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JEZ 0612
Mitochondrial Contribution to Metabolic Changes
in the Digestive Gland of Mytilus galloprovincialis
During Anaerobiosis
Istituto di Biologia e Genetica, Università di Ancona, 60100 Ancona, Italy
The activities of several enzymes and the levels of metabolites have been measured in the digestive gland of Mytilus galloprovincialis exposed to increasing hypoxia from 7 to
168 hr. A sharp decrease of pyruvate kinase was observed after 7 hr. The anoxic enzyme showed
increased Km for phosphoenolpyruvate and decreased apparent Ki for alanine. Glyoxalase I was
constant after up to 72 hr of exposure and then decreased. Phosphoenolpyruvate carboxykinase
and alanopine dehydrogenase decreased. The metabolites alanine and succinate increased with
hypoxia time, whereas D- and L-lactic and aspartic acids were undetectable and constant respectively. Mitochondrial formation of pyruvate from D-lactate was demonstrated in intact mitochondria isolated from the digestive gland of Mytilus galloprovincialis . The significance of the observed
enzyme and metabolite changes in hypoxia is discussed in comparison with other invertebrate
organisms. The role of mitochondria in the overall adaptive strategy of Mytilus galloprovincialis is
discussed. J. Exp. Zool. 286:107–113, 2000. © 2000 Wiley-Liss, Inc.
Exposition to intermittent severe hypoxia can
be a frequent occurrence for mollusks as well as
barnacles, sea anemones, and polychaete worms.
In fact, several marine invertebrates are exposed
to daily episodes of oxygen lack when the tides
recede. Hypoxia as well as the restoration of oxygen supply can happen suddenly, thus cellular response must be fast and easily reverted. There is
not enough time for large-scale reorganization of
the amounts or types of cellular proteins. Thus
biochemical modifications rather than gene expression modifications are the basis for survival
in these harsh conditions (de Zwaan, ’77, ’83;
Kreutzer et al., ’85; Storey, ’85).
For intertidal marine invertebrates, resistance
to severe hypoxia results as part of an adaptive
strategy that maximize the survival time of the
organism in the anoxic interval, ready to restart
with respiration as soon as oxygen becomes available again. Adaptive strategy seems to involve:
(1) metabolic rate depression; (2) activity modifications of some glycolytic enzymes; and (3) production of low toxicity metabolic wastes. Metabolic rate
depression occurs by transition to a hypometabolic
state throughout the coordinated reduction of the
activity of some key regulatory enzymes. Facultative anaerobes do not show a Pasteur effect, but a
“reverse Pasteur effect” occurs and the glycolytic
rate in hypoxia is reduced below that seen in the
normoxic situation (Ebberink and de Zwaan, ’80;
Lutz et al., ’84; Storey, ’85; Kelly and Storey, ’88;
Storey et al., ’90). The mechanism involves covalent but reversible enzyme modifications, such as
phosphorylation or dephosphorylation, and seems
to be conserved in phylogenetic lines (Storey and
Storey, ’90). Indeed, studies of the glycolytic response to the transition from normoxia to hypoxia
show the same pattern in turtles, goldfish, and
marine mollusks (Shoubridge and Hochachka, ’80;
Kelly and Storey, ’88; Storey et al., ’90).
Throughout the anoxic time, the metabolism of
bivalve mollusks is sustained only by endogenous
fermentable fuel reserves. Depression of the metabolic rate, when oxygen is depleted, allows energy
demands to be met by the ATP output using alternative pathways of fermentative ATP production.
Anaerobic ATP from substrate-level phosphorylations is greatly enhanced; instead of two ATP per
glucose fermented to lactate, five/seven ATP per glucose fermented to succinate/propionate are produced
(von Brandt, ’46; Hochachka, ’80; Brinkhoff et al.,
’83; Hole et al., ’95). Besides the increase in ATP,
these alternative pathways produce low toxicity
Abbreviations used: ADH, alanopine dehydrogenase; DHAP,
dihydroxyacetonephosphate, GI, glyoxalase I; MG, methylglyoxal;
PEP, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; PK, pyruvate kinase.
