properties of aminotransferases from...

Annals of West University of Timişoara, ser. Biology, 2014, vol XVII (2), pp.111-122

111

PROPERTIES OF AMINOTRANSFERASES FROM

TELADORSAGIA CIRCUMCINCTA

Noorzaid MUHAMAD

a, David C. SIMCOCK

bcd, Heather V. SIMPSON

e,

Kevin C. PEDLEYb, Simon BROWN

cf

aUniversity Kuala Lumpur, Royal College of Medicine Perak, Ipoh, Perak, Malaysia, bInstitute of Food, Nutrition and Human Health, Massey University, New Zealand,

cDeviot Institute, Deviot, Tasmania 7275, Australia,

dFaculty of Medicine, Health and Molecular Sciences, James Cook University, Australia,

eInstitute of Veterinary and Biomedical Sciences, Massey University, New Zealand,

fSchool of Human Life Sciences, University of Tasmania, Launceston, Tasmania 7250,

Australia

Corresponding author e-mail: [email protected] Received 30 August 2014; accepted 4 November 2014

ABSTRACT Activities of several aminotransferases were measured in L3 and adult Teladorsagia

circumcincta, but most of these had maximal activity of less than 8 nmol min-1

mg-1

protein. Only aspartate aminotransferase (AspAT) and alanine aminotransferase

(AlaAT) activities exceeded this value and some kinetic properties of these enzymes

were characterised. For L3 AspAT, the apparent Kms were 1.2 mM, 0.13 mM, 0.11 mM

and 0.04 mM for aspartate, α-ketoglutarate, glutamate and oxaloacetate, respectively,

and the apparent Vmaxs were 960 nmol min-1

mg-1

protein for aspartate deamination

and 420 nmol min-1

mg-1

protein in the direction of glutamate deamination. For L3

AlaAT, the apparent Kms were 5.2 mM, 0.5 mM, 0.5 mM and 1.2 mM for alanine, α-

ketoglutarate, glutamate and pyruvate, respectively, and apparent Vmaxs were 107

nmol min-1

mg-1

protein for alanine deamination and 48 nmol min-1

mg-1

protein for

alanine formation. Both enzymes required exogenous pyridoxal 5′-phosphate for

optimal activity. The equilibrium constants for the AspAT and AlaAT reactions were

consistent with those estimated from the estimated kinetic parameters. From these

parameters we infer that T. circumcincta AlaAT is present predominantly as a

mitochondrial enzyme favouring pyruvate formation while AspAT is predominantly a

cytosolic enzyme favouring glutamate formation.

KEY WORDS: aminotransferase, enzyme kinetics, nematode, parasite.

INTRODUCTION

Teladorsagia circumcincta is a nematode parasite of sheep that secretes or

excretes NH3/NH4+ (Muhamad et al., 2012) and urea (Muhamad et al., 2013), but

appears not to secrete or excrete amino acids (Muhamad, 2006), although secretion or

excretion of all three has been reported for other helminths (Barrett, 1991). We have

characterised the kinetics of some of the enzymes that might be involved in generating

these compounds (Muhamad et al., 2005; Muhamad et al., 2011). However, it is also

MUHAMAD et al: Some properties of aminotransferases from Teladorsagia circumcincta

112

important to consider the intracellular transfer of nitrogenous groups that provides

alternative connections between the metabolites involved in these reactions (Muhamad

et al., 2005; Muhamad et al., 2011). Arguably, transaminations are among the most

important of these and, of those, the reactions catalysed by aspartate aminotransferase

(AspAT, E.C. 2.6.1.1)

aspartate + α-ketoglutarate ⇌ glutamate + oxaloacetate

and alanine aminotransferase (AlaAT, E.C. 2.6.1.2)

alanine + α-ketoglutarate ⇌ glutamate + pyruvate

are of particular significance, although other aminotransferases have been detected in

nematodes (Barrett, 1991).

