitochondria is as

important in non-photosynthesizing plant cells as it is in mammalian cells (Skulachev, 1996; Skulachev,

1997; Møller, 2001). In animal cells, high glucose concentrations can lead to activation of NADPH

oxidase, which is one of the sites responsible for glucose toxicity (Bonnefont-Rousselot, 2002). However,

it has been proposed that ROS release by mitochondria via the respiratory chain is a causal link between

high glucose levels and the main pathways responsible for cell damage (Russell et al., 1999; Nishikawa

et al., 2000; Russell et al., 2002). In this regard, our group recently demonstrated that mt-HK activity

plays a central role in preventing mitochondrial ROS generation through a steady-state ADP re-cycling in

rat brain (da-Silva et al., 2004). High glucose levels in plants may not trigger mitochondrial ROS

formation as in mammals for the following reasons: (1) plant mt-HK activities are not strongly inhibited by

hexose-6-phosphate as they are in mammals (Renz and Stitt, 1993; Galina et al., 1995; da-Silva et al.,

2001; da-Silva et al., 2004; Meyer et al., 2006); (2) plant mt-HK activities are strongly inhibited by ADP

(Fig. 1B and Galina et al., 1995; da-Silva et a., 2001). These observations may explain the plant cell

tolerance to mitochondrial ROS formation due to high sugar concentration availability compared to

mammalian cells (Nishikawa et al., 2000; Brownlee, 2001; Couée et al., 2006). Thus, the ADP re-cycling

mt-HK activity would play a key preventive antioxidant role by keeping lower ΔΨm and ROS generation

levels in mitochondria.

In the present work we observed mt-HK activity (Table I) in isolated PTM, in agreement with a

recent report (Graham et al., 2007). The mt-HK activity found in PTM is highly dependent on oxygen

consumption, because the activation of mitochondrial respiration by succinate restores the activity levels

of the enzyme (Fig. 1A). On the other hand, the activity of mt-HK is able to promote an acceleration of



12

oxygen consumption after the conversion of ADP to ATP (Fig. 2A). This would ensure that the

hexokinase could respond rapidly to changes in the cellular demand for glucose-6-P, which is known to

be a key intermediate in several metabolic pathways sensitive to ADP/ATP ratio, including glycolysis,

sucrose synthesis, pentose phosphate pathway, and cellulose biosynthesis.

As previously observed for maize mt-HK, mt-HK activity from PTM is also much more sensitive

to ADP inhibition in the micromolar range when the substrate is glucose than with fructose (Fig. 1B)

(Galina et al., 1995; Galina et al., 1999; da-Silva et al., 2001). This result suggests that the affinity of mt-

HK for glucose and fructose is different. In fact this was shown in Figure 1 and is in accordance with the

kinetic behavior of mt-HK from maize seedling roots (Galina et al., 1999).

Because atractyloside and oligomycin impair the decrease of ΔΨm and the preventive role in

H2O2 formation sustained by the mt-HK reaction, we can conclude that this mt-HK activity is able to

promote modulation of electron flux in ETS via a mechanism of ADP re-cycling through ANT:FoF1ATP

synthase complex (Fig. 2 and Fig. 4). This mechanism of control of H2O2 formation was previously

observed in rat brain mitochondria which contains large amounts (more than 80% of total tissue content)

(Wilson, 2003; da-Silva et al., 2004) of mt-HK activity, but it had not been described so far for plant

mitochondria.

Besides the classical antioxidant enzymes (SOD, catalase, and ascorbate-glutathione

peroxidase), two systems have been identified as participants in controlling pro- and anti-oxidant

balance in plants, the alternative oxidase (AOX) and the uncoupling protein (PUMP). Ultimately, these

two systems work to decrease the long-lived ubisemiquinone (UQH●) concentration which, in turn, is

capable of directly reducing O2 (Skulachev, 1996; Skulachev, 1997). In isolated PTM, the mt-HK activity

achieves the same effect on the ubiquinone pool by decreasing the ΔΨm and increasing the O2

consumption after mt-HK activation (Figs. 2, 4 and 5). These observations are in accordance with the

respiratory control theory in which the ΔΨm is decreased by the presence of ADP (Figs. 2B and C). It is

known that a small decrease in the ΔΨm leads to a large reduction in the rate of H2O2 generation

(Korshunov et al., 1997). Our findings are in line with these data because the activation of mt-HK activity

by glucose reduces ΔΨm and practically abolished the rate of H2O2 generation (Figs. 2 and 3). In

addition, the mt-HK system reduces H2O2 generation by 50 % in PTM at glucose concentrations as low

as 20 μM (Fig. 5A). At saturating glucose concentration, the mt-HK activity level of 30 % (about 20 mU)

of the total amount recovered in PTM is sufficient to reduce the rate of H2O2 generation by 50 % (Figs.