*Correspondence to: Prof. Giovanni Principato, Istituto di Biologia
e Genetica, Via Ranieri, Montedago, 60100 Ancona, Italy. E-mail:
Received 4 December 1998; Accepted 16 April 1999
metabolic wastes that are weak rather than strong
acids as L-lactate. Gastropod and bivalve mollusks
are generally lacking in lactic dehydrogenase. The
redox equilibrium is ensured by alternative pathways for NADH oxidation that produce octopine
or alanopine by a reductive condensation catalyzed
by the corresponding dehydrogenases (Hochachka,
Specimens of the mussel Mytilus galloprovincialis
were directly obtained by drawing from a cliff off
the coast of Senigallia (Ancona, Italy) during September/October 1997. To impose severe hypoxia,
mussels were simply exposed to air at room temperature (20–23°C) for intervals from 7 to 168 hr.
Preparation of cytosolic and
mitochondrial fractions
Digestive glands were dissected from aerobic or
anoxic mussels and quickly homogenized by Potter Elvehjem (1:2, w/v) in 100 mM Tris-HCl buffer,
pH 8.0, containing 5 U/ml bacitracine, 0.009 TIU/
ml aprotinine and 0.1 mM phenylmethylsulfonyl
fluoride. After centrifugation at 48,000g for 10 min
at 4°C, supernatants were further centrifuged at
125,000g for 40 min at 4°C and stored at –20°C
until used for enzyme assays. Some other glands
were homogenized in 1:2 (w/v) 0.8 M perchloric
acid and the mixture centrifuged at 38,000g for
15 min at 4°C. Supernatants were stored at –20°C
until used for metabolite assays.
Mitochondria were isolated in according to
Ballantyne and Storey (’84), with the following
modifications: digestive glands of five-six mussels
were homogenized in 1:20 (w/v) ice-cold 5 mM TrisEDTA buffer, pH 7.4, containing 0.25 M sucrose.
The homogenate was centrifuged at 1,500g for 8
min at 4°C, the supernatant was collected and recentrifuged at 9,000g for 15 min at 4°C.
The mitochondrial pellet was then resuspended
in 1:4 (w/v) ice-cold 24 mM Tris-HCl buffer, pH
7.6, containing 1 mM EDTA, 0.25 M sucrose and
0.5 mg/ml bovine serum albumin fatty acids-free,
and recentrifuged at 9,000g for 15 min at 4°C.
The final pellet was resuspended in a small volume of the latter solution at a mitochondrial protein content of 10–20 mg/ml.
Enzyme assays
Pyruvate kinase (PK) was measured by the
continous coupled assay of Bergmeyer (’74) in the
presence of final concentrations of 1 mM ADP, 9.4
mM magnesium chloride, and 94 mM potassium
chloride. The phosphorylation reaction was evaluated by the NADH oxidized during the reduction
of pyruvate by lactate dehydrogenase. NADH utilization was monitored at 340 nm on a Varian uv/
vis recording spectrophotometer attached to a circulating water bath at 25°C.
The kinetics at different alanine concentrations
in the presence of 1 mM ADP were not linear in
the range of PEP 0.05–1.6 mM and alanine
0.05–15 mM. However, at the highest PEP concentrations double reciprocal plots approximate
competitive kinetics and apparent Ki values
were calculated from Dixon plots according to
Plaxton and Storey (’84).
Phosphoenholpyruvate carboxykinase (PEPCK)
was assayed according to Urbina (’87). The carboxylation reaction was measured by coupling the
formation of oxaloacetate to NADH oxidation by
malate dehydrogenase. Alanopine dehydrogenase
(ADH) activity was determined by following the
NADH oxidation during the formation of alanopine from alanine and pyruvate (Fields et al., ’80).
Glyoxalase I (GI) was determined according to
Regoli and Principato, (’95), by following the formation of S-D-lactoylglutathione from the hemimercaptal adduct of methylglyoxal (MG) and
glutathione at 240 nm (e = 3.37 mM–1 cm–1).
Preliminary experiments indicate that enzymes
activities were unchanged (e.g., Glyoxalase I) or
decreased less than 20% (e.g., Pyruvate kinase)
in supernatants stored at –20°C. Statistical evaluation (ANOVA) was carried out on the activities
of at least three independent determinations for
each hypoxia time.
