The toxic and lethal effects of the trehalase inhibitor trehazolin in locusts are caused by hypoglycaemia
1 Institut für Zoologie, Molekulare Physiologie, Johannes
Gutenberg-Universität, 55099 Mainz, Germany
2 Lead Discovery Research Laboratories, Sankyo Co. Ltd, 1-2-58 Hiromachi,
Shinagawa-ku, Tokyo 140-8710, Japan
* Author for correspondence (e-mail: gwegener{at}uni-mainz.de)
Accepted 10 January 2003
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Summary |
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Key words: trehalose, glucose, haemolymph, insect, trehalase localisation, flight muscle, Locusta migratoria
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Introduction |
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Trehalose is split into two glucose units by the enzyme trehalase (EC
3.2.1.28), which is present in many insect tissues (for a review, see
Becker et al., 1996). In
locusts, the enzyme has been found in thoracic ganglia
(Strang and Clement, 1980
),
brain and skeletal muscle. Trehalase activity is very low in locust
haemolymph, and it is doubtful whether this activity is due to a `true'
trehalase (Vaandrager et al.,
1989
). High activity has been found in flight muscle
(Candy, 1974
;
Worm, 1981
;
Vaandrager et al., 1989
; the
present study), and trehalose has been identified as an important fuel in
locust flight (Mayer and Candy,
1969
; Jutsum and Goldsworthy,
1976
; Van der Horst et al.,
1978
; Candy,
1989
).
In locust flight muscle, trehalase is bound to membranes that appear in the
microsomal fraction upon cell fractionation, but the exact cellular location
is not known. The trehalase reaction is irreversible under physiological
conditions, thus the enzyme would hydrolyse all available trehalose. In
resting locusts, trehalase in flight muscle shows low activity, although the
concentration of its substrate in the haemolymph is very high. With the onset
of flight, when ATP turnover in flight muscle increases dramatically (for a
review, see Wegener, 1996),
trehalose utilization rises by more than 10-fold
(Van der Horst et al., 1978
),
and this requires a corresponding increase in trehalase activity. Hence, the
activity of trehalase in locust flight muscle must be regulated, but the
mechanism of control has remained obscure despite several attempts to
understand this problem (Candy,
1974
; Worm, 1981
;
Vaandrager et al., 1989
; for a
review, see Becker et al.,
1996
). Insect trehalase activity has not been found to be
modulated by hormones, second messengers, allosteric effectors or reversible
interconversions. It has, however, been shown that trehalase in homogenates of
locust flight muscle appears in two forms, an overt form that is active
without further treatment and a latent form that is inactive but can be
activated in vitro by detergents or other means that destroy the
structural integrity of membranes (Candy,
1974
; Worm, 1981
;
Vaandrager et al., 1989
; for a
review, see Becker et al.,
1996
). Also, Candy
(1974
) observed an increase in
the overt form but no changes in the total activity of flight muscle trehalase
after a short flight. The mechanism of this phenomenon has remained
obscure.
Trehazolin is a natural pseudosaccharide (amino sugar) and a potent and
specific inhibitor of trehalases (for a review, see
Kobayashi, 1999). It was
discovered and isolated as a product of the actinomycete
Micromonospora by Ando et al.
(1991
). Trehazolin has
antifungal as well as insecticidal activity but does not affect mice when
injected at a dose of 100 mg kg1
(Ando et al., 1995a
). The
effects of trehazolin (and of related trehalase inhibitors) on trehalases have
been thoroughly studied. Trehazolin is a tight-binding competitive inhibitor
that seems to mimic the structure of the transition state of the substrate
(Ando et al., 1995b
). The
effects of trehazolin and other competitive trehalase inhibitors on
physiological processes such as development, metamorphosis, metabolism and
flight performance in insects have also been investigated in detail in various
species. Injection of the trehalase inhibitor validoxylamine in last instar
silkworms (Bombyx mori) caused severe developmental disruption. Using
1H- as well as 13C- and 31P-NMR spectroscopy
on silkworm haemolymph, Kono et al.