5C and 7B). The half-maximal glucose concentration needed to activate mt-HK (about 100 μM) causes

an almost complete inhibition of H2O2 generation (Figs. 1C and 5A). This indicates that the mt-HK does

not need to be saturated with glucose to cause a significant impairment in ROS generation. That

saturation with glucose is not required is confirmed by comparing the rate of H2O2 generation and the

activity levels of mt-HK treated with its competitive inhibitor, NAG, which promotes a displacement of

glucose concentration required for inhibition to a higher values (i.e. a decrease by more than 80% in

H2O2 generation is achieved only at about 1 mM glucose) (Fig. 5B). A threshold plot of the activity levels



13

of mt-HK and the rate of H2O2 generation reveals that 50 % inhibition in ROS generation requires only

30 % of the total mt-HK activity bound to the PTM membrane (which corresponds to approximately 12 %

of total potato tuber HK activity) (Table I). This estimate leads us to propose that only 6 % of total

glucose phosphorylation occurring at the surface of the PTM membrane would lead to almost total

blockage of H2O2 generation. According, the data on rate of H2O2 release observed in potato tuber slices

are in agreement with the expectation that a small activation of mt-HK by glucose is sufficient to inhibit its

release in slices (Figs. 8B and C). Interestingly, the decrease in ROS generation was accompanied by a

corresponding increase in the oxygen consumption when glucose was added to the incubation medium

of potato tuber slices (Fig 8A).

The effect of glucose in decreasing the H2O2 release by potato tuber slices and in isolated PTM

could be due to a tightly bound mitochondrial HK that guides the ADP delivery to FoF1ATP synthase via

ANT in an efficient channeling to the mitochondrial matrix (Fig 7). Almost two orders of magnitude more

activity from an unbound HK form than from mt-HK is needed to reduce the rate of H2O2 release in

isolated PTM (Fig. 7B). These data suggest that the access to ADP is substantially increased by mt-HK

in PTM and in potato tuber slices. Several lines of evidence indicate that, in addition to its activity, the

localization of mt-HK is relevant for mitochondrial respiration (Moore and Jöbsis, 1970; BeltrandelRio

and Wilson, 1991; Galina et al., 1995). This possibility would be explained in mammalian mitochondria

by the mt-HK binding site in the VDAC-ANT complex. According to our previous study, mt-HK

localization seems to be critical for performing its preventive antioxidant activity, as the ADP would be

rapidly delivered through the VDAC-ANT complex to the FoF1ATP synthase, which phosphorylates it at

the expense of ΔΨm (da-Silva et al., 2004). Although the binding mechanism of mt-HK to plant

mitochondrial outer membranes is not yet fully established (Rezende et al., 2006), the data presented in

Figure 7 is similar to data from previous study in rat brain mitochondria which demonstrated that the mt-

HK activity is more effective in stimulating respiration in the presence of glucose than its nonbindable

chymotrypsin-treated mt-HK form (Moore and Jöbsis, 1970; BeltrandelRio and Wilson, 1991).

Comparison of ADP-recycling activity of mt-HK with the activity of St-UCP as preventive

antioxidant systems in PTM (Fig. 6) shows that mt-HK is able to decrease the ΔΨm, accelerate oxygen

consumption and block H2O2 release to the same extent as observed with St-UCP activated by 30 μM

linolenic acid (Fig. 6C). In contrast, when the St-UCP activity is inhibited by the presence of 1 mg/mL faf-

BSA and 1 mM ATP, the mt-HK activity is even more potent than St-UCP in preventing ROS generation

(Fig. 6B).

These similarities between mt-HK and St-UCP in the response with regard to ROS formation

may indicate that potato tubers have complementary mechanisms against oxidative damage induced by

respiration in heterotrophic plant tissues. In conditions of higher oxidative metabolism fueled by hexoses

may lead to increased ROS formation, the mt-HK activity would play a predominant role as a preventive

mechanism. On the other hand, when the rate of fatty acid β-oxidation is increased, the UCP becomes

the main mechanism to prevent the accumulation of ROS. In plant tissues, we cannot exclude the



14

possible operation of AOX in detoxifying the formation of superoxide anions. However, in potato tuber

under no abiotic stress, the AOX activity levels are low (Calegario et al., 2003; Considine et al., 2003). In

our experimental conditions the cyanide-resistant respiration is less than 3% when using succinate or

NADH as oxidizable substrates, and about 10% when using pyruvate plus malate as substrates (data not

shown).

CONCLUSION

Thus, besides its involvement in general plant sugar metabolism, in glucose sensing (Rolland et

al., 2006) and in the regulation of programmed cell death (Kim et al., 2006), we propose that mt-HK

plays a specific role in generating ADP to support oxidative phosphorylation, thereby avoiding an ATP

synthesis–related limitation of respiration and subsequent H2O2 release in plants.

MATERIALS AND METHODS

Chemicals and Biological Materials

ADP, ATP, FCCP, horseradish peroxidase, rotenone, safranine O, MHP, NAG, yeast

hexokinase, fatty acid-free bovine serum albumin, β-NAD+, linolenic acid, oligomycin and G6PDH from

Leuconostoc mesenteroides, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Percoll was

from Amersham Biosciences (USA). Amplex Red was purchased from Invitrogen. All other reagents

were analytical grade. Potato tubers (Solanum tuberosum) were purchased from a local supermarket.

