Control of heat production by the
Ca2+-ATPase of rabbit and
trout sarcoplasmic reticulum
Leopoldo
De Meis
Instituto de Ciências Biomédicas, Departamento de
Bioquímica Médica, Universidade Federal do Rio de
Janeiro, Cidade Universitária, Rio de Janeiro 21941-590,
Brazil
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ABSTRACT |
The sarcoplasmic
reticulum Ca2+-ATPase of rabbit
skeletal muscle can convert the energy derived from a
Ca2+ gradient into heat (L. de
Meis, M. L. Bianconi, and V. A. Suzano. FEBS
Lett. 406: 201-204, 1997). In this report, it is
shown that this conversion varies depending on the temperature and on
whether rabbit (endotherm) or trout (poikilotherm) sarcoplasmic
reticulum vesicles are used. The gradient doubled the yield of heat
produced during ATP hydrolysis and the calorimetric enthalpy of ATP
hydrolysis (
Hcal) value
found with both rabbit and trout varied between
10 and
12
kcal/mol in leaky vesicles (no gradient) and between
20 and
22 kcal/mol with intact vesicles (gradient). For the rabbit, the
difference of
Hcal measured
with and without gradient was detected in the range of 30-35°C
and disappeared when the temperature was decreased below 30°C. For
the trout, the difference was detected between 20 and 25°C and
disappeared below 20°C. The effect of the gradient on the
Hcal for ATP
hydrolysis was modified by DMSO, trifluoperazine, and heparin sodium.
calorimetric enthalpy of adenosine 5'-triphosphate
hydrolysis
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INTRODUCTION |
IN ANIMALS LACKING brown adipose tissue, the principal
source of heat during nonshivering thermogenesis is derived from the hydrolysis of ATP by the sarcoplasmic reticulum
Ca2+-ATPase of skeletal muscles
(3, 32, 36). The evidence supporting this proposal is as follows.
1) Calorimetric
measurement of rat soleus muscle (7) indicated that 25-45% of the
heat produced in resting muscle is related to
Ca2+ recirculation between
sarcoplasm and sarcoplasmic reticulum. 2) In cold-acclimated ducklings,
70% of the total heat production is derived from muscle (24, 25). In
these birds, the development of muscular nonshivering thermogenesis was
associated with a 30-50% increase of both sarcoplasmic reticulum
Ca2+-ATPase and
ryanodine-sensitive Ca2+ release
channel. 3) In billfish (marlin,
swordfish), ocular muscles are transformed into specialized heater
tissues (2, 4-6, 38). During the daily fluctuations in
temperature, the swordfish reduces the temperature changes experienced
by the brain and retina by warming these tissues with the heater organ.
The heater tissues are composed of modified muscle cells in which the
contractile filaments are virtually absent and the cell volume is
packed with mitochondria and a highly developed sarcoplasmic reticulum.
Activation of thermogenesis seems to be associated with the
ATP-dependent cycling of Ca2+ at
the sarcoplasmic reticulum.
The Ca2+-ATPase of the
sarcoplasmic reticulum of skeletal muscle is able to interconvert
different forms of energy. During
Ca2+ transport, the chemical
energy derived from ATP hydrolysis is converted into osmotic energy.
After Ca2+ has accumulated inside
the reticulum, a Ca2+ gradient is
formed across the membrane, and this promotes the reversal of the
catalytic cycle of the enzyme, during which osmotic energy is converted
back into chemical energy (1, 9, 13, 21, 30, 33, 34). During cycle
reversal, Ca2+ leaves the
reticulum through the Ca2+-ATPase,
and this is coupled with the synthesis of ATP from ADP and
Pi. The
Ca2+ release is referred to as
active Ca2+ efflux. Not all the
Ca2+ that leaves the vesicles is
active; a part of it leaks through the
Ca2+-ATPase without promoting
synthesis of ATP, and this is referred to as uncoupled
Ca2+ efflux (12, 17, 19, 20, 22,
27-29, 31). In a previous report (14), it was shown that heat is
produced when Ca2+ leaks from the
vesicles through the uncoupled efflux. In this process, the
Ca2+-ATPase converts osmotic
energy into heat. These experiments were performed at 35°C using
vesicles derived from rabbit skeletal muscle. In this report, the
effect of temperature and drugs on the heat released during ATP
hydrolysis was studied using the Ca2+-ATPase of two animals that in
physiological conditions have different body temperatures, the rabbit,
an endothermic animal, and the trout, a poikilothermic animal.
