Comp. Biochem. Physiol. Vol. 74B, No. 4, pp. 791 to 792, 1983 0305-0491/83/040791-02503.

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H E X O K I N A S E A N D CARNITINE P A L M I T O Y L T R A N S F E R A S E
ACTIVITIES IN F L I G H T M U S C L E S OF G R A S S H O P P E R
VALANGA NIGRICORNIS(BURM.)
C. W. LEE and C. H. TAN*
Department of Biology, University of Agriculture Malaysia, Serdang,
Selangor, Malaysia

(Received 21 July 1982)

Abstract--1. The activities of hexokinase and carnitine palmitoyltransferase (CPT) activities were
assayed in flight muscle mitochondria of Valanga nioricornis.
2. Kinetic analysis of the enzyme activities yielded the following information: for hexokinase, its Km
(glucose) is 150 #M while its Vm~x is 2.5 #mol/min/mg protein; the corresponding data for CPT are K,,
(palmitoylCoA) of 39/zM and VmaXof 22.2 nmol/min/mg mitochondrial protein.
3. Under conditions of equivalent rates of ATP production the hexokinase-CPT activity ratio is 64,
suggesting that although V. nigricornis is a "mixed" fuel utilizer, it appears that carbohydrate oxidation
constitutes the predominant energy source.