Isolation of Potato Tuber Mitochondria by Self-generated Percoll Gradient (PTM)

PTM were obtained as previously described (Neuburger et al., 1982) using a cold extraction

buffer containing: 10 mM Hepes/Tris pH 7.4; 0.3 M manitol; 2 mM EGTA ; 5 mM EDTA; 0.3 mM PMSF;

20 mM β-mercaptoethanol and 0.1 g % (w/v) faf-BSA. The homogenate was strained through eight

layers of cheesecloth and centrifuged at 3,000 x g at 4 °C for 3 min. The supernatant was centrifuged at

12,000 x g at 4 °C for 10 min. The supernatant (S12) was used to determine enzymatic activities. The

mitochondrial pellet (P12) was resuspended in 5 mL of ice-cold extraction buffer and layered in Hitachi

P50 centrifuge tubes containing 35 mL of cold extraction buffer containing 28 % (v/v) Percoll and

centrifuged at 40,000 x g at 4 °C for 30 min. After centrifugation, three major bands were obtained in

sequence; the first band (fraction 1) at the top and the middle band (fraction 2) were used for further

PTM isolation or for enzymatic activity determinations. Mitochondrial activity markers were found to be

enriched in fraction 2. The mitochondrial fraction was removed and diluted with extraction buffer without

Percoll and centrifuged twice at 12,000 x g at 4 °C for 10 min. The final pellet was resuspended in 0.6

mL of extraction buffer and kept in an ice-water bath. The final protein concentration varied from 10 to 20

mg/mL.



15

Enzyme Assays and Kinetic Parameters

The activity of mt-HK was determined by a coupled assay according to Galina et al., (1995).

Briefly, mt-HK activity was determined by NADH formation following the absorbance at 340 nm at 28oC.

The assay medium contained: 20 mM Tris-HCl pH 7.4, 5 mM glucose, 6 mM MgCl2, 1 mM β-NAD+, 1

U/mL G6PDH, 2 mM PEP, 0.1% (v/v) Triton X-100 and 10 U/ml pyruvate kinase. The reaction was

started by adding 1 mM ATP. The final mitochondrial protein concentration varied from 0.05 to 0.1

mg/mL. When mt-HK activity was measured in intact PTM, Triton X-100 was omitted from the reaction

medium which was in this case the same as the respiration buffer used to measure oxygen consumption.

Glucose 6-P dehydrogenase (G6PDH) activity was assayed in a reaction medium containing 50

mM Tris-HCl buffer, pH 7.4, 6 mM MgCl2, 0.1% (v/v) Triton X-100 and 0.5 mM β-NADP+. The reaction

was started by adding 1 mM glucose 6-P, and G6PDH activity was determined by measuring the

absorption of β-NADPH formation at 340 nm.

FoF1 ATPase activity was determined by measuring the release of Pi from ATP in two different

reaction media: (1) in the absence or (2) in the presence of 5 mM NaN3. Both contained 20 mM Tris-HCl

pH 8.0, 5 mM MgCl2, 2 mM ATP and 1 μM FCCP. The reaction was started by the addition of PTM

protein and the difference between the activities in media (1) and (2) was considered as an authentic

FoF1 ATPase. The PTM protein concentration varied from 0.06 to 0.1 mg/mL.

The kinetic parameters were estimated by non-linear regression analysis applied to the

Michaelis-Menten equation using the program package supplied by Origin software (Galina et al., 1999).

Oxygen Uptake Measurements

Oxygen uptake was measured in an oximeter fitted with a water-jacket Clark-type electrode

(Yellow Springs Instruments Co., Model 5300, USA) or in Oxytherm System for Photosynthesis &

Respiration Measurements in Liquid-Phase – Hansatech Inst. (England).The PTM (0.2 mg/mL) were

incubated with 1 to 1.5 mL of the standard respiration buffer containing 0.3 M mannitol, 10 mM Tris-HCl

pH 7.2, 3 mM MgSO4, 10 mM NaCl, 5 mM KH2PO4, 0.3 mM β-NAD+ and 0.1% (v/v) faf-BSA. The cuvette

was closed immediately before starting the experiments. Respiratory control ratio (RCR) values were

obtained with isolated PTM, after complex I inhibition by 1 μM rotenone and complex II activation by 10

mM succinate. Other additions are indicated in the figure legends.

ΔΨm Determination

The mitochondrial membrane potential was measured by using the fluorescence signal of the

cationic dye safranine O, which is accumulated and quenched inside energized mitochondria (Akerman

and Wikströn, 1976). PTM (0.2 mg protein/mL) were incubated in the standard respiration buffer

supplemented with 15 µM safranine. FCCP (2 µM) was used as a positive control to collapse ΔΨm.

Fluorescence was detected with an excitation wavelength of 495 nm (slit 5 nm) and an emission



16

wavelength of 586 nm (slit 5 nm) using a Hitachi Model F-3010 spectrofluorimeter (Tokyo). Data were

reported as arbitrary fluorescence units (AFUs). Other additions are indicated in the figure legends.