(1993
) demonstrated a more
than twofold increase in trehalose but only minor changes in other metabolites
(glucose was not detected in the spectra). The mechanism of the toxic effects
of trehalase inhibitors in insects, however, could not be elucidated. In the
present study, the effects of trehazolin on trehalase activity of locust
flight muscle in vitro and in vivo are investigated, as well
as its toxicity in intact adult locusts. A hypothesis that the lethal effect
of trehazolin (and similar trehalase inhibitors) is due to severe
hypoglycaemia that appears to cause a failure of the nervous system is
suggested and tested. Trehalase inhibitors are advocated as valuable tools in
studies on insect physiology as well as cellular and molecular aspects of
trehalase function and control.
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Materials and methods |
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Experimental treatment of locusts
Trehazolin was dissolved in distilled water at various concentrations and 5
µl was injected into the haemolymph using a 10 µl Hamilton syringe. The
needle was inserted into the thorax cavity through the soft membrane behind
the base of the hind leg. Controls were injected with 5 µl distilled water.
Controls and experimental animals were kept singly in small plastic containers
with access to food and water.
For collecting haemolymph, the injection puncture was widened with a needle, and the emerging fluid was absorbed in a graded capillary tube. Up to 30 µl haemolymph per insect was collected in a plastic vessel, which was incubated at 100°C for 10 min, cooled on ice and centrifuged at 10 000 g for 5 min. The supernatant was diluted with 19 parts distilled water and kept at 40°C until assayed.
Feeding glucose to locusts injected with trehazolin
Twenty male locusts were separated and fed well for 72 h. The animals were
weighed and individually injected with 50 µg trehazolin in 5 µl. They
were kept singly and randomly divided into two groups of 10. One group was fed
glucose every 2 h while the other was fed tapwater. Glucose (in doses of 50
µl 10% glucose in tapwater) was offered to individual locusts from the tip
of a pipette and was readily taken up by the animals, as was the tapwater
given to the controls. Surviving experimental animals and controls were
weighed after 24 h and their behaviour was further observed for up to 72
h.
Chemicals
Chemicals and enzymes were purchased from Roche Diagnostics (Mannheim,
Germany), Sigma (Deisenhofen, Germany), Merck (Darmstadt, Germany), Roth
(Karlsruhe, Germany) and Serva (Heidelberg, Germany). Trehazolin was prepared
as described by Ando et al.
(1991).
Assay of trehalase activity
Trehalase activity was followed by a spectrophotometric assay at 340 nm and
25°C. In a 500 µl total volume, the assay comprised 120 mmol
l1 sodium acetate (pH 6.5), 10 mmol l1
MgCl2, 0.6 mmol l1 NADP+, 1 mmol
l1 ATP, 0.42 U (1 U = 1 µmol substrate transformed
min1) glucose-6-phosphate dehydrogenase, 0.67 U hexokinase,
trehalase sample and 20 mmol l1 trehalose (to start the
reaction after a pre-incubation period of 5 min). The assays to differentiate
between overt and latent trehalase activity are described below (see Tissue
processing). One Unit of trehalase activity is equivalent to the hydrolysis of
1 µmol trehalose min1 at 25°C.
Assay of glucose and trehalose in haemolymph
Glucose was measured at 30°C by a specific spectrophotometric test
adapted from Kunst et al.
(1984). In a 500 µl total
volume, the assay comprised 150 mmol l1 triethanolamine
buffer (pH 7.6), 1 mmol l1 ATP, 0.6 mmol
l1 NADP+, 8 mmol l1
MgCl2, 0.42 U glucose-6-phosphate dehydrogenase and 0.67 U
hexokinase.