Metabolite assays
Supernatants from perchloric acid precipitation
were neutralized by addition of 1:1 (v/v) 1 M potassium carbonate solution. Metabolites were
quantified by coupled enzyme assays (Bergmeyer,
’74), following the NADH oxidation in the assay
Alanine and aspartate levels were evaluated in
10 mM sodium phosphate buffer, pH 6.8, containing 0.15 mM NADH and 20 mM α-ketoglutarate.
Alanine was measured by the addition of 3 U/ml
lactate dehydrogenase and 0.8 U/ml glutamicpyruvic transaminase. Aspartate was measured
by addition of 12 U/ml malate dehydrogenase and
2 U/ml glutamic-oxalacetic transaminase. Succinate was determined in 100 mM triethanolamine
buffer, pH 7.4, containing 100 mM magnesium
chloride, 5 mM EDTA, 0.15 mM NADH and 25
µl/ml of coenzyme A solution (5 mM coenzyme A,
50 mM PEP, 10 mM ATP). The reaction was
started by adding 7.5 U/ml lactate dehydrogenase,
2 U/ml pyruvate kinase and 0.1 U/ml succinic
thiokinase. D-and L-lactate were measured in 400
mM bicine buffer, pH 8.5, containing 4 mM Lglutamate and 6.8 mM NAD+. The reaction started
by addition of 4.5 U/ml D- or L-lactate dehydrogenase and 1.12 U/ml glutamic-pyruvic transaminase. D- or L-lactate concentration were calculated
by comparison with standard curves (0.4–6.4 mM).
Mitochondrial pyruvate formation from D- and
L-lactate was measured by adding 30 µg of protein
from freshly prepared mitochondrial suspension
(within 1 hr from isolation) to 1 ml of 20 mM HepesTris buffer, pH 7.3, containing 0.2 M sucrose, 10
mM potassium chloride, 3 mM magnesium chloride,
2 µg rotenone, 2 mM arsenite. Pyruvate formation
was monitored by adding 3 U/ml L-lactate dehydrogenase, 0.2 mM NADH, 5 mM D- or L-lactate
(final concentrations) and following NADH oxidation at 340 nm. Protein concentration was determined according to Lowry et al. (’51) by using bovine
serum albumin as standard.
Table 1 shows the activities of PK, GI, PEPCK
and ADH in the digestive gland of Mytilus galloprovincialis over a time course of hypoxia. All the
enzymes show time-dependent activity variations
with respect to control (zero time) values. For all
the enzymes the activity changes in response to
hypoxia are already evident in the first 7 hr. PK
activity shows the strongest variation by decreasing to about 20% of the control value. GI and ADH
TABLE 1. Activities of pyruvate kinase (PK), glyoxalase I
(GI), phosphoenolpyruvate carboxykinase (PEPCK), and
alanopine dehydrogenase (ADH) in digestive gland of mussel
Mytilus galloprovincialis over a time course of anoxia1
hypoxia (hr)
0.671 a
0.153 b
0.108 b
0.082 c
0.087 c
0.096 c
0.121 b
0.132 ab
0.140 b
0.106 b
0.125 b
0.062 c
0.037 c
0.0185 ac
0.0109 ad
0.0119 ad
0.0097 ad
0.0123 ad
0.0069 bd
0.0153 a
0.0126 a
0.0115 a
0.0074 b
0.0067 b
0.0072 b
0.0058 b
Enzyme activities were assayed spettrofotometrically at constant temperature (25°C) as described in Materials and Methods. Statistical
evaluation (ANOVA) was carried out on the activities of at least three
independent determinations for each hypoxia time.
*Means followed by the same letters (within each column) are not
significantly different at P < 0.05.
activity decrements are about 20–30%, whereas
PEPCK shows an activity increase of approximately 40% in the same period of time.