Determination of Mitochondrial H2O2 Release

The H2O2 released from PTM was determined by the Amplex Red oxidation method, as previously

described (Smith et al., 2004). Briefly, mitochondria (0.2 mg protein/ml) were incubated in the standard

respiration buffer supplemented with 10 µM Amplex Red and 5 U/mL horseradish peroxidase. Fluorescence

was monitored at excitation and emission wavelengths of 563 nm (slit 5 nm) and 587 nm (slit 5 nm),

respectively. Calibration was performed by the addition of known quantities of H2O2.

Potato Tuber Slice Assays

Small square pieces were cut (8 mm diameter, 2 mm thickness) perpendicular to the stolon-apex

axis of potato tuber (Tiessen et al., 2002). The tuber slices were taken from the middle of the tuber,

avoiding the outer 3 mm and the tuber skin. The slices were incubated for 2 hours in 10 mM MES-KOH,

pH 6.5, in different conditions as shown in figure legend. In oxygen consumption measurements we used

16 slices (0.3 to 0.45 g total slices) in a Hansatech oxygraph chamber of 1 mL and the oxygen

consumption rate was monitored. For H2O2 generation 20 slices were placed in 10 mL of the incubation

buffer in an orbital shaker at 28oC. At different times, aliquots of 0.5 mL were removed and added to 1.5

mL respiration buffer containing 10 μM Amplex Red and 5 U/mL horseradish peroxidase and the

fluorescence measured was taken as the amount of H2O2 accumulated in the incubation buffer of potato

tuber slices.

Protein determination

The protein concentration was determined as described by Lowry (1951), using BSA as standard.

Statistical Analysis

Data were plotted with Origin 7.0 and analyzed by one-way ANOVA and a posteriori Tukey´s test. P

values < 0.05 were considered statistically different.

ACKNOWLEDGMENTS

We are grateful for Dr Martha Sorenson for kind and valuable comments in improving the paper. This

work is dedicated to Leopoldo de Meis in honor of his 70th birthday.



17

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23

FIGURE LEGENDS

Figure 1. Mitochondrial Hexokinase Activity is Dependent on Respiration, Kinetic Properties of ADP

Inhibition of mt-HK Activity and Hexose Phosphorylation. A, The time course of G6P production by mt-

HK in PTM after addition of 5 mM glucose and 1 mM ATP is shown in traces 1, 2 and 3. Isolated PTM,

0.2 mg/ml, was added to respiration buffer containing 1 U/mL G6PDH and 1 mM β-NAD+. The reaction

temperature was 25oC. When indicated (↓), 10 mM succinate (Succ) or 50 mM mannoheptulose (MHP)

was added to the respiration medium. In trace 3, 0.3 mM ADP was included before starting the mt-HK

reaction with ATP and glucose. In trace 2, the reaction course was as in trace 1, but when indicated

MHP was added. B, Inhibition of the mt-HK activity by ADP in the presence of 5 mM glucose (closed

circles) or fructose (open circles). The maximal activity (Vo/Vi) of 1.0 was 160 ± 23 (mean ± SE, n = 5)

for glucose or 107 ± 6 (mean ± SE, n = 4) for fructose (nmol Hex6P.min-1.mg ptn-1). In C, the glucose

(closed circles) dependence for mt-HK activity revealed a KM of 148 ± 12 μM. The fructose (open circles)

dependence revealed a KM of 1375 ± 380 μM. The Vmax was 150 or 121 nmol G6P.min-1.mg ptn-1 for

glucose or fructose, respectively. The figure shows a representative experiment. The mean values ± SE

are from at least four independent preparations.

Figure 2. The Rate of Oxygen Consumption and Membrane Potential (ΔΨm) are Modulated by mt-HK

Activity. A, Oxygen uptake was measured using the respiration buffer described in Materials and

Methods. Numbers indicates the rate of oxygen consumption (nmol O2. min-1. mg ptn-1). B, The ΔΨm was

measured with fluorescence quenching of safranine O. The numbers indicated are the final ADP

concentration, in micromolar, added to the cuvette in each pulse. After the inclusion of 200 μM ADP, a

pulse of 5 mM glucose (Glc) was added followed by 50 mM mannoheptulose (MHP) to inhibit mt-HK

activity. When indicated, 100 μM atractyloside (Atr) was added to the reaction medium. In C is shown a

higher magnification of the ΔΨm trace in 2B. The figure shows a representative experiment. The

membrane potential and the oxygen consumption measurements were repeated with at least fifteen

independent PTM preparations.

Figure 3. Effect of mt-HK Activity on H2O2 Release by PTM. H2O2 release in two independent traces

shows the effect of 1mM ADP (left trace) or 5 mM glucose (Glc) (right trace) during a time course of

H2O2 release. The H2O2 release was detected by Amplex Red and peroxidase, after induction of

respiration by 10 mM succinate and 1 mM ATP when indicated by vertical lines. In the right trace, 50 mM

mannoheptulose (MHP) was added when indicated. The PTM was 0.2 mg/mL and the reaction was

performed at 25oC. The figure shows a representative experiment. The H2O2 release by PTM was

measured with at least ten independent PTM preparations.