The assay of trehalose was based on the trehalase assay (see above), with a haemolymph sample replacing the substrate trehalose, and 0.4 U ml1 commercial trehalase from pig kidney (Sigma) replacing the trehalase sample. Care was taken to sufficiently dilute haemolymph samples from locusts that had been injected with trehazolin because otherwise the trehazolin in these samples may inhibit the trehalase in the assay. The extinction at 340 nm was read before starting the reaction by adding trehalase and again after 90 min incubation at 37°C. Two controls were run in which either the haemolymph sample or the trehalase was replaced with distilled water. All data are means ± S.E.M. and were analysed for statistical differences by Student's two-sided t-test.
Tissue processing, membrane extraction and purification of
trehalase
Locusts were immersed in liquid nitrogen and stored at 80°C
until use. The thoraces were isolated and dissected while still frozen. The
flight muscles were carefully freed from adhering parts of gut and fat body,
collected on ice, and weighed. The tissue was homogenised for about 3 min at
600 r.p.m. in nine parts (v/w) of buffer A (50 mmol l1
maleate buffer, pH 6.5) using a Potter homogeniser (Teflonglass; Braun,
Melsungen, Germany). This crude homogenate was used to differentiate between
overt, latent and total trehalase activity. Overt activity was measured in the
absence of detergent, and total activity was measured after addition of 30
mmol l1 of the zwitterionic detergent CHAPS
{3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate} to the
homogenate. In locusts not treated with trehazolin, latent trehalase activity
is the total activity minus the overt activity, according to the equation:
total trehalase activity = overt trehalase activity + latent trehalase
activity (see Table 1 and
Discussion).
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It should be mentioned that homogenates of freshly prepared flight muscles contain less overt trehalase activity than homogenates of flight muscles dissected from frozen locusts. This observation is in line with previous reports that flight muscle trehalase can be activated by repeated freezing and thawing. However, in order to process all animals used in an experiment in the same way and to determine their trehalase activity in the same series of measurements, we have routinely frozen and stored the locusts.
To extract trehalase for kinetic analysis in the absence and presence of trehazolin, the crude homogenate of locust flight muscle was centrifuged at 4°C and 40 000 g in a Sorvall RC5C for 45 min. The sediment was resuspended with four parts of a detergent buffer (buffer B: 300 mmol l1 sodium acetate, pH 6.5, containing 30 mmol l1 CHAPS) and again centrifuged as before. The supernatant (membrane extract) was stored at 40°C. Trehazolin was added before the enzyme assays were started.
Purification of trehalase was performed at 4°C. Media contained 0.1 mmol l1 phenylmethylsulfonyl fluoride (PMSF) and 0.01% sodium azide and were degassed by sonication. The crude muscle homogenate was centrifuged at 100 000 g for 1 h (Beckman L8-55 centrifuge). The sediment was resuspended in four volumes of homogenisation buffer (buffer A) in a Potter homogeniser (800 r.p.m., 3 min) and incubated with 30 mmol l1 CHAPS at room temperature for 30 min. After another centrifugation at 100 000 g, approximately 95% of the trehalase activity was found in the supernatant, which was chromatographically separated on chelated Cu2+ ions (iminodiacetic acid-epoxy-activated Sepharose 6B) using a Pharmacia chromatography unit. The Cu2+-chelate column was equilibrated with buffer C (0.1 mol l1 sodium acetate, pH 6.5, 0.1% Triton X-100), loaded with the solubilised trehalase and washed with 0.5 mol l1 KCl in the same buffer to remove non-specifically bound proteins. Trehalase was eluted by a gradient of 01 mol l1 glycine in buffer C at 40 ml h1.
Fractions containing more than 2% of the initial trehalase activity were
combined and subjected to affinity chromatography using a lectin concanavalin
A column (Con-ASepharose 4B; Sigma) according to Jahagirdar et al.