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METHODS |
Preparation of intact and permeated vesicles.
Vesicles derived from the longitudinal sarcoplasmic reticulum of rabbit
hindleg white skeletal muscle and from rainbow trout (Salmo gairdnerii) dorsolateral
muscles were prepared as previously described (8, 26). Electrophoretic
analysis revealed that the two preparations have practically no
junctional proteins (8, 31). The efflux of
Ca2+ measured in the rabbit and
trout vesicles was not altered by ryanodine, indicating that they did
not contain significant amounts of ryanodine-sensitive
Ca2+ channels. The two vesicle
preparations also did not exhibit the phenomenon of
Ca2+-induced
Ca2+ release found in the heavy
fraction of sarcoplasmic reticulum. The fish used were adapted to the
temperature range of 10-18°C. Rabbit permeated vesicles were
prepared by incubating the vesicles at pH 9.0 in the presence of 2 mM
EGTA at room temperature for 20 min. After that, the pH was readjusted
to 7.0; the ATPase activity of the vesicles was maintained, but the
permeability of the membrane was increased and the vesicles were no
longer able to accumulate Ca2+
(23). To ensure that the vesicles did not retain any
Ca2+ in the lumen, the divalent
cation ionophore A-23187 (5-10 µM) was included in the assay
medium in addition to the treatment with EGTA at pH 9.0.
ATPase activity,
Ca2+ uptake, ATP
synthesis, and heat of reaction.
The methods for measuring the ATPase activity using
[
-32P]ATP, calcium
uptake using 45Ca, and ATP
synthesis from ADP and
32Pi
are described elsewhere (10). Heats of reaction were measured using an
OMEGA isothermal titration calorimeter from Microcal (Northampton, MA)
(14, 41). The calorimeter cell was filled with a reaction medium
containing ATP, and the reference cell was filled with Milli-Q water.
After equilibration at the desired temperature, the reaction was
started by injecting the vesicles into the reaction cell, and the heat
change due to the ATP hydrolysis was recorded. The calorimetric
enthalpy of ATP hydrolysis
(
Hcal) was
calculated by dividing the amount of heat released by the amount of ATP
hydrolyzed. The units used were moles for ATP hydrolyzed and
kilocalories for the heat released. A negative value indicates that the
reaction was exothermic.
Figures 1-7 are resentative experiments, and the values of Tables
1-3 are means ± SE of the principal finidings described in this study.
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RESULTS |
Ca2+
transport by rabbit and trout vesicles.
The activity of the Ca2+-ATPases
increased with the temperature, and after 40-min incubation at
25°C, the amounts of Ca2+
retained by the rabbit and trout vesicles were practically the same
(Figs. 1 and
2 and Table
1). The trout
Ca2+-ATPase is unstable at
temperatures higher than 25°C and is inactivated after a few
minutes of incubation at 35°C (8). The rabbit ATPase, however, was
stable for >1 h at 35°C. The physiological body temperature of
the trout varies between 20 and 25°C, whereas that for the rabbit
is 37°C. Thus, despite the fact that the two enzymes can pump
similar amounts of Ca2+ at
25°C, at the physiological body temperature the rabbit sarcoplasmic reticulum was able to pump more
Ca2+ and at a faster rate than the
reticulum of the trout (Table 1 and Figs.
1A and
2A). After formation of the
gradient, both the rabbit and the trout
Ca2+-ATPases were able to
synthesize a small amount of ATP, and in all conditions tested, the
rate of synthesis was 45 to 25 times smaller than the rate of ATP
hydrolysis (Table 1).

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Fig. 1.