Both AspAT and AlaAT catalyse the transfer of an amino group from an

amino acid donor (aspartate or alanine, respectively) to a ketoacid acceptor (α-

ketoglutarate) generating an amino acid (glutamate) and a different ketoacid

(oxaloacetate or pyruvate, respectively). The reactions are readily reversible, as

is apparent from ∆rG´0 = 4.96 kJ mol

-1 for AspAT (Kishore et al., 1998) and

∆rG´0 = 0.46 kJ mol

-1 for AlaAT (Tewari et al., 1998). Both AspAT and

AlaAT require pyridoxal 5′-phosphate (PLP), which can be lost from the

enzyme and incubation of the enzyme suspension with PLP increases the

observed rate of reaction as the PLP is reincorporated into the apoenzyme

(Martinez-Carrion et al., 1967; Scardi et al., 1963). Aminotransferases, including AlaAT and AspAT, have been detected in

several helminth species (Barrett, 1991), but the only data for T. circumcincta appears

to be the estimate of AlaAT activity given by Walker and Barrett (1991). Most of the

reports for other species also provide only an estimate of the activity. For example,

Grantham and Barrett (1986) surveyed the activity of many aminotransferases in

Heligmosomoides polygyrus and Panagrellus redivivus without providing any other

indication of the kinetics of each reaction. This is not enough to begin to understand

the interactions between the reactions producing nitrogenous compounds (Muhamad et

al., 2005; Muhamad et al., 2011). In a small number of cases more data are available.

For example, Sharma and Singh (1977) reported apparent Kms for aspartate of 9 mM

and 2.9 mM aspartate for AspAT from male and female, respectively, Stephanurus

dentatus and activity was greatest at pH 8 for both genders. Burenina (2001) reported

the apparent Kms for alanine and α-ketoglutarate of both the mitochondrial and

cytoplasmic isozymes of AlaAT from the trematode Eurytrema pancreaticum. Lowe

and Rowe (1985) reported the Kms for aspartate and α-ketoglutarate of the single form

of AspAT they could identify in the protozoon Trichomonas vaginalis, later they

reported a second isoform that differed only in the Vmax for substrates other than


113

aspartate (Lowe & Rowe, 1986). Here we describe some of the kinetic properties of

AspAT and AlaAT from T. circumcincta.

MATERIAL AND METHOD

Parasite culture and homogenate preparation

Pure strains of Teladorsagia circumcincta were maintained by regular passage

through sheep to provide L3. Larvae were concentrated by centrifugation at

approximately 1000 × g, washed and then resuspended in 100 mM KH2PO4-KOH pH

7.5. Where necessary adult T. circumcincta were obtained from the abomasal contents

of donor sheep as described previously (Simcock et al., 2006). It was not practicable to

culture sufficient L3 or obtain enough adults to purify the enzymes and so all the work

described here was carried out using nematode homogenates. In order to homogenise

the nematodes, the suspension was frozen at -20°C and then ground manually at 4°C as

described previously (Walker et al., 2007).

Enzyme activities

The activity of AspAT and AlaAT were assayed by coupling the reaction with

an appropriate NAD+-linked dehydrogenase. Each reaction was monitored

spectrophotometrically by following the oxidation of NADH at 340 nm (ε340 = 6220

M−1

cm−1

) using 50 µg mL−1

protein at 30°C. All assays were carried out using either

an Ultrospec III (LKB Instruments) or a CE 599 (Cecil Instruments Ltd., Cambridge,

England) spectrophotometer from which the data were collected by computer using

software and hardware developed in-house as previously reported (Walker et al.,

2007). For enzymes other than AspAT and AlaAT, for which the aminotransferse

activities were very low, the rates were estimated discontinuously over 30-60 min. In

all cases data from at least three homogenates were analysed (n ≥ 3), except for the pH

dependence experiments where the data represent only two separate homogenates (n =

2).

The activity of AspAT was determined in the direction of aspartate

deamination by coupling the reaction to malate dehydrogenase (MDH, E. C. 1.1.1.37)

so that the oxaloacetate produced was reduced to malate and the concentration of

NADH declined. Unless otherwise noted, the assay mixture consisted of 10 mM

aspartate and 0.2 mM α-ketoglutarate, 200 µM NADH, 200 µM PLP, 1 U mL-1

MDH,

50 µg mL-1

homogenate protein, 100 mM Tris-HCl pH 7.5. In the direction of aspartate

formation by AspAT, the α-ketoglutarate generated was reductively aminated to

glutamate by glutamate dehydrogenase (GDH, E. C. 1.4.1.3) and the concentration of

NADH declined. Unless otherwise specified, the assay mixture consisted of 1 mM

glutamate, 2 mM oxaloacetate, 200 µM NADH, 200 µM PLP, 1 U mL-1

GDH, 50 µg

mL-1

homogenate protein, 100 mM Tris-HCl pH 7.5. Teladorsagia circumcincta has

considerable endogenous activities of both MDH (Simcock et al., 2012) and GDH


114

(Muhamad et al., 2011) in addition to the exogenous enzyme provided. Further details

of the assays are provided in the figure legends.