(1990). The column was washed
with 0.25 mol l1 NaCl in Con-A buffer (0.1 mol
l1 sodium acetate, pH 7.0, 1 mmol l1
CaCl2, 1 mmol l1 MnCl2 and 0.1% Triton
X-100). Elution (at 50 ml h1) was achieved with 0.2 mol
l1
-methyl-D-mannoside in Con-A buffer. As before,
trehalase fractions were combined. They were concentrated by ultrafiltration
using N2 at 200300 kPa pressure (Amicon unit, Millipore
cellulose filter, 30 kDa). The
-methyl-D-mannoside was removed by gel
filtration on Sephadex G25. The preparation was finally dialysed against 80%
(v/v) glycerol in Con-A buffer and stored at 40°C. Protein was
determined with bovine serum albumin as standard
(Bradford, 1976
).
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Results |
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Virtually all trehalase activity was found in the total membrane fraction of locust flight muscle after centrifugation of the muscle homogenate at 100 000 g for 1 h. Trehalase was purified from this fraction (see Materials and methods) about 700-fold, with a 24% yield, to a specific activity of 12.5 U mg1 protein. Trehalase activity followed MichaelisMenten kinetics (not shown) with a Km of approximately 1 mmol l1. The effects of trehazolin on purified trehalase were similar to those with membrane extracts. The apparent Ki was 8 nmol l1 and the IC50 was 120 nmol l1 trehazolin.
Inhibition of locust flight muscle trehalase by trehazolin
in vivo
Total trehalase activity in homogenates of flight muscle was approximately
4 µmol min1 g1 at 25°C (4 U
g1). Only a fraction of the total trehalase activity was
active if the homogenates had not been incubated with detergent. This overt
trehalase activity accounted for less than 18%; hence, more than 82% was
present in a latent form that needed detergent for activation
(Table 1). Interestingly,
trehazolin injected into locusts strongly inhibited the overt form of
trehalase but had much less effect on total activity. Thus, 50 µg
trehazolin per locust completely blocked the overt trehalase in flight muscle
homogenates, while total activity was only reduced by 35%. However, total
trehalase after activation by detergent is fully susceptible to inhibition by
trehazolin.
Toxicity of trehazolin in locusts
Eleven male locusts were each injected with 50 µg trehazolin. These
insects showed reduced motor activity. They moved sluggishly, if at all, and
failed to show the normal avoidance or flight reflexes when prodded. Six
locusts had died after 24 h, indicating that the LD50 (24 h) is
approximately 50 µg locust1 (see below).
Approximately two hours before they died, the locusts started to tumble, with poor co-ordination of body and extremities. The animals fell to their sides about 3045 min before death, and, lying on their side or back, some of them showed intense contractions of their jumping legs. During the final 510 min, an intense tremor was observed, with rapid clonic cramps of the hind legs. We classed the locusts as dead when no movements could be elicited.
Trehazolin causes severe hypoglycaemia in locusts
In order to understand the mechanisms of the toxic action of trehazolin in
locusts we followed changes in sugar content of locust haemolymph. Adult
locusts (of both sexes) were injected with 10 µg trehazolin in 5 µl
distilled water (experimental animals), while controls received only 5 µl
water. 20 µl of haemolymph (one sample per locust) was collected at six
intervals after the injection and assayed for trehalose and glucose. Trehalose
is the main blood sugar of locusts, corresponding to 21.0±1.1 g
l1 haemolymph in controls (N=5), whereas glucose
accounted for 0.50±0.03 g l1 haemolymph, i.e. only
2.4% of the trehalose content.
As was expected from work in other insect species, trehazolin caused a marked increase in the trehalose concentration of locust haemolymph, which rose from 21 g l1 (=61.35±3.1 mmol l1) in controls by over 80% to 37.9 g l1 (=110.9±3.8 mmol l1) 24 h after the injection of trehazolin (Fig. 2A).