Rabbit: heat released during ATP hydrolysis and
Ca2+ uptake at 35°C ( ) and
25°C ( ). Assay medium composition was 50 mM MOPS-Tris buffer, pH
7.0, 0.1 mM CaCl2, 1 mM ATP, 4 mM
MgCl2, and 10 mM
Pi. Medium was divided into 3 samples. One was used for heat measurements
(C). To the other 2 samples, trace
amounts of either 45Ca or
[ -32P]ATP were
added for measurement of Ca2+
uptake (A) and ATPase activity
(B). Three reactions were started
simultaneously by addition of either 10 µg/ml (35°C; ) or 40 µg/ml (25°C; ) rabbit intact sarcoplasmic reticulum vesicles.
In D, data of
B and
C were replotted.
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Fig. 2.
Trout: Ca2+ uptake
(A) and heat released (C) during ATP
hydrolysis at 25°C ( ) and 17°C ( ). Assay medium
composition and other experimental conditions were as described in Fig.
1 using either 20 µg/ml (25°C; ) or 40 µg/ml (17°C; )
trout intact sarcoplasmic reticulum vesicles. In
D, calorimetric enthalpy of ATP
hydrolysis
( Hcal) was
calculated by dividing amount of heat released measured in
C by amount of ATP hydrolyzed in
B.
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Table 1.
Ca2+ uptake, ATPase activity, and ATP synthesis by
rabbit and trout sarcoplasmic reticulum vesicles at different
temperatures
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Heat production in the presence and absence of a
Ca2+ gradient.
Both in the presence and in the absence of a
Ca2+ gradient, the amount of heat
released was proportional to the amount of ATP hydrolyzed (Figs.
1-3). This could be visualized by either
plotting the heat released as a function of the amount of ATP
hydrolyzed (Figs. 1D and 3) or
calculating the
Hcal at each
incubation interval (Fig. 2D). In an
earlier report (14), it was shown that the heat released for each ATP
molecule hydrolyzed by rabbit intact vesicles at 35°C was larger
than that measured with leaky vesicles. We now show that the difference
between intact and leaky vesicles varied depending on the temperature
of the assay and the source of the vesicles used (Fig.
4 and Table 2). For the rabbit, the value of
Hcal measured
at 35°C with intact vesicles was double that measured with leaky
vesicles. This difference was no longer detected when the temperature
was decreased to 25°C, as if in the rabbit the mechanism that
converts osmotic energy into heat production were turned off when the
temperature was decreased to a level far from the physiological body
temperature. For the trout vesicles (poikilotherm), formation of a
transmembrane Ca2+ gradient at
25°C leads to a change of the
Hcal for ATP
hydrolysis to a value similar to that measured with the rabbit vesicles
at 35°C. The difference of
Hcal values
measured with trout vesicles in the presence and absence of a
Ca2+ gradient was also abolished
when the temperature of the medium was decreased, in this case to a
value below 17°C. The
Hcal measured
with leaky vesicles did not vary with the temperature or with the
source of the vesicles used (Figs. 3 and 4 and Table 2).

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Fig. 3.
Heat released during ATP hydrolysis by rabbit and trout leaky vesicles
(no gradient) at different temperatures. Ionophore A-23187 (5 µM) was
included in assay medium. Other additions to assay medium and
experimental conditions were as described in Figs. 1 and 2. and
, Rabbit leaky vesicles at 35 and 25°C, respectively; and
, trout vesicles at 25 and 17°C, respectively.
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Fig. 4.
Effects of gradient and temperature on
Hcal of ATP
hydrolysis measured with trout ( , ) and rabbit ( , )
vesicles. Assay medium composition and other experimental conditions
were as described in Figs. 1-3 for intact (solid symbols) and
leaky vesicles (open symbols).
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Controls.
Pi was included in the assay
medium in most of the experiments described in this report. During
transport, this anion diffuses through the membrane and forms calcium
phosphate crystals inside the vesicles (15). The difference in heat
release measured with intact and leaky vesicles was not related to the
formation of calcium precipitates inside the vesicles because
1) the
Hcal values
found without added Pi or in the
presence of 10 mM Pi were the same
(data not shown and Ref. 14) and 2)
the amount of calcium phosphate precipitate formed inside the vesicles
is proportional to the amount of
Ca2+ accumulated by the vesicles
(9, 15, 30). At 25°C, the amounts of
Ca2+ accumulated by the vesicles
of trout and rabbit were practically the same (Table 2), but only in
the trout did the accumulation of
Ca2+ lead to an increase of heat
production during ATP hydrolysis.
