(Received for publication, July 5, 1995; and in revised form, October 25, 1995)
From the
The nucleotide vesicular transport has been studied with the
fluorescent substrate analogues, the
(1,N-ethenoadenosine) nucleotides. The transport
experiments were carried out with granular preparations from bovine
adrenal medulla, and
-ATP,
-ADP, and
-AMP were
quantified after separation by high performance liquid chromatography.
The granular concentration increase of all three nucleotides was
time-dependent. The concentration dependence of
-nucleotide
transport to chromaffin granules did not follow the Michaelis-Menten
kinetics and presented a similar three-step curve with cooperativity.
This shape can be considered to be the result of the addition of three
sigmoidal curves with their corresponding kinetic parameters.
-ATP
exhibited K values of 0.25, 1, and 3 mM and V
values of 0.02, 0.04 and 0.19
nmol
min
mg of protein
,
for the first, second, and third curves for each step, respectively.
-ADP exhibited K values of 0.15, 0.9, and 3.6 mM and V
values of 0.025, 0.035, and 0.3
nmol
min
mg of protein
,
respectively for the first, second, and third curves.
-AMP
exhibited K values of 0.2, 1.2, and 3.2 mM, and V
values of 0.01, 0.04, and 0.055
nmol
min
mg of protein
,
also for the first to third steps. The Hill numbers for
-ATP,
-ADP, and
-AMP were not constant but a function of the
transport saturation. The nonhydrolyzable ATP analogues AMPPNP,
ATP
S, and ADP
S were activators of
-nucleotide transport
at concentrations under 1 mM and inhibitors at higher
concentrations. Atractyloside and N-ethylmaleimide partially
inhibited the nucleotide granular transport. High extragranular ATP
concentrations specifically induced the exit of the previously
transported granular
-ATP.
Studies of vesicular storage mechanisms that allow the
functioning of neural and non-neural secretory tissues are essential to
understand the various steps in cellular communication. It is well
known that secretory vesicles containing catecholamines or
acetylcholine also contain nucleotides such as ATP and ADP, their
intragranular levels reaching 0.15 to 0.2 M in some cases
(Winkler and Carmichael, 1982; Zimmermann, 1994). More recently,
diadenosine polyphosphates: ApA, Ap
A, and
Ap
A have also been found (Rodriguez del Castillo, 1988;
Pintor et al., 1992b, 1992c; Schlütter et al., 1994). The co-stored vesicular components are also
co-released to the extracellular media (Pintor et al., 1991,
1992a; Zimmermann, 1994).
Chromaffin granules from adrenal medulla have been the most employed model by which to study the mechanisms of vesicular storage, both for aminergic and nucleotidic components. The vesicular transport of catecholamines has been characterized from a physicochemical, pharmacological, and molecular biology approach in this preparation (Njus and Radda, 1978; Knoth et al., 1980; Schermann and Henry, 1983; Liu et al., 1992; Henry et al., 1994). Furthermore, the nucleotide vesicular transport has been characterized using radiolabeled nucleotides and similar specificity, and affinity values have been reported in chromaffin granules and in the cholinergic vesicles from the torpedo electric organ (Aberer et al., 1978; Luqmani, 1981; Grüninger et al., 1983). Molecular biology approaches by which to characterize the nucleotide vesicular transporter have been also made (Schläfer et al., 1994). The fact that ATP and other nucleotide triphosphates are also substrates for the vesicular ATPase that generate the gradient necessary for their own transport makes the experimental approach to nucleotide transport difficult (Pollard et al., 1976; Cidon et al., 1983; Apps and Percy, 1987; Nelson, 1992).
The existence of kinetic and allosteric cooperativity was recently demonstrated, for the first time, in the equilibrative nucleoside transporter from neural tissues (Casillas et al., 1993). However, no allosteric regulation for aminergic or nucleotidic vesicular transporters has yet been reported. It is possible, though, that such mechanisms exist, which could take into account the rather complex energetic and metabolic situations related to the intracellular ATP levels, as occurs with the key regulatory enzymatic steps in cellular metabolism (Ricard and Cornish-Bowden, 1987).
In this work,
the nucleotide vesicular transport has been characterized by using the
fluorescent (1,N-ethenoadenosine) nucleotides
(
-ATP,
-ADP, and
-AMP) as substrate analogues. The
-adenine nucleotides can be followed, once internalized, by
monitoring their fluorescence (Secrist et al., 1972;
Rotllán et al., 1991). Due to the
existence of multiple enzymes that can modify the phosphate moiety of
nucleotides and their analogues, the
-ATP,
-ADP, and
-AMP have been quantified in every assay after their separation by
HPLC chromatography and fluorescent detection. This technique proved to
be efficient and accurate in studying the vesicular nucleotide
transport, allowing its deeper kinetic characterization. The complexity
of the saturation kinetics for
-ATP,
-ADP, and
-AMP
transport can be interpreted by postulating the existence of
cooperative mnemonic mechanisms.