24

Figure 4. Effects of Substrates, Uncouplers and Inhibitors on PTM Membrane Potential (ΔΨm) and H2O2

Release. After addition of 10 mM succinate, the agents were added sequentially in the order indicated

below each bar, separated by slashes (/), to evaluate ΔΨm (A and B) and H2O2 release (C). The

concentrations were: 5 μM FCCP; 150 – 200 μM ADP; 1 mM ATP; 1 - 4 mM glucose; 150 μM

atractyloside; 2 μg/mL oligomycin; 50 mM mannoheptulose; 50 mM N-acetylglucosamine. The

concentration of mitochondrial protein varied between 0.2 and 0.3 mg / mL. A Decrease in membrane

potential is represented as percent of depolarization related to the maximal polarization observed after

addition of succinate. The 100% rate of mitochondrial H2O2 release (225.1 ± 14 pmol.min -1.mg ptn-1)

was that following the addition of 10 mM succinate and 1 mM ATP in at least 6 independent potato tuber

mitochondrial preparations. The addition of glucose was performed after the ΔΨm return to the

hyperpolarization state from state 3 to state 4. Asterisks represent statistically significant differences

between groups, determined by one-way ANOVA and Tukey´s test. (*) p < 0.0001 relative to

Suc/ADP/glucose. (**) p < 0.0001 relative to Suc/ATP/glucose. The values are mean ± SE of percent

values of the numbers (n) of experiments.

Figure 5. Glucose Concentration Dependence for Activate the mt-HK Activity and Inhibition of H2O2

Release by PTM. In A is shown the rate of H2O2 release (open circles) and mt-HK activity (closed circles)

in the absence of NAG, an inhibitor of mt-HK activity. In B is shown the rate of H2O2 release (open

triangles) and mt-HK activity (closed triangles) in the presence of 25 mM NAG. The PTM H2O2 release

was induced by the addition of 10 mM succinate and 1 mM ATP. The final protein concentration of PTM

was 0.2 mg/mL in the respiration buffer. In C is shown a plot of mt-HK activity against the rate of H2O2

release from the data in A and B. The maximal rate of H2O2 release was 210 pmol. min-1. mg ptn-1

measured in the absence of added glucose; the maximal rate of mt-HK activity was 105 nmol. min-1. mg

ptn-1 measured at 10 mM glucose. The values shown in C are means ± SE of at least three independent

PTM preparations.

Figure 6. Comparison of the Preventive Antioxidant Role of mt-HK and PUMP Activation in PTM. A

shows the FCCP titration the rate of H2O2 release (open triangles), rate of oxygen consumption (open

stars) and membrane potential (open circles). The closed symbols plotted on a dotted vertical line

represent the values of these parameters as percentage of maximum, when mt-HK is fully active in the

absence of uncoupler. This activation is equivalent to the uncoupling effect of 700 nM FCCP. In B is

shown the effect of linolenic acid titration, an activator of PUMP, on the same parameters measured in

the presence of 1 mg/mL faf-BSA (which sequesters free fatty acids) and 1 mM ATP (an antagonist of

plant UCP). In C is shown the same experiment as in B, but without faf-BSA and ATP. The mt-HK

activity was induced by 5 mM glucose in the presence of 1 mM ATP. The symbols plotted once dotted

vertical line represent the values of these parameters as percentage of maximum, when mt-HK is fully

active and PUMP is inhibited. The values shown are means ± SE of at least three independent PTM



25

preparations. The error bars for bioenergetics parameters when mt-HK is active shown in A were similar

in B and C, but were omitted for clarity.

Figure 7. Specific Localization of mt-HK Is Important to Prevent H2O2 Release in PTM. In A, left and

middle traces, show different fructose or glucose concentrations being added during a time course of

H2O2 release in PTM to induce the mt-HK activity to different activity levels (the final concentrations are

indicated on the traces). Oligomycin was added to a final concentration of 2 μg/mL. In the right graph,

0.5 mM fructose was added as a saturating substrate for exogenously added soluble yeast-HK. Different

amounts of yeast-HK were added to the respiration buffer which contained 0.2 mg/mL PTM. The

numbers in different traces indicate the final mU attained in the 2 mL reaction mixture. In B is plotted the

percentage of maximum rate of H2O2 release by PTM against the activity level of mt-HK obtained with

different glucose concentrations (Figure 4A) (closed circles) or yeast-HK (open circles). The mt-HK

activity was induced by 0.5 mM (closed star) or 20 mM (open star) fructose in PTM to measure the H2O2

release without yeast-HK. The figure shows a representative experiment. The measurements of H2O2

release by PTM were repeated at least with three independent PTM preparations.