INTRODUCTION tivities of hexokinase and CPT activities in flight

muscle mitochondria of the insect to obtain a quali-
Different energy substrates are used during insect
tative insight into the nature of its fuel utilization.
flight. Whilst the Hymenoptera, Diptera and Dictyop-
tera depend exclusively on carbohydrates as the sole
metabolic fuel (for a review, see Sacktor, 1975), the
Lepidoptera generally oxidise lipids during flight
(Domroese & Gilbert, 1964), although some species
are known to utilise carbohydrates as well. Among
the Orthoptera, however, both carbohydrates and
lipids serve as fuels for flight, e.g. in Locusta migra-
toria and Shistocerca gregaria, trehalose constitutes
the predominant substrate utilized initially during the
first half hour of flight (Jutsum & Goldsworthy,
1976); during sustained flight, however, diglycerides
mobilized from the fat body become increasingly im-
portant (Mayer & Candy, 1969). In yet other insects
like the tsetse fly and Colorado potato beetle, proline
oxidation provides the energy for flight.
Several approaches have been used to investigate
fuel utilization by flight muscles (for a review, see
Crabtree & Newsholme, 1975). One such approach is
the assay of key enzymes of various metabolic path-
ways. For example, the activities in vitro of hexo-
kinase (EC 2.7.1.1) and carnitine palmitoyl-transferase
(EC 2.3.1.23), key enzymes of glycolysis and fatty acid
oxidation, respectively, have been widely used in com-
parative studies to provide good qualitative indica-
tions of the maximum utilization in vivo of glucose
and fatty acids (Crabtree & Newsholme, 1972, 1975).
V. nigricornis (Orthoptera) is a potentially serious
pest of oil palm and rubber as well as their cover
crops in Malaysia. In its natural habitat, the grass-
hopper performs "trivial" flights of durations not
exceeding 30 sec, although it has been observed to fly
for longer periods (1-2 min) when pursued by man.
Fuel utilization of this non-migratory acridid during
flight is unknown. Therefore, we have assayed the ac-
* To whom correspondence should be addressed.
791
MATERIALS AND METHODS
Adult V. nigricornis were caught in the field and reared in
the laboratory under crowded conditions at 30°C with a
daily illumination of 8 hr and were fed on young leaves of
cover crops. Adults of both sexes and of undetermined age
were used in all experiments.
For the isolation of flight muscle mitochondria, flight
muscles from about 20 insects were dissected out, pooled
and washed in an ice-cold medium consisting of 250 mM
sucrose, 5raM Tris-HC1, pH 7.2 (0°C), 1 mM EGTA
[-ethylene glycolbis-(aminoethylether)-tetraacetate] and
0.5% (w/v) fatty acid-free BSA. The tissues were then hom-
ogenised in 10vol of the fresh medium with two quick
passes of a motor driven Potter-Elvehjem tissue grinder.
The resulting homogenate was centrifuged at 600g for
5 rain (0°C) and mitochondria in the supernatant were pel-
leted by recentrifugation at 12,000 g for 5 rain (0°C). The
mitochondrial fraction was "washed" in fresh isolation
medium and recentrifuged; this was repeated twice before
use. The mitochondria were sonicated for 3 x 20see
periods at 0°C at full setting of the MSE 150 watts ultra-
sonic disintegrator. The 12,000 g supernatant was used di-
rectly for determination of hexokinase activities.
CPT activities were determined as described (Tan &
Robinson, 1982), while hexokinase activity in the crude
muscle extract was assayed according to the procedure of
Berthiller et al. (1970). Protein was estimated by the biuret
reaction using bovine serum albumin as standard (Gornall
et al., 1949).
RESULTS AND DISCUSSION
The transfer of acyl groups across the inner mito-
chondrial membrane involves distinct carnitine acyl-
transferases specific for fatty acids of various chain-
lengths (Kopec & Fritz, 1971). Since long-chain fatty
acids (C16 and C18) constitute the major lipid sub-
strate oxidised during flight of the locust (Jutsum &
792
~J f+
2°l °'10.
J
~s.
0 (Hymenoptera).
0
Pllmitoyl -CoA concentration(uM)
Fig. 1. Carnitine palmitoyltransferase activity in sonicated
flight muscle mitochondria assayed at different concen-
trations of palmitoyl-CoA. Each point represents the mean
+ SE for 3 determinations.
IIi
o
0i
o11 0:2 o~3 d4
o-glucose conclnfrafion (11#t)
Fig. 2. Hexokinase activity in crude cytoplasmic extract of
flight muscles: effect of glucose concentration. Each point
represents mean +SE for 4 determinations.
Goldsworthy, 1976), we have accordingly assayed the
activity of C P T in flight muscle mitochondria of V.
nigricornis. Figure 1 shows the C P T activities at dif-
ferent substrateconcentrations; maximal activity was
obtainable at 100 #M palmitoyl-CoA. Kinetic analysis
of the data yielded an apparent K s for this substrate
of 39.2 #m and a Vmax of 22.2 nmol/min/mg mitochon-
drial protein. The corresponding data for hexokinase
are: Km (glucose) of 125 #M, and Vmax of 2.5/~mol/
min/mg protein (Fig. 2).
It is clearly evident that V. nigricornis can poten-
tially utilise both carbohydrate and lipid as energy
substrates. A comparison of the two enzyme activities
under equivalent rates of ATP production (Crabtree
& Newsholme, 1975), revealed a hexokinase-CPT ac-
tivity ratio of 64. This indicates that although the
insect can oxidise lipid, the relatively higher hexo-
C. W. LEE and C. H. TAN
kinase activity suggests that carbohydrate oxidation is
predominant and probably constitutes the primary
energy source supporting flight. In other "mixed" fuel
utilizers, variable activity ratios have been reported
(Crabtree & Newsholme, 1975): in the migratory
locusts, L. migratoria and S. gregaria, the ratios are
1.3 and 2.4, respectively; among some species of Lepi-
doptera such as Plusia gamma and Noctua pronuba,
the respective values are 25 and 16. In contrast,
insects that depend exclusively on carbohydrate oxi-
dation have much higher activity ratios: 100 in Perip-
laneta americana (Dictyoptera); 175 in Calliphora
erythrocephala (Diptera); and, 88 in Apis hortorum
The present study has yielded some preliminary
insight into the nature of fuel utilization, hitherto un-
known, in I/:. nigricornis: the insect may be regarded
as a "mixed" fuel utilizer, but having a predominant
capability to oxidise carbohydrate. In the absence of
information regarding fuel utilization in vivo derived
from flying experiments, this finding is significant.
Moreover, previous attempts to induce flight by the
insect have failed (S. S. Lee and G. J. Goldsworthy,
personal communication).
Acknowledgement--We are grateful to Mohd. Nordin
Kassim for his assistance during this study.
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