The purity of chromaffin granule preparations was controlled, measuring the presence of mitochondrial and plasma membrane enzyme markers as described elsewhere (Torres et al., 1992).
The crude
granular fraction (P), only for comparative purposes, and
the purified chromaffin granules were employed in the transport
experiments. These fractions were resuspended, immediately before their
use, in a buffer containing 0.3 M sucrose, 5 mM MgCl
, and 10 mM Tris-HCl, pH 7.2 (buffer B),
in a volume of 1 ml per each g of original fresh adrenomedullary
tissue. These preparations contain a very reproducible quantity of
protein, 8.6 ± 0.6 mg/ml and 3.8 ± 0.7 mg/ml (mean
± S.D.; n = 10), respectively, for the
nonpurified (P
) and purified chromaffin granule fractions.
Transport experiments were done routinely with 100 µl of the
granular preparations that corresponded to 0.1 g of the original
adrenomedullary tissue, resuspended in the assay buffer B. The final
volume of transport experiments was 200 µl containing the
etheno-nucleotides in concentrations ranging from 50 µM to
6 mM in the saturation experiments. In the time function
experiments, a single concentration of -nucleotides was employed
and is specified in the text when required. When other compounds were
added, special care concerning the osmotic conditions of the assay was
taken. When indicated, the transport assays were carried out in the
presence of 5 µM atractyloside (Sigma) to inhibit the
mitochondrial adenine nucleotide translocase (Klingenberg et
al., 1975) and/or 5 µM Ap
A (Boehringer,
Germany) to inhibit the cytosolic adenylate kinase (Lienhard and
Secemski, 1973). When the ionophores valinomycin (20 µM),
nigericin (10 µM) with 5 mM KCl or carbonyl
cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (Sigma) were
employed, the granular preparations were preincubated with them for a
3-min period. Experiments were done at 25 °C and started with the
addition of the granular preparation. The concentration dependence
experiments were usually carried out at 10 min experimental time. The
transport was stopped by the addition of cold (0 °C) buffer A. The
volume of the stop solution added was dependent on the additional
processing to isolate the granules as follows. (i) 0.8 ml of stop
solution was added and then the mixture was layered over 4 ml of 1.6 M sucrose and centrifuged at 100,000
g for 45
min. (ii) 13 ml of stop solution were added and then the mixture was
centrifuged directly at 20,000
g for 30 min. The
washing procedure was repeated twice. This stop procedure gave the
lowest blank values and was used preferentially.
In both stop
procedures, an aliquot of the supernatant, to control the extragranular
metabolism of -nucleotides, was taken, and the pellet was
resuspended in 1 ml of a mixture of 0.1 ml of ethanol and 0.9 ml of the
HPLC mobile phase buffer, described under HPLC Procedures. The
granular resuspension was submitted to a freeze-thaw cycle and then
centrifuged at 100,000
g for 30 min, and the
supernatants were taken for additional analysis to quantify the
-nucleotides internalized. The samples were stored at -80
°C until their processing by HPLC. Blanks were done maintaining the
granular preparations at 0 °C and then processed as the assay
samples. The values obtained for the zero time blanks were subtracted
from the assays.
Controls of chromaffin granule integrity were done
by measuring the presence of catecholamines in the extragranular
incubation media of transport experiments by electrochemical detection
as described elsewhere (Castro et al., 1990). Dopamine
-monooxygenase activity was measured as already described
(Miras-Portugal et al., 1980). Proteins were determined by the
Bradford method.
The separation of the -adenine
nucleotides was performed using ion pair chromatography, as described
previously for adenine nucleotides (Pintor et al., 1992c).
Briefly, the mobile phase conditions were as follows: 10 mM KH
PO
, 2 mM tetrabutylammonium,
and 15% acetonitrile, pH 7.5, with a column Novapak C-18 cartridge
column (4-µm particle size, 15-cm length, and 0.39-cm internal
diameter) from Waters. The eluents from the column were excited at a
306 nm, and the emission at 410 nm was recorded. The peak areas were
transformed to concentrations by correlation with commercial standards.
because the V/S representation was not hyperbolic and appeared to be the result of the addition of various Hill type curves with two or more inflexion points and plateaus.