The activity values during the hypoxia exposure up to 168 hr show further variations for all
the examined enzymes. Between 7 and 48 hr, PK
activity decreases to about 10% of the initial
value, then shows the tendency to increase so that
its value after 168 hr is about 20% of the control
After the initial rise, PEPCK activity decreases
rapidly after 24 hr, remains constant until 96 hr
and then again decreases averaging 50% of the
aerobic activity at 168 hr.
GI activity slowly decreases until 72 hr and then
falls at 96–168 hr averaging 20% of the control
value. Km values of GI using hemimercaptal as
substrate are 0.53 mM for the aerobic enzyme and
0.60 mM after 96 hr indicating that the decrease
in activity is probably due to the decrease in
amount of enzyme rather than to an intervention
of a different enzymatic form.
On the contrary, changes in kinetic parameters
clearly result for PK. In Table 2 Km values of PK
for the substrate PEP are reported together with
apparent Ki values for the inhibitor alanine, calculated after 0-7-24 hr of anoxic exposure. Km values increase nearly 10-fold after 7 hr and double
after 24 hr. Apparent Ki values decrease by more
than 10-fold after 7 hr and by more than 20-fold
after 24 hr. These two kinetic parameters show
only small variations during the following exposure to hypoxia up to 168 hr.
The activity of ADH continuously decreases by
increasing the time of hypoxia exposure. After 168
hr the remaining activity is about 30% of the initial value. Figure 1A shows the effect of anoxia
exposure on the levels of aspartate and alanine,
Figure 1B shows the levels of D- and L-lactate
and succinate in the digestive gland of Mytilus
TABLE 2. Km and apparent Ki values of pyruvate kinase for
the substrate phosphoenolpyruvate and the allosteric
inhibitor alanine, respectively1
Apparent Ki
0.05 a
0.49 b
0.84 c
14.4 a
1.19 b
0.54 b
Statistical evaluation (ANOVA) was carried out on the activities of
at least three independent determinations for each hypoxia time.
*Means followed by the same letters (within each column) are not
significantly different at P < 0.05.
Fig. 1. Mean alanine and aspartate (A), succinate and DL-lactate (B) levels in the digestive gland of the mussel
Mytilus galloprovincialis during anoxia exposure. Statistical
evaluation (ANOVA) has been made for data relative to succinate (3–6 samples per experimental point). a: significantly
different from the aerobic control value at P < 0.05; b: P <
0.01. Data relative to alanine were not compared by ANOVA
since each experimental point was the mean of two samples.
The concentrations of the end products alanine
and succinate continuously increase with hypoxia
exposure and their levels are almost doubled after 168 hr. The organ content of aspartate is unaffected even up to 168 hr of exposure to hypoxia,
whereas D- and L-lactate levels are undetectable.
In Figure 2 the amount of pyruvate that is
formed by the mitochondrial oxidation of D-and
L-lactate is shown. D-lactate is formed from
methylglyoxal by means the glyoxalase enzyme
This paper deals with metabolic modifications
in the digestive gland of Mytilus galloprovincialis
during anaerobiosis. Mussels were exposed to air
for intervals from 7 to 168 hr at room temperature (20–23°C). In these conditions M. gallo-
Fig. 2. Possible contribution to the synthesis of alanopine
by mitochondrial pyruvate formation from D-lactate in
Mytilus galloprovincialis. (1) Formation of pyruvate through
glycolysis. (2) MG is formed from an intermediate of glycolysis, DHAP, by means of triosephosphate isomerase (Richard,
’91). (3) D-lactate is formed from MG by means of the glyoxalase enzyme system (glyoxalase I and glyoxalase II) (Regoli
and Principato, ’95). (4) The pyruvate production by D-lactate oxidation needs the presence of intact mitochondria. The
enzyme involved is probably α-D-hydroxyacid dehydrogenase
(Tubbs and Greville, ’61). (5) Formation of alanine from pyruvate by glutamic-pyruvic transaminase. (6) Reductive condensation of pyruvate and alanine with formation of alanopine
by means of alanopine dehydrogenase (Fields et al., ’80).
provincialis closes valves hermetically and can
survive for several days.
Variations of enzyme activities and metabolite
levels in response to lack of oxygen are observed.
These metabolic changes start at the beginning
of hypoxia, after as soon as 7 hr.