The difference of
Hcal for ATP
hydrolysis measured with leaky and intact vesicles does not seem to be
related to Ca2+ efflux through the
ryanodine and/or the Ca2+
channel regulated by inositol 3-phosphate for two reasons.
1) The rabbit and trout vesicle
preparations used were derived primarily from the longitudinal
sarcoplasmic reticulum. As previously shown (8, 31), electrophoretic
analysis of the two preparations revealed that the dominant protein
found in both rabbit and trout vesicles was the
Ca2+-ATPase, with no
ryanodine-sensitive Ca2+ channels.
2) The
Ca2+ transport of the vesicles was
not sensitive to inositol 3-phosphate, ryanodine, or caffeine, nor did
it exhibit the phenomenon of activation of
Ca2+ efflux by external
Ca2+, i.e.,
Ca2+-induced
Ca2+ release (data not shown).
Effect of DMSO.
In early reports (11, 16, 18), it was shown that DMSO promotes the
coupling of enzyme units that are leaky and abolished the difference of
Hcal for ATP
hydrolysis measured with intact and leaky vesicles (14). These
measurements were done only with rabbit vesicles at 35°C. We now
show that DMSO only modified the
Hcal of ATP
hydrolysis in the temperature range in which the gradient enhanced the
yield of heat released during ATP hydrolysis (Table 2). Thus it had no
effect 1) on the
Hcal measured
with rabbit intact vesicles (gradient) below 25°C,
2) on trout vesicles at temperatures
below 20°C, and 3) at all
temperatures when the vesicles were leaky (data not shown).
Effect of uncoupling drugs.
Heparin and trifluoperazine inhibit the
Ca2+ uptake and the synthesis of
ATP measured with rabbit vesicles at 35°C (Figs.
5 and 6). The two drugs
modifiy the mechanism of Ca2+
transport in different ways (12, 19, 37). Heparin promotes a small
increase of the Ca2+ efflux rate
(19) and inhibits the rate of ATP hydrolysis (Fig. 5), whereas
trifluoperazine promotes a large increase of
Ca2+ efflux (12, 22) and enhances
the rate of ATP hydrolysis (Fig. 6C). With heparin, the inhibition of
Ca2+ uptake is mostly due to a
decrease of the ATPase activity, whereas with trifluoperazine it is
promoted by the large increase of the Ca2+ efflux (12, 19). In a recent
report, it was shown that at 35°C heparin increases the yield of
heat produced during ATP hydrolysis by rabbit intact vesicles (14). The
effects of heparin and trifluoperazine on
Ca2+ uptake and ATP hydrolysis at
25°C with both rabbit and trout vesicles were the same as those
previously measured with rabbit at 35°C (Figs. 5 and 6). The effect
on the
Hcal
for ATP hydrolysis, however, was found to vary depending on the temperature and the vesicle preparation used (Fig.
7A
and Table 3). For the rabbit and in
presence of a gradient, the
Hcal for ATP
hydrolysis became more negative after the addition of 2-4 µg/ml
heparin, but this was only detected at 35°C. At 30°C and at
25°C, 2-4 µg/ml heparin did not modify the
Hcal measured
with and without a gradient, despite the fact that it decreased both
the amount of Ca2+ uptake and the
rate of ATP hydrolysis (Table 3). For the trout vesicles at 25°C,
the effect of heparin was opposite to that found with rabbit at
35°C, the
Hcal value
measured with intact vesicles becoming less negative with increasing
heparin concentrations, reaching the same value as that found with
leaky vesicles (Fig. 7A). Both the
rabbit and trout vesicles were no longer able to accumulate
Ca2+ when the heparin
concentration was raised from 4 to 10 µg/ml. In these concentrations,
the vesicles were still able to hydrolyze ATP, and the
Hcal for ATP
hydrolysis was the same as that measured with leaky vesicles in the
range of 15-35°C (Table 3). The effect of heparin in rabbit skeletal muscle is antagonized by
K+ (19, 37).