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The effect of trehazolin on haemolymph glucose was even more dramatic than that on trehalose, although in the opposite direction (Fig. 2B). Haemolymph glucose decreased precipitously, from 0.50±0.03 g l1 (2.8 mmol l1) in controls, by 50% within 2 h and reached 0.041±0.07 g l1 (0.23 mmol l1), i.e. 8.2% of the control level, after 24 h. At this time, the glucose content in the haemolymph was only 0.11% of the trehalose content. Hypoglycaemic effects of trehazolin or other trehalase inhibitors have not been reported before.
Feeding glucose to locusts neutralizes the toxic effects of
trehazolin
To study whether hypoglycaemia was causing the toxic and lethal effects of
trehazolin, we fed glucose to locusts as a possible antidote to trehazolin. 20
male locusts were randomly divided into two groups of 10, and each locust was
injected with 50 µg trehazolin. The 10 animals of the control group (which
received only water) had a mean body mass of 1.40±0.08 g, and the
survivors had not lost mass after 24 h. However, food uptake in all control
animals was markedly reduced, and four of the 10 locusts did not consume any
food. However, all animals accepted tapwater when this was offered from a
pipette in 50 µl portions every 2 h for 24 h. The first animal died 4 h
after the injection of trehazolin, the second after 10 h; after 24 h, five
locusts had died, and only three were alive 36 h after the injection
(Fig. 3). This is in line with
the previous observation that trehazolin is toxic in locusts, with an
LD50 (24 h) of 50 µg locust1. Based on body
mass, the LD50 (24 h) was 36 µgg1.
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Glucose (10%), instead of water, was offered to the 10 locusts in the experimental group at 2 h intervals for 24 h. Their mean mass was 1.35±0.10 g, and this was not significantly changed after 24 h. Nine locusts took the glucose eagerly and did not show any adverse effects of trehazolin in their behaviour. The animals were active and ingested food in a similar manner to untreated locusts; their movements were fully coordinated and their avoidance and flight reflexes unaffected. After 24 h, these locusts appeared completely normal, and none of them died in the following two days of the observation period. Hence, feeding glucose to locusts injected with a potentially lethal dose of trehazolin can fully relieve the toxic effects of trehazolin. This indicates that the lethal effect of trehazolin is due to its hypoglycaemic action (see Discussion). The one locust that did not accept the glucose offered behaved unusually from the start of the experiment: it hardly moved after the injection of trehazolin, did not show avoidance reflexes and died within 6 h. We assume that it must have been either diseased or injured by the injection. Hence, the unusual behaviour of this one locust does not invalidate our conclusion that glucose can neutralize the toxic effects of trehazolin.
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Discussion |
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We have studied the effect of trehazolin on trehalase from locust flight muscle, a tissue particularly rich in trehalase, but, given that trehazolin is a substrate analogue, there can be little doubt that trehalases from all locust tissues will be inhibited by trehazolin. So far, trehalases from all sources (invertebrates and vertebrates) have been found to be susceptible to trehazolin or similar competitive inhibitors.
Of interest is our observation that trehazolin (and probably other
competitive trehalase inhibitors) selectively affects the overt form of flight
muscle trehalase, as this offers the possibility to differentiate between
overt and latent trehalase. The fact that a trehalase inhibitor differentiates
between overt and latent trehalase has not been reported before. Although the
activation of insect trehalases by treatments that interfere with membrane
structures had been noticed early on (Zebe
and McShan, 1959; Gussin and
Wyatt, 1965
; Gilby et al.,
1967
), the molecular basis of this phenomenon has remained elusive
(for a review, see Becker et al.,
1996
).
Latent trehalase, which is catalytically inactive in vitro, must be derived from a trehalase form that is protected from inhibition by trehazolin in the intact locust. The fact that latent trehalose becomes active and susceptible to trehazolin if treated with detergent suggests that enzyme latency also occurs in vivo and is based on a structural barrier that prevents equally the substrate and the inhibitor from reaching the active site of the enzyme (see below). The above-mentioned properties make trehazolin a useful tool in physiological experiments as it facilitates studies of location and function of trehalases in live insects as well as manipulation of glucose and trehalose in insect haemolymph.