The activity of AlaAT was determined in the direction of alanine deamination

by coupling the reaction to lactate dehydrogenase (LDH, E. C. 1.1.1.27) so that the

pyruvate produced was reduced to lactate and the decline in the concentration of

NADH was monitored at 340 nm. Unless otherwise noted, the assay mixture consisted

of 10 mM alanine and 2 mM α-ketoglutarate and 200 µM NADH, 200 µM PLP, 1 U

mL-1

LDH, 50 µg mL-1

homogenate protein, 100 mM Tris-HCl pH 7.5. In the direction

of alanine formation by AlaAT, the α-ketoglutarate generated was reductively

aminated to glutamate by GDH and the reaction was monitored continuously by

following the decline in the the absorbance at 340 nm as a measure of the

concentration of NADH. Unless otherwise specified, the assay mixture consisted of 5

mM glutamate and 2 mM pyruvate and 200 µM NADH, 200 µM PLP, 40 mM NH4Cl,

1 U mL-1

GDH, 50 µg mL-1

homogenate protein, 100 mM Tris-HCl pH 7.5.

Teladorsagia circumcincta has considerable endogenous activities of both MDH

(Simcock et al., 2012) and GDH (Muhamad et al., 2011) in addition to the exogenous

enzyme provided. Further details of the assays are provided in the figure legends.

The protein concentration of homogenates was determined using the Bradford

method (Bradford, 1976) and bovine serum albumin as the standard. No detergents

were employed to solubilise the nematode homogenate as they can interfere with

protein assays (Sapan et al., 1999).

Data analysis

While it is likely that the kinetics of L3 T. circumcincta AspAT and AlaAT can

be modelled using the same mechanisms as those of other species (Cascante & Cortés,

1988; Creighton & Murthy, 1990), we have not attempted to employ such models in

the analysis presented here. Instead, apparent Kms and Vmaxs were obtained by fitting

the standard Michaelis-Menten expression to the activity data for each substrate. This

approach is consistent with that adopted previously (Muhamad et al., 2011). Estimates

of apparent Vmax depend on the enzyme concentration, so comparisons between L3 and

adult should be made with caution, but such comparisons can be made more reliably

for estimates involving the same lifecycle stage. Estimates of apparent Km are

independent of enzyme concentration, so comparisons between values for L3 and adult

preparations can be made with more confidence. All nonlinear regression and the other

analyses described below were carried out using R (Ihaka & Gentleman, 1996).

RESULTS

In the direction of aspartate deamination the apparent Kms for aspartate and α-

ketoglutarate of L3 AspAT were 1.2 ± 0.2 mM and 0.13 ± 0.02 mM, respectively, and

the apparent Vmax was 960 ± 50 nmol min-1

mg-1

protein (Figure 1, A and B). In the

direction of glutamate deamination the apparent Kms for glutamate and oxaloacetate


115

were 0.11 ± 0.02 mM and 0.04 ± 0.01 mM, respectively, and the apparent Vmax was

420 ± 20 nmol min-1

mg-1

protein (Figure 1, C and D). For L3 AlaAT, the Kms were 5.2

± 0.5 mM, 0.5 ± 0.1 mM, 0.5 ± 0.1 mM and 1.2 ± 0.2 mM for alanine, α-ketoglutarate,

glutamate and pyruvate (n = 3 in each case), respectively, and Vmaxs were 107 ± 6 nmol

min-1

mg-1

protein for alanine deamination and 48 ± 4 nmol min-1

mg-1

protein for

alanine formation (n = 6 in each case).