Figure 8. H2O2 Release and Oxygen Consumption in Potato Tuber Slices. A shows the rate of O2

consumption in potato tuber slices. B shows the time course of H2O2 release in potato tuber slices.

Twenty slices were maintained in 10 mM MES-KOH, pH 6.5 medium with different media that contained:

no added sugars (closed circles); 5 mM glucose (open circles); 5 mM glucose plus 50 mM NAG (closed

triangles); 5 mM sucrose (open triangles) and no added glucose plus 1 μM FCCP (open square). C

shows the rate of H2O2 release from potato tuber slices under these same conditions. The values

represent means ± S.E. of four independent experiments.

* At the 0.05 level, the population means are significantly different.



26

Table I Mitochondrial location of potato tuber particulate hexokinase activitya

Fractions Enzyme activities

(mU/mg ptn)

F1FOATPase Hexokinase Glucose 6-P

dehydrogenase

Total

protein in

fraction

(mg)

Total HK

activity per

fractiond

(mU)

(% of total)

Homogenate

6.4 ± 2.2

(n=7)

9.7 ± 2.4

(n=8)

16.4 ± 1.9

(n=4)

N.D.

S12b 8.4 ± 2.5

(n=7)

2.6 ± 0.6

(n=8)

25.5 ± 1.2

(n=6)

2150 ± 97

(n=3)

5200

(82%)

P12c 22.4 ± 8.1

(n=6)

32.1 ± 3.5

(n=8)

30.8 ± 3.8

(n=6)

36 ± 7

(n=5)

1156

(18%)

P12 after Percoll Gradient

Fraction 1

5.7 ± 0.5

(n=4)

20.2 ± 7.7

(n=7)

32.2 ± 2.9

(n=4)

13 ± 1.6

(n=3)

262

(4%)

Fraction 2

83.9 ± 22.6

(n=7)

75.8 ± 16

(n=8)

0.3 ± 0.1

(n=6)

10 ± 1

(n=3)

758

(12%)

a The F0F1 ATPase, hexokinase and Glucose 6-P dehydrogenase activities were measured

as described in Experimental procedures. The reaction time was 15 min and the temperature

was 28oC. The activities represent mean ± S.E. of, at least 3 independent preparations, as shown in

parenteses. b S12 is the supernatant of 12,000 g/ 10 min c P12 is the particulate pellet (12,000 g/10 min) d Total HK activity (100%) was considered to be the sum of the HK activities recovered in S12 plus P12

fractions.



Camacho-Pereira et al. Figure 1

Figure 1. Mitochondrial Hexokinase Activity is Dependent on Respiration, Kinetic Properties of ADP Inhibition of mt-HK

Activity and Hexose Phosphorylation. A, The time course of G6P production by mt-HK in PTM after addition of 5 mM

glucose and 1 mM ATP is shown in traces 1, 2 and 3. Isolated PTM, 0.2 mg/ml, was added to respiration buffer containing

1 U/mL G6PDH and 1 mM β -NAD+. The reaction temperature was 25oC. When indicated (↓ ), 10 mM succinate (Succ) or 50 mM mannoheptulose (MHP) was added to the respiration medium. In trace 3, 0.3 mM ADP was included before starting

the mt-HK reaction with ATP and glucose. In trace 2, the reaction course was as in trace 1, but when indicated MHP was

added. B, Inhibition of the mt-HK activity by ADP in the presence of 5 mM glucose (closed circles) or fructose (open

circles). The maximal activity (Vo/Vi) of 1.0 was 160 ± 23 (mean ± SE, n = 5) for glucose or 107 ± 6 (mean ± SE, n = 4) or fructose (nmol Hex6P.min-1.mg ptn-1). In C, the glucose (closed circles) dependence for mt-HK activity revealed a KM of

148 ± 12 µµµµM. The fructose (open circles) dependence revealed a KM of 1375 ± 380 µµµµM. The Vmax was 150 or 121 nmol G6P.min-1.mg ptn-1 for glucose or fructose, respectively. The figure shows a representative experiment. The mean values

± SE are from at least four independent preparations.

MHP

Succ

Glc

trace 1

trace 2

trace 3

60 sec

ATP

50 mABS340

0 10 100 1000

0,2

0,4

0,6

0,8

1,0

Fraction of maximal HK activity (Vo/Vi)

ADP, µµµµM

Glucose

Fructose

0 1000 2000 3000 10000

0,2

0,4

0,6

0,8

1,0

CB

Vmax 0.93489 0.02047

k 148.13775 12.07055

Vmax 1.13023 0.10836

k 1375.22003 381.95563

Hexose, µµµµM

Glucose

FructoseA

Glc 6-P

1.0

0.8

0.6

0.4

0.2

1.0

0.6

0.8

0.4

0.2




ADP

Glc

MHPGlc

Suc

0.15 mM ADP

Suc

0.15 mM ADP

5 mM Glc

B C

0.3

AU

F

8 A

UF

2 min. 2 min.

A

Mito

ADP, µµµµM AtrMHP

Glc

Glc

Atr50

150100

200

121

62

198

40 n

mo

l O

2

2 min.