Hypertrehalosaemic and hypoglycaemic effects of trehalase
inhibitors
The main blood sugar in locusts is not glucose but trehalose, as had
already been shown in the 1950s when this sugar was rediscovered in insects
(for reviews, see Wyatt, 1967;
Becker et al., 1996
). Trehalose
has two major advantages over glucose as a blood sugar: (1) as a disaccharide,
trehalose is less osmotically active than glucose and can, therefore, be
tolerated at higher levels (w/v) in blood than glucose and (2) trehalose is
non-reducing and hence not involved in the non-enzymatic glycosylation of
proteins, which is a major factor in glucose toxicity (as in diabetes
mellitus; for a review, see Cohen,
1986
).
Trehazolin caused a marked increase in trehalose in the haemolymph of
locusts. This effect can be regarded as typical for trehalase inhibitors of
this kind, as it has been demonstrated in all insects tested so far and also
with trehalase inhibitors other than trehazolin. For instance, validoxylamine
A was recently shown to bring about a marked and long-lasting increase in
haemolymph trehalose in migratory locusts
(Kono et al., 1999).
Glucose is much less prominent in locust haemolymph, accounting <2.5% of
trehalose on a mass basis. The low glucose content in insect haemolymph is
probably the reason why the inhibitor-induced dramatic decrease in haemolymph
glucose has escaped notice for so long. In previous studies, the effects of
trehalase inhibitors on haemolymph constituents were usually analysed by
NMR-spectroscopy (Kono et al.,
1993,
1994a
,b
,
1999
;
Takahashi et al., 1995
). This
powerful method can detect many compounds simultaneously, yet is not sensitive
enough to follow a decrease in glucose content.
Thus, it is suggested that trehalase inhibitors, such as trehazolin and validoxylamine A, bring about a marked increase in haemolymph trehalose in locusts and cause severe hypoglycaemia, and this will hold true for other insects that are similarly susceptible to trehalase inhibitors.
Haemolymph glucose is derived from trehalose and is essential for
locust survival
Our hypothesis that the toxic and lethal effects of trehazolin are due to
lack of glucose has been substantiated by feeding glucose to
trehazolin-injected locusts. Not only did the glucose result in the survival
of the locusts (Fig. 3) but it
also prevented all behavioural impairment by trehazolin. This observation
leads to interesting conclusions about the metabolic physiology of locusts
(and probably other insects). (1) Feeding glucose will stimulate rather than
reduce the synthesis of trehalose and yet enables the locusts to tolerate
lethal doses of trehazolin. The inhibitor-induced non-physiologically high
levels of haemolymph trehalose do not therefore contribute noticeably to the
acute toxicity of trehazolin in locusts. (2) Although trehalose is far more
prominent than glucose in haemolymph, glucose is absolutely necessary for
locust survival. This would require haemolymph levels of glucose to be
regulated more strictly than those of trehalose, which has indeed been
reported (Mayer and Candy,
1969; Strang and Clement,
1980
). (3) The fraction of trehalase in vivo that gives
rise to the overt form in vitro must be intimately involved in the
production of haemolymph glucose, and haemolymph trehalose appears to be the
major, if not the only, source of glucose in insects. (4) Trehalose and
trehazolin are both hydrophilic, and there is no indication that they are
transported into locust cells. We therefore assume that trehazolin and
trehalose have access to the same metabolic compartments. Thus, the
observation that trehazolin in live locusts had not reached the fraction of
trehalase that corresponds to the latent enzyme activity in vivo
(after 24 h of incubation; see Table
1) would mean that this fraction of trehalase (in vivo
latent trehalase) is separated from its substrate in vivo and is
hence not active in live locusts (see below). (5) Unlike vertebrates, in which
blood glucose is produced by the liver, locusts (and probably other insects)
do not have an organ specialised for glucose production. (6) Locusts must
possess vital organs (cells) that require glucose for proper functioning. This
is backed up by reports that fuel other than glucose, such as lipids and amino
acids, which are prominent in insect haemolymph
(Mullins, 1985
), is not
depleted in insects injected with trehalase inhibitors (Kono et al.,
1993
,
1994a
,b
,
1999
). It is not known which
organ failure proves to be fatal, but the behaviour of the trehazolin-poisoned
locusts preceding death suggests that the central nervous system is the most
likely candidate. Failure of the nervous system can be triggered by subjecting
insects to anoxia (Walter and Nelson,
1975
), and this brings about behavioural responses (reviewed by
Wegener, 1993
) similar to
those in locusts succumbing to trehazolin. Our hypothesis that failure of the
nervous system brings about the lethal effect of trehazolin is in line with
the observation that glucose is a much better substrate for isolated thoracic
ganglia from locusts than is trehalose
(Strang and Clement, 1980
; for
reviews, see Strang, 1981
;
Wegener, 1987
). The results
further suggest that trehazolin inhibits the trehalase of the nervous tissue,
so that glucose cannot be produced locally from haemolymph trehalose.