FIG. 1. Apparent kinetics of AspAT in the direction of aspartate deamination (A and B) or glutamate

deamination (C and D). In (A) and (B), the assay mixture consisted of the indicated concentration of

aspartate and 0.2 mM α-ketoglutarate (A) or 10 mM aspartate and the indicated concentration of α-

ketoglutarate (B) and 200 µM NADH, 200 µM PLP, 1 U mL-1 MDH, 50 µg mL-1 homogenate protein, 100

mM Tris-HCl pH 7.5. In (C) and (D), the assay mixture consisted of 1 mM glutamate, 2 mM oxaloacetate,

200 µM NADH, 200 µM PLP, 1 U mL-1 GDH, 50 µg mL-1 homogenate protein, 100 mM Tris-HCl pH 7.5.

The data (± SEM) were obtained from n = 2-7 homogenates.

A few measurements were made using the enzymes from adult T.

circumcincta. For adult AspAT, the Kms were 2.6 ± 0.6 mM (n = 3) and 0.2 mM (n =

2) for aspartate and α-ketoglutarate, respectively, and the Vmax was about 185 nmol

min-1

mg-1

protein. For adult AlaAT, the Kms were 0.5 mM (n = 2) and 0.1 mM (n = 1)

for alanine and pyruvate, respectively, and the Vmax in the direction of alanine

deamination was about 79 nmol min-1

mg-1

protein.


116

The PLP prosthetic group of an aminotransferase is often labile, but it can be

reincorporated when added to an enzyme suspension (Martinez-Carrion et al., 1967).

In the case of AspAT, the rate of glutamate deamination was increased to 375 nmol

min-1

mg-1

protein after incubation with 0.1 mM PLP (Figure 2A). The activity of

AspAT was almost completely inhibited by 1 mM PLP. The activity of L3 AlaAT was

more than doubled by incubation with PLP (Figure 2B) and the enzyme was more

sensistive than AspAT to concentrations of PLP in the range 0.2 mM to 0.5 mM

(Figure 2, A and B).

FIG. 2. The effect of added pyridoxal 5′-phosphate (PLP) on the rate of aspartate (A) or alanine (B)

deamination. The assays were carried out using 3 mM aspartate, 1 mM α-ketoglutarate, 200 µM NADH, 1

U mL-1 MDH, 50 µg mL-1 homogenate protein, 100 mM Tris-HCl pH 7.5. The homogenate was incubated

with the indicated concentration of PLP in the reaction mixture for 2 minutes before the reaction was

initiated by the addition of α-ketoglutarate.

The aspartate deamination activity of L3 AspAT was greatest at pH 7.4 and

declined at lower pH with an apparent pKa of 6.2 and at higher pH with an apparent

pKa of 8.7 (Figure 3). The low activity of L3 AlaAT made it relatively difficult to

assess the pH sensitivity of the enzyme, but the rate of alanine deamination appeared to

be greatest at about pH 7.5.

We also attempted to measure the activity of several other aminotransferases

using α-ketoglutarate as the ketoacid acceptor, but in L3 T. circumcincta the rates were

less than about 2 nmol min-1

mg-1

protein using valine, histidine, leucine or glycine,

and no activity could be detected using cysteine, isoleucine or ornithine (the lower

limit of detection was about 0.1 nmol min-1

mg-1

protein). Greater activity (up to about

8 nmol min-1

mg-1

protein) was observed for serine and valine using pyruvate as the

ketoacid acceptor. In adult nematode homogenates rates of less than about 2 nmol min-

1 mg

-1 protein were observed using cysteine, valine, isoleucine, histidine and lysine

using α-ketoglutarate as the ketoacid acceptor.


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FIG. 3. The pH dependence of the rate of aspartate deamination by L3 AspAT. The assays were carried out

using 5 mM aspartate, 1 mM αααα-ketoglutarate, 200 µµµµM NADH, 200 µµµµM PLP, 1 U mL-1 MDH, 50 µµµµg mL-1

homogenate protein, 100 mM Tris at the indicated pH. The homogenate was incubated in the reaction

mixture for 2 minutes before the reaction was initiated by the addition of αααα-ketoglutarate. DISCUSSIONS

We have reported that the activity of AspAT and AlaAT are much greater than

that of any other aminotransferase we tested, for several of which we were unable to

detect any activity. In both respects our observations are similar to those of Grantham

and Barrett (1986) for H. polygyrus and P. redivivus, although we have also

characterised the kinetics of AlaAT and AspAT in T. circumcincta. Several issues arise

from our observations. First, the data probably represent the activity of a mixture of

isozymes because in other organisms, both AspAT and AlaAT are usually present as

cytoplasmic and mitochondrial isozymes and each of these isozymes can occur in

several isoforms. Second, the presence of two or more isozymes may yield parameter

estimates that are not internally consistent. Finally, we consider the metabolic

significance of the data we have reported.