72

25

Figure 2. The Rate of Oxygen Consumption and Membrane Potential (∆∆∆∆Ψm) are Modulated by mt-HK

Activity. A, Oxygen uptake was measured using the respiration buffer described in Materials and Methods.

Numbers indicates the rate of oxygen consumption (nmol O2 . min-1. mg ptn-1). B, The ∆∆∆∆Ψm was measured

with fluorescence quenching of safranine O. The numbers indicated are the final ADP concentration, in

micromolar, added to the cuvette in each pulse. After the inclusion of 200 µµµµM ADP, a pulse of 5 mM glucose

(Glc) was added followed by 50 mM mannoheptulose (MHP) to inhibit mt-HK activity. When indicated, 100

µµµµM atractyloside (Atr) was added to the reaction medium. In C is shown a higher magnification of the ∆∆∆∆Ψm

trace in 2B. The figure shows a representative experiment. The membrane potential and the oxygen

consumption measurements were repeated with at least fifteen independent PTM preparations.




Figure 3. Effect of mt-HK Activity on H2O2 Release by PTM. H2O2 release in two independent traces shows

the effect of 1mM ADP (left trace) or 5 mM glucose (Glc) (right trace) during a time course of H2O2 release.

The H2O2 release was detected by Amplex Red and peroxidase, after induction of respiration by 10 mM

succinate and 1 mM ATP when indicated by vertical lines. In the right trace, 50 mM mannoheptulose (MHP)

was added when indicated. The PTM was 0.2 mg/mL and the reaction was performed at 25oC. The figure

shows a representative experiment. The H2O2 release by PTM was measured with at least ten independent

PTM preparations.

ATP

Succ

ADP

60 sec

H

2O

2

350 pmol

MHP

Glc

ATP

Succ



Suc

Suc / FCCP

Suc / ADP

Suc / ADP / Glc At

r

OligoMHPNAG

0

2

4

6

8

10

12

14

50

100

150

* **

Decrease of ∆Ψ

∆Ψ

∆Ψ∆Ψ

mform

ed by succinate.

(% of Maxim

um)

*

Suc/ADP/Glc Suc / ATP

Suc / FCCP

Suc / ATP / Glc At

r

OligoMHP

NAG

(5)(5)(5)(5)

(10)

(5)(5)(5) (7)

(10)

Suc/ATP/Glc

CB

Suc/ATP/Glc

A

(10)

Suc

Suc / ATP

Suc / FCCP

Suc / ATP / Glc A

tr

OligoMHP

NAG

0

20

40

60

80

100 (4)(5)(6)(5)

(11)

(8)

(10)

****** Rate of mitochondrial H

2O

2 release.

(% of Maxim

um)

**


Figure 4. Effects of Substrates, Uncouplers and Inhibitors on PTM Membrane Potential (∆Ψm) and H2O2 Release. After addition of 10 mM

succinate, the agents were added sequentially in the order indicated below each bar, separated by slashes (/), to evaluate ∆Ψm (A and

B) and H2O2 release (C). The concentrations were: 5 µM FCCP; 150 – 200 µM ADP; 1 mM ATP; 1 - 4 mM glucose; 150 µM atractyloside;

2 µg/mL oligomycin; 50 mM mannoheptulose; 50 mM N-acetylglucosamine. The concentration of mitochondrial protein varied between

0.2 and 0.3 mg / mL. A, Decrease in membrane potential is represented as percent of depolarization related to the maximal polarization

observed after addition of succinate. The 100% rate of mitochondrial H2O2 release (225.1 ± 14 pmol.min -1. mg ptn-1) was that following

the addition of 10 mM succinate and 1 mM ATP in at least 6 independent potato tuber mitochondrial preparations. The addition of glucose

was performed after the ∆Ψm return to the hyperpolarization state from state 3 to state 4. Asterisks represent statistically significant

differences between groups, determined by one-way ANOVA and Tukey´s test. (*) p < 0.0001 relative to Suc/ADP/glucose. (**) p <

0.0001 relative to Suc/ATP/glucose. The values are mean ± SE of percent values of the numbers (n) of experiments.




Figure 5. Glucose Concentration Dependence for Activate the mt-HK Activity and Inhibition of H2O2 Release by PTM. In A is shown the

rate of H2O2 release (open circles) and mt-HK activity (closed circles) in the absence of NAG, an inhibitor of mt-HK activity. In B is

shown the rate of H2O2 release (open triangles) and mt-HK activity (closed triangles) in the presence of 25 mM NAG. The PTM H2O2

release was induced by the addition of 10 mM succinate and 1 mM ATP. The final protein concentration of PTM was 0.2 mg/mL in the

respiration buffer. In C is shown a plot of mt-HK activity against the rate of H2O2 release from the data in A and B. The maximal rate of

H2O2 release was 210 pmol. min-1. mg ptn-1 measured in the absence of added glucose; the maximal rate of mt-HK activity was 105

nmol. min-1. mg ptn-1 measured at 10 mM glucose. The values shown in C are means ± SE of at least three independent PTM

preparations.