Where is trehalase localized in locust flight muscle?
Trehalases in flight muscle of locusts and other insects with synchronous
flight muscles, such as cockroaches and Lepidoptera, are membrane-bound
enzymes that can be activated by treatments that interfere with the structural
integrity of membranes. This has been known since the 1950s (e.g.
Zebe and McShan, 1959;
Gussin and Wyatt, 1965
;
Gilby et al., 1967
;
Candy, 1974
;
Worm, 1981
;
Vaandrager et al., 1989
), but
the important question of how and where trehalase is bound to muscle cell
membranes has remained unanswered despite many attempts at answering it (for a
review, see Becker et al.,
1996
). Also not known are the physiological (in vivo)
equivalents of overt and latent trehalases and their possible roles in the
control of trehalase activity in flight muscle. Trehazolin could be useful for
a novel approach to answering some of these questions.
To simplify the discussion of our working hypothesis, the following terms to describe the different forms of trehalase activity will be used. Trehalase that cannot be sedimented by centrifugation at 100 000 g for 60 min is termed soluble trehalase (trehalase s). Trehalase s is fully active and cannot be further activated by detergents or repeated freezethawing. Trehalase that can be sedimented at 100 000 g is called membrane-bound (or particulate) trehalase (trehalase p). The activity of trehalase p in vitro can be subdivided into an overt fraction (trehalase povert) and a latent fraction (trehalase platent). Trehalase povert has originated from a fraction of trehalase in vivo that we will hence call in vivo-overt trehalase. In vivo-overt trehalase is thought to be located in or attached to plasma membranes of muscle cells such that the active site is accessible for the substrate trehalose and the inhibitor trehazolin (if present) from the haemolymph. This would explain how, by inhibiting the in vivo-overt trehalase, injection of trehazolin eliminates trehalase povert and that only in vivo-overt trehalase can be catalytically active to produce sufficient haemolymph glucose in locusts. This view is supported by the lack of evidence for a trehalose transporter and trehalose transport in locust flight muscle.
The in vivo-latent trehalase would appear in the in
vitro- latent fraction (trehalase platent) after
homogenisation of flight muscle. The active site of in vivo-latent
trehalase must be shielded from trehalose and trehazolin alike, with the
effect that this fraction would be neither catalytically active nor
susceptible to inhibition by trehazolin in live locusts. The observations of
Candy (1974) and our own
preliminary studies suggest that in vivo-latent trehalase can be
transformed into in vivo-overt trehalase and that this is essential
for the control of trehalase activity in locust flight muscle in
vivo. Trehalase could therefore be a prototype of a novel mechanism for
regulating enzyme activity. The exact localization of both overt and latent
trehalase in locust flight muscle and the molecular mechanism of their
proposed transformation will be studied with more direct methods.
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Acknowledgments |
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References |
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