Both AlaAT and AspAT are found in both the cytoplasm and the mitochondria

of mammalian cells (Braunstein, 1973). As we have argued elsewhere (Brown et al.,

2014), the fact that several isoforms of AspAT, for example, are found in the

mitochondrial matrix and different isoforms of the enzyme are found in the cytoplasm

(Huynh et al., 1980), complicates the estimation of enzyme purity. Not only did we not

have enough nematodes to purify the two separate isozymes, we certainly did not have

enough to purify the individual isoforms of each isozyme. Moreover, at least some of

these isoforms differ in the mode of binding PLP to the enzyme (Martinez-Carrion et

al., 1967) and may be formed spontaneously in vitro from a single isoform during

storage (Huynh et al., 1980).

The AlaAT and AspAT we have considered here may comprise complex

mixtures of isoforms each of the cytoplasmic and mitochondrial isozymes. The

apparent Km of the mixture will be between those of the pure isozymes, but the kinetics


118

of such mixtures tend to resemble those of a homogeneous population of enzymes

unless the Kms of the isoforms are sufficiently different (Brown et al., 2014). For

example, we have estimated that mitcyt

mmKK=β (or β

-1) must be at least 20 for the

isoforms to be apparent from the kinetics of the mixture (Brown et al., 2014). This is

rarely the case for AspAT (the pig heart enzyme is one example where β > 20

(Braunstein, 1973: 427), although some preparations of these isozymes give β < 20

(Nisselbaum & Bodansky, 1966)). However, the Kms for aspartate and α-ketoglutarate

form distinct clusters corresponding to the mitochondrial and cytoplasmic isozymes

and the values we have reported fall clearly among the cytoplasmic isozymes (p =

0.69) (Figure 4A). We infer from this that the mitochondrial AspAT isozymes are less

concentrated or less active in the samples assayed than the corresponding cytoplasmic

isozymes, consistent with reports for Ascaris lumbricoides (Chu & Chang, 1975). A

similar analysis carried out for the AlaAT isozymes is less clear, but the T.

circumcincta data do appear to be consistent with the cytoplasmic isozymes being at a

lower concentration or less active than the corresponding mitochondrial isozymes (p =

0.75) (Figure 4B). We should point out that χ2 tests of the contingency matrices yield

p < 0.001 for AspAT and p = 0.003 for AlaAT, although the relatively small number of

points prompts us to urge caution. The other commonly used quality measure is

Matthew’s (1975) correlation coefficient, which is related to Cramér’s (1946:282-283)

mean square contingency (φ). All of the quality measures recommended by Vihinen

(2012) indicate that the discriminants shown in Figure 4, A and B, are moderately

useful (Table 1), although we urge caution given the small number of points in the case

of AlaAT (Figure 4B).

The internal consistency of our kinetic parameter estimates can be confirmed

by comparing the equilibrium constant (K′) estimated from the Haldane relation with

the measured value. While such consistency might be considered inevitable, it is not

always the case (Duff et al., 2012). For the ping pong bi bi mechanism characteristic of

aminotransferases (Braunstein, 1973), the relevant Haldane relation is

mBmA

mQmP

r

f

KK

KK

V

VK

2

max

max

=′ ,

where KmA and KmB are the Kms for the substrates (A and B) and KmP and KmQ are those

for the products (P and Q) and the superscripts f and r indicate the forward and reverse

reactions, respectively (Cleland, 1963). Using the parameter estimates for T.

circumcincta AlaAT, the Haldane relation yields K′ = 1.1 ± 0.4, which is similar to the

measured value (adjusted to 30°C) of K′ = 1.29 (Tewari et al., 1998). For AspAT, the

Haldane relation yields K′ = 0.15 ± 0.06, similar to the value at 30°C of K′ = 0.145 ±

0.016 given by Kishore et al. (1998).