K+ had no effect on the heat
produced during ATP hydrolysis measured with either leaky vesicles
(data not shown) or with intact vesicles (Table 3), but it did
antagonize the effects of heparin on the
Hcal for ATP
hydrolysis and transport of Ca2+
in both rabbit at 35°C and trout vesicles at 25°C (Table 3).

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Fig. 5.
Inhibition of Ca2+ uptake
(A), ATP synthesis
(B), and ATP hydrolysis
(C) by heparin sodium. Assay medium
composition and other experimental conditions were as described in
Figs. 1 and 2 using intact rabbit vesicles at either 35°C ( ) or
25°C ( ) and trout intact vesicles at 25°C ( ). On figure,
100% refers to activity measured in absence of heparin sodium.
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Fig. 6.
Inhibition of Ca2+ uptake
(A), ATP synthesis
(B), and ATP hydrolysis
(C) by trifluoperazine. Assay medium
composition and other experimental conditions were as described in
Figs. 1 and 2 using intact rabbit vesicles at either 35°C ( ) or
25°C ( ) and trout intact vesicles at 25°C ( ). On figure,
100% refers to activity measured in absence of trifluoperazine.
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Fig. 7.
Effects of heparin sodium (A) and
trifluoperazine (B) on
Hcal of ATP
hydrolysis. Assay medium composition and other experimental conditions
were as described in Figs. 1 and 2 using intact rabbit vesicles at
either 35°C ( ) or 20°C ( ) and trout intact vesicles at
25°C ( ).
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The effect of trifluoperazine on the
Hcal for ATP
hydrolysis has not been measured previously. With the rabbit at
35°C and the trout at 25°C, it was found that the
Hcal for ATP
hydrolysis measured in the presence of a gradient became less negative
with trifluoperazine, reaching the same value as that measured with
leaky vesicles (Fig. 7B). At
25°C, trifluoperazine inhibited the
Ca2+ uptake measured with rabbit
vesicles (Fig. 6) but had no effect on the
Hcal for ATP
hydrolysis (Fig. 7B).
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DISCUSSION |
The data reported show that the amount of heat produced during ATP
hydrolysis by the Ca2+-ATPase
increases when a gradient is formed across the sarcoplasmic reticulum
membrane. The gradient-dependent heat production, however, seems to be
arrested when the temperature of the medium is decreased more than
5°C below the physiological body temperature, i.e., below 30°C
for the rabbit and below 20°C for the trout. The enhancement of
heat production associated with the gradient could therefore play a
physiological role in the maintenance of the body temperature but would
not be a good emergency system to raise the body temperature after
rapid cooling of the animal to an extreme point that leads to a large
variation of the body temperature. More experiments with different
animal species are needed to establish whether or not the temperature
limits observed in Table 2 and Fig. 4 are specific characteristics of
rabbit and trout or represent a common feature of endotherms and
poikilotherms.
When ATP is hydrolyzed by the
Ca2+-ATPase, part of the chemical
energy released is dissipated into the surrounding medium as heat. The
other part is used to pump Ca2+
across the vesicle membrane, and in this process, chemical energy is
converted into osmotic energy. When
Ca2+ flows back from the vesicle
lumen to the medium driven by the Ca2+ gradient, the osmotic energy
can be used to synthesize ATP, or it can be converted into other forms
of energy that cannot be detected with the methods used in this report.
An example is molecular work in which a protein cycles between two or
more different conformations (35, 39, 40). The data described in this
and a previous report (14) suggest that the energy derived from the
gradient can also be converted into heat. However, it seems that
osmotic energy cannot be transformed spontaneously into heat and that a
device is needed for this conversion. For the sarcoplasmic reticulum, the device is probably the
Ca2+-ATPase itself, which, in
addition to interconverting chemical into osmotic energy, could also
convert osmotic energy into heat. The following data support this
proposal. 1) In leaky vesicles, Ca2+ is translocated across the
membrane during ATP hydrolysis and afterward flows back to the medium
through the permeabilized membrane. If the simple diffusion of
Ca2+ through any kind of pore in
the membrane would lead to heat production, then the same
Hcal value
should have been found with intact and permeabilized vesicles (Table
2). 2) Trifluoperazine greatly
enhances the Ca2+ efflux from both
rabbit and trout vesicles (12; Fig. 6). If the simple diffusion of
Ca2+ through the membrane were to
produce heat, then in steady-state conditions the
Hcal for ATP
hydrolysis should become more negative and not less negative as
observed in Fig. 7B with
40 and 70 µM trifluoperazine.