According to expectations, we found a notable
drop in PK activity from 75 to 90% of the control
value in the period from 7 to 48 hr of hypoxia.
The kinetic properties of the enzyme are profoundly modified as demonstrated by the consistent decreases of Vmax (more than 10-fold) and
apparent Ki for alanine (more than 20-fold). Km
values for PEP instead are increased (about 20fold). These results are in agreement with those
found in Mytilus edulis and in Busycotypus
canaliculatum where the presence, in hypoxia, of
a phosphorylated form of PK has been demonstrated (Siebenaller, ’79; Holwerda et al., ’83;
Plaxton and Storey, ’84). Dephosphorylation occurs as soon as oxygen becomes available again
with the complete restoration of PK activity typical of aerobiosis. It is possible that also in our
case the modified PK is phosphorylated. The small
increases of PK activity in the final hr of hypoxia
could be due to dephosphorylation linked to cell
damage as demonstrated in other organisms in
similar conditions (Storey and Storey, ’90).
The decreased velocity together with the decreased affinity for the substrate PEP and the increased inhibition by alanine (that increases),
indicates that the effective intracellular PK activity must be very low, much less than 10% with
respect to control activity, with consequent PEP
Because of PK inhibition the glycolytic flux in
anoxia could be channeled in alternative pathways
(metabolic branches). We found a measurable
amount of PEPCK in Mytilus galloprovincialis,
thus PEP can become substrate of this enzyme
and go into succinate-producing pathways that
yield additional ATP from substrate-level phosphorylations (Storey and Storey, ’90). This metabolic
shunt is of special importance in the adaptation
to severe hypoxia of helminth worms (Hochachka,
’80), but seemed not to be as important in mollusks such as oysters, where PEPCK activity is
undetectable (Hochachka, ’80).
Another glycolytic shunt that can be increased
by slowing down the carbon flux through PK leads
to increased methylglyoxal formation. In conditions of reduced glycolytic flux, Richard demonstrated that DHAP could be transformed into MG
by an elimination reaction catalyzed by triosephosphate isomerase (Richard, ’91). Methylglyoxal formation from glycolytic intermediates can also
occur non-enzymatically (Phillips and Thornalley,
’93). Methylglyoxal formation is then unavoidable.
This α-ketoaldehyde is a known toxic metabolite
that is efficiently inactivated to D-lactate in the
presence of GSH and glyoxalase I. The activity of
this enzyme protects cells from damage as long
as its activity is maintained.
GI activity is physiologically high in M. galloprovincialis and apparently is not greatly modified
over anaerobiosis up to 72 hr. Activity values are
higher than that of the inhibited activity of pyruvate kinase. The observed small decrease in GI
activity in the last hr of anoxia is probably not
due to enzyme modification, because of the constant Km. Lack of GSH can also be excluded because its concentration is constant throughout
anoxia time (data not shown). The large amount
of glyoxalase II activity present in M. galloprovincialis (Regoli and Principato, ’95) indicates
the presence for an active glyoxalase metabolism.
The importance of the glyoxalase shunt is probably high in hypoxia since the ratio PK/GI is 3.75
before hypoxia but is 1.19 after 7 hr of hypoxia
and decrease to 0.70 after 72 hr. Therefore there
are the right conditions for D-lactate formation
in hypoxia.
Contrary to expectations, however, D-lactate is
undetectable and does not accumulate in the digestive gland of M. galloprovincialis, suggesting
a possible further metabolization. Indeed our data
demonstrate that D-lactate can be transformed
into pyruvate, probably by means of α-D-hydroxyacid dehydrogenase (EC (Tubbs and
Greville, ’61) or another unknown enzyme. In our
experimental conditions, in vitro, L-lactate was
used as well as D-lactate for mitochondrial pyruvate formation. However since we found neither
lactic dehydrogenase nor lactic acid in the digestive gland of M. galloprovincialis, it seems that
L-lactate cannot be formed and that D-lactate is
formed by the glyoxalase enzyme system. The reaction catalyzed by glyoxalases is stereospecific
and only D-lactate is produced from methylglyoxal
(Ekwall and Mannervik, ’73).