C

0 10 100 1000 10000

B

Glucose, µµµµMGlucose, µµµµM

% of maxim

al mt-HK activity

A

0

20

40

60

80

100

% of maxim

al rate of ROS form

ation

% m

t-HK activ

ity with

25 m

M NAG

0 10 100 1000 10000

0

20

40

60

80

100

% ra

te of R

OS fo

rmatio

n with

25 m

M NAG

0 20 40 60 80 100

0

20

40

60

80

100

% of maxim

al H

2O

2 release rate

% of maximal mt-HK activity

in Potato Tuber Mitochondria



Figure 6. Comparison of the Preventive Antioxidant Role of mt-HK and PUMP Activation in PTM. A shows the

FCCP titration the rate of H2O2 release (open triangles), rate of oxygen consumption (open stars) and

membrane potential (open circles). The closed symbols plotted on a dotted vertical line represent the values of

these parameters as percentage of maximum, when mt-HK is fully active in the absence of uncoupler. This

activation is equivalent to the uncoupling effect of 700 nM FCCP. In B is shown the effect of linolenic acid

titration, an activator of PUMP, on the same parameters measured in the presence of 1 mg/mL faf-BSA (which

sequesters free fatty acids) and 1 mM ATP (an antagonist of plant UCP). In C is shown the same experiment as

in B, but without faf-BSA and ATP. The mt-HK activity was induced by 5 mM glucose in the presence of 1 mM

ATP. The symbols plotted once dotted vertical line represent the values of these parameters as percentage of

maximum, when mt-HK is fully active and PUMP is inhibited. The values shown are means ± SE of at least

three independent PTM preparations. The error bars for bioenergetics parameters when mt-HK is active shown

in A were similar in B and C, but were omitted for clarity.


A B C

0,00 0,1 1 10

0

20

40

60

80

95

100

Bioenergetic m

itochondrial Parameters

(% of maxim

al)

FCCP, µ µ µ µM

0,0 1 10 1000

20

40

60

80

98

99

100

Linolenic acid, µµµµM

0,0 1 10 1000

20

40

60

80

98

99

100

0.0 0.1 0.0 0.0



0 10 100 1000

0

20

40

60

80

100

Rate of MItochondrial H

2O

2 release

(% of maxim

al rate)

HK activity, mU

Oligo

Oligo

0.5 mM Glc

10 mM

Fruct

0.5 mM

Fruct

Succ +

ATP

B

60 sec

3150

1575

630

378 189

Yeast-HK (mU)

Succ +

ATP

0.5 mM

Fruct

H

2O

2

700 pmol

A


Figure 7. Specific Localization of mt-HK Is Important to Prevent H2O2 Release in PTM. In A, left and middle traces, show different

fructose or glucose concentrations being added during a time course of H2O2 release in PTM to induce the mt-HK activity to different

activity levels (the final concentrations are indicated on the traces). Oligomycin was added to a final concentration of 2 µg/mL. In the

right graph, 0.5 mM fructose was added as a saturating substrate for exogenously added soluble yeast-HK. Different amounts of

yeast-HK were added to the respiration buffer which contained 0.2 mg/mL PTM. The numbers in different traces indicate the final mU

attained in the 2 mL reaction mixture. In B is plotted the percentage of maximum rate of H2O2 release by PTM against the activity

level of mt-HK obtained with different glucose concentrations (Figure 4A) (closed circles) or yeast-HK (open circles). The mt-HK

activity was induced by 0.5 mM (closed star) or 20 mM (open star) fructose in PTM to measure the H2O2 release without yeast-HK.

The figure shows a representative experiment. The measurements of H2O2 release by PTM were repeated at least with three

independent PTM preparations.



0 30 60 90 120

Potato Tuber Slices

H2O

2 , pmol/ g of fresh weight

35.0

52.5

17.5

CB

minutes

A

0.0

Control G

lc

Glc + NAG

Sucrose

FCCP

0,0

0,4

0,8

1,2

1,6

*

**

Rate of H2O2 release

pmol / 30 m

in . g of fresh weigth

*

Control

Glc

Glc + NAG

Sucrose

FCCP

0

5

10

15

20

25

30

*

Rate of O2 consumption,

nmol/ m

in.g tissue

Figure 8. H2O2 Release and Oxygen Consumption in Potato Tuber Slices. A shows the rate of O2 consumption in potato tuber slices.

B shows the time course of H2O2 release in potato tuber slices. Twenty slices were maintained in 10 mM MES-KOH, pH 6.5 medium

with different media that contained: no added sugars (closed circles); 5 mM glucose (open circles); 5 mM glucose plus 50 mM NAG

(closed triangles); 5 mM sucrose (open triangles) and no added glucose plus 1 µM FCCP (open square). C shows the rate of H2O2

release from potato tuber slices under these same conditions. The values represent means ± S.E. of four independent experiments.

•At the 0.05 level, the population means are significantly different.


1.6

1.2

0.8

0.4

0.0



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