3) In previous reports (12, 19, 37),
it was shown that heparin and trifluoperazine uncoupled the reversal of
the Ca2+ pump in different
manners. At the concentration range used in Figs. 5 and 7, heparin
promoted the same increase of Ca2+
efflux as that induced by ADP during reversal of the pump.
Trifluoperazine, on the other hand, promotes an increase of the efflux
orders of magnitude larger than that measured with either heparin or
ADP. At present, we do not know why heparin modifies the
Hcal for ATP
hydrolysis in rabbit and trout differently (Fig. 7 and Table 3).
Perhaps this is related to differences in the protein structure of the
two Ca2+-ATPase isoforms. In spite
of this difference, however, if the simple diffusion of
Ca2+ through the membrane were to
produce heat, then it would be expected that heparin should have the
same effect on the
Hcal value
measured with rabbit and trout vesicles.
A possible interpretation of the data presented in this and previous
reports (12, 14, 16-22) is that during transport in intact
vesicles, the different
Ca2+-ATPase units could operate in
four different modes as follows. 1)
Most of the enzyme units would work in the forward mode totally coupled, cleaving ATP and accumulating
Ca2+ inside the vesicles. In this
mode, one part of the energy derived from the hydrolysis of ATP would
be dissipated as heat (10-12 kcal/mol), and the other part would
be converted into osmotic energy. 2)
A small part of the enzyme units would work in the reverse direction
and synthesize ATP in a process coupled with a slow
Ca2+ efflux. In this condition,
there would be no heat production. 3) A few enzyme units would be
partially uncoupled, working in the reverse direction but without
synthesizing ATP, and the slow Ca2+ release would produce heat.
In this form, the ATPase would convert osmotic energy into heat, and
this would account for the difference of heat production measured
during the hydrolysis of ATP by intact and leaky vesicles in Table 2.
This enzyme form is thermosensitive and becomes incapable of converting
osmotic energy into heat below 30 or 20°C depending on the animal
species studied. As shown previously (16, 18, 20), DMSO couples these
leaky enzyme units, arresting the heat production and increasing both
the amount of Ca2+ retained by the
vesicles and the amount of ATP synthesized. In the rabbit, heparin
increases the fraction of enzyme units that are partially uncoupled and
produces heat. 4) The enzyme would be totally uncoupled as observed with trifluoperazine (12). Now
Ca2+ leaves the vesicles at a fast
rate in a process that is not coupled to either ATP synthesis or heat
production (Figs. 6 and 7B).
Finally, the effects of DMSO, heparin, and trifluoperazine on the
Hcal of ATP
hydrolysis raise the possibility that in living animals drugs may
change the yield of heat derived from the transport of
Ca2+ and the hydrolysis of ATP by
the sarcoplasmic reticulum, thus characterizing a new mechanism of
thermoregulation in which the drugs act directly at the source of heat
production, changing the amount of heat generated from each molecule of
ATP cleaved during the transport of
Ca2+ and not indirectly through
hormones, receptors, and nervous system that enhance the metabolic
activity of the cell.
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ACKNOWLEDGEMENTS |
I thank Valdecir A. Suzano for technical assistance.
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FOOTNOTES |
This work was supported by grants from PRONEX-Financiadora de Estudos e
Projetos, Conselho Nacional de Desenvolvimento Científico e
Tecnológico, and Fundaçao de Amparo à Pesquisa do
Estado do Rio de Janeiro.
Address for reprint requests: L. de Meis, Instituto de Ciências
Biomédicas, Departamento de Bioquímica Médica,
Universidade Federal do Rio de Janeiro, Cidade Universitária, RJ
21941-590, Brazil.
Received 13 November 1997; accepted in final form 23 February
1998.
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