The relative changes of PK and GI activities in
hypoxia indicate that the glycolytic carbon flux after dihydroxyacetone phosphate could be channeled
in two similar branches, one giving phosphoenolpyruvate and the other giving methylglyoxal
(Fig. 2). Both branches can be a source of pyruvate
necessary for alanine and alanopine formation.
By comparing the activities of PEPCK and PK
it is possible to determine that about 10% of PEP
could be carboxylated to oxaloacetate and be a
source of the increasing succinate. Then PEPCK
shunt decreases the glycolytic pyruvate and the
high intracellular alanine levels strongly inhibits
PK to activity values lower than those measured
in vitro.
In anoxic mitochondria D-lactate formed from
methylglyoxal is reduced to pyruvate (Fig. 3).
This pathway can play an important role for
the increased alanine and alanopine biosynthesis. In terms of energy, glycolysis yields ATP
but the metabolism of methylglyoxal throughout
glyoxalase I produces the high-energy thioester
S-D-lactoylglutathione. In addition, the mitochondrial D-lactate → pyruvate reaction liberates 2H
that represent an additional and potentially important reducing power available.
Our results indicate the metabolic role of the
glyoxalase pathway are: (1) contributing to respiration in the presence of oxygen by forming pyruvate, and (2) contributing with glycolysis in
anaerobiosis to form the pyruvate that is necessary to produce alanine and alanopine in M.
Fig. 3. Possible contribution of mitochondrial D-lactate oxidation in the formation of succinate in the digestive gland of
Mytilus galloprovincialis.
galloprovincialis. In fact, in the digestive gland
of M. galloprovincialis, as in the oysters, NADH
reoxidation for the maintenance of the cytoplasmic redox balance is associated with the reductive condensation of pyruvate and alanine into
alanopine catalyzed by alanopine dehydrogenase.
Therefore in catalyzing NADH reoxidation this
reaction is formally analogous to that catalyzed
by lactic dehydrogenase in vertebrate anaerobic
glycolysis. In M. galloprovincialis L-lactic acid production and consequent acidification is avoided
since alanine and alanopine are weak acids. Their
accumulation produces a reduced cytoplasmic
acidification and, at the same time, stores pyruvate for the restart of respiration as soon as oxygen becomes available again.
A consistent increase in both alanine and succinate levels is found in M. galloprovincialis during severe hypoxia. Similar results have been
found in oysters, where the rise of these end products correlates with the decrease of aspartate levels (Hochachka, ’80). On the contrary, aspartate
levels in the digestive glands of M. galloprovincialis remain constant throughout exposure to hypoxia. However, the other possibility, the decrease
of glutamate, seems to be excluded by preliminary
data (not shown). The existence of an unknown
aspartate and/or glutamate supply is possible. The
source of amino groups could be ammonia formed
during protein degradation and fixed by glutamic
In Figure 3 the forward and reverse flows of
carbons in Krebs cycle that might contribute to
succinate accumulation in anaerobiosis are shown.
NADH and CO2 are produced by the oxidation of
α-ketoglutarate to succinyl-CoA, while NADH is
consumed by the oxaloacetate reduction to malate.
The source of two H equivalents necessary to re-
duce fumarate to succinate is unknown, but it
probably could derive from D-lactate oxidation to
pyruvate, at least in M. galloprovincialis.
The observed increase of succinate (Krebs cyclelinked), and the involvement in producing pyruvate, suggests that mitochondria are involved in
the anaerobic metabolism of M. galloprovincialis.
The mitochondrial production of pyruvate is possible only in the presence of intact mitochondria.
In conclusion our hypothesis is that in Mytilus
galloprovincialis both glucose (deviation at the
PEP branchpoint) and aspartate contribute in
raising the amount of succinate. Furthermore the
forward flow of carbons from α-ketoglutarate or
metabolites convertible to a-ketoglutarate (e.g.,
glutamate) supplies another possible route for succinate formation. By these reactions mitochondria
are maintained metabolically active in anaerobiosis. The adaptive strategy of M. galloprovincialis
allows mitochondria to remain undamaged and
ready to restart with respiration as soon as oxygen becomes available again.
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