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FIG. 4. Discrimination between mitochondrial (□) and cytoplasmic (○) isozymes of AspAT (A) and AlaAT

(B) using published values of Km for α-ketoglutarate, aspartate and alanine. The dashed curve in each panel

is the boundary between the two isozymes calculated using Fisher’s linear discriminant analysis (using lda

in R) based on those data in the literature for which the isozyme is unambiguously identified (Braunstein,

1973: 427; Burenina, 2001; DeRosa & Swick, 1975; Di Cola et al., 1976; Hopper & Segal, 1962; Huynh et

al., 1980; Kuramitsu et al., 1985; Lowe & Rowe, 1985; Martins et al., 2002; McAllister et al., 2013; Michuda

& Martinez-Carrion, 1969; Nisselbaum & Bodansky, 1966; Orlacchio et al., 1979; Ruscák et al., 1982; Segal

et al., 1962; Swick et al., 1965; Umemura et al., 1994), the values for T. circumcincta (▲) are superimposed

on the plot (but were not used in estimating the curves).

TABLE 1. Quality measures of the linear discriminants shown in Fig. 4.

AspAT AlaAT

Accuracy (%) 88.2 85.7 Negative predictive value (%) 75.0 100.0

Positive predictive value (%) 95.4 76.9

Sensitivity (%) 87.5 100.0 Specificity (%) 90.0 72.7

Matthews correlation coefficient 0.739 0.748

p (χ2) <0.001 0.003

The only previous report of AlaAT activity in T. circumcincta is that of

Walker and Barrett (1991). They reported that Vf/V

r = 0.42 for T. circumcincta,

similar to the values they reported for Nippostrongylus strongyloides (0.50),

Trichostrongylus colubriformis (0.76) and Haemonchus contortus (1.03).

However, Walker and Barrett (1991) used high alanine concentrations in

estimating the rate of alanine deamination (Vf). Such concentrations can be

inhibitory in some species (Bulos & Handler, 1965; Splittstoesser et al., 1976),

although we observed no such substrate inhibition of this reaction by alanine or

glutamate. Nevertheless, the data of Walker and Barrett (1991) might represent

an underestimate of Vf/V

r for T. circumcincta, because we observed that

23.2maxmax

=rf VV , similar to the value of 2.29 for AspAT. However, the internal

consistency of the kinetic parameters we have demonstrated leads us to


120

conclude that our estimates are reasonable, despite this apparent inconsistency

with the data of Walker and Barrett (1991) for AlaAT.

We have shown that AspAT and AlaAT have 2.2maxmax ≈rf VV , but AlaAT

has a K′ that is about seven times that of AspAT. We do not know the value of the

mass action ratio (Q) of either enzyme in the cytosol and mitochondria of T.

circumcincta, but if the mitochondrial isoform of AlaAT is the predominant form of

the enzyme (Figure 4B and Table 1), which is characteristic of gluconeogenic tissues

(liver and kidney) in mammals, then the reaction might tend to favour the production

of pyruvate from alanine (DeRosa & Swick, 1975). This implies that QAlaAT < K′AlaAT,

consistent with the depletion of pyruvate in the mitochondrial matrix by the pyruvate

dehydrogenase complex and pyruvate carboxylase. However, Anderson and Garfinkel

(1971) estimated that QAlaAT ≈ K′AlaAT, which implies that AlaAT tends to operate close

to equilibrium and that the reaction can proceed in either direction. In contrast, QAspAT

< K′AspAT (Anderson & Garfinkel, 1971), which prompts us to conclude that the

reaction would tend to favour the formation of glutamate. If this is the case and if the

cytosolic isozyme is the predominant form of AspAT in T. circumcincta (Figure 4A

and Table 1), then this would be consistent with the transfer of reducing equivalents

into the mitochondrial matrix by the malate-aspartate shuttle (Kovacevic, 1972).

CONCLUSIONS

From the kinetic properties of T. circumcincta AlaAT, we infer that the

enzyme is present predominantly as a mitochondrial isozyme favouring pyruvate

formation, consistent with the depletion of pyruvate in the mitochondrial matrix by the

pyruvate dehydrogenase complex and pyruvate carboxylase. In contrast, the kinetic

properties of T. circumcincta AspAT indicate that this enzyme is predominantly a

cytosolic isozyme favouring glutamate formation, which may be consistent with the

transfer of reducing equivalents into the mitochondrial matrix by the malate-aspartate

shuttle.

ACKNOWLEDGEMENTS

This work was funded by Beef + Lamb New Zealand, AGMARDT, Universiti

Malaysia Sarawak and the Public Service Department, Malaysia.

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