As
the kinetics do not fit in a simple Hill equation, the differential
method of Kurganov(1982) was employed to calculate the n value with respect to the saturation
where V, V`, and V" were the
transport velocities obtained at a substrate concentration of
[S],
[S]
/
, and
[S]
, respectively, and
is a constant multiplier which was higher than unity, in our
case the
value was 2. A similar kinetic analysis was done with
complex enzymatic behaviors (Kagan and Dorozhko, 1973).
The rapid destruction of
-ATP in the extragranular incubation media of crude granular
fractions (P
) indicated the necessity for using purified
granular preparations. Thus, the chromaffin granules purified on
density gradient were employed to carry out the transport experiments. Fig. 1shows the extragranular and intragranular distribution of
-adenine nucleotides as a function of the experimental time. In
the extragranular media (Fig. 1A), the
-ATP
concentration with purified fractions was maintained more than 80%,
even after 2 h of incubation period. The
-ADP and
-AMP are
present in the commercially available
-ATP and represent,
respectively, 6.1% and 0.4% of the total. The
-ADP increased very
slowly during the incubation period and even to a lower extent was the
production of
-AMP. The percentage distribution of the nucleotides
after a 10-min incubation was 0.5, 10, and 89.5%, respectively, for
-AMP,
-ADP, and
-ATP. These data were necessary to
establish the optimum situation for the concentration dependence
studies. When the experiments were carried out in the presence of
Ap
A, 10 µM or 5 µM atractyloside,
no changes were observed in
-ADP and
-AMP production at the
extragranular level.
Figure 1:
HPLC
chromatographic profiles of -ATP transport to purified chromaffin
granules as a function of time. Samples of purified chromaffin granules
containing 0.38 mg of protein were incubated in the presence of 6
mM
-ATP, with 5 µM atractyloside, as
described under ``Experimental Procedures.'' The consecutive
HPLC chromatographic profiles represented correspond to the
experimental times of 0 min, 10 min, 30 min, 1 h, and 2 h. The
transport was stopped by addition of 13 ml of cold stop solution buffer
A and processed as indicated under ``Experimental
Procedures.'' The fluorescence of
-nucleotides is expressed
in arbitrary units. A, HPLC chromatograms of the
-nucleotide levels in the extragranular media. B, HPLC
chromatograms of the
-nucleotide content in the granular pellet.
The
-nucleotides in the intragranular samples exhibited a slightly
shorter time of retention when compared to the extragranular samples.
This is due to the extremely high levels of endogenous nucleotides
(mM) that are not detectable by fluorescence, but induce a
saturation on the HPLC chromatographic column. Addition of external
standards corresponds exactly to the
-adenine nucleotides in each
experimental situation. These chromatograms represent a very
reproducible experiment.
Fig. 1B shows the intragranular
levels of etheno-nucleotides. -ATP constituted the overwhelming
majority of intragranular nucleotides.
-ADP and
-AMP were
also stored and appeared at a higher ratio than outside the granule.
The presence of
-ADP at such levels will be further emphasized in
the results of the
-ADP transport. The intragranular transport of
-ATP was maintained linear until 30 min incubation time in the
experiment reported in Fig. 1and in general with high
extragranular nucleotide concentration; with the lowest
-ATP
concentration used (50 µM), the lineal period was reduced
to 15 min. The total of the intragranular
-nucleotides never
surpassed 2% of that present in the incubation media. This ratio also
assures the linearity of the transport process.
Figure 2:
Concentration dependence of -ATP
transport to chromaffin granules. The transport experiments were
accomplished with 0.38 mg of protein of purified chromaffin granules
and incubated with
-ATP concentrations ranging from 50 µM to 6 mM, for 10 min at 25 °C as described under
``Experimental Procedures.'' Both the extragranular and the
intragranular
-nucleotide levels were quantified after separation.
To avoid a crowded figure, only the HPLC chromatograms corresponding to
the
-ATP concentrations of 0.3, 0.4, 0.6, 0.8, 1, 1.2, 1.6, 2, 4,
and 6 mM, in a consecutive order have been represented. A, HPLC chromatograms of the extragranular levels of
-AMP,
-ADP, and
-ATP, as a function of
-ATP
concentration. B, HPLC chromatograms of the intragranular
levels of
-AMP,
-ADP, and
-ATP, as a function of
-ATP concentration.
At the intragranular level, as shown in Fig. 2B, the -ATP comprised the majority of the
nucleotides present, but a significant fraction was as
-ADP and
also
-AMP. The nucleotide distribution percentages were completely
different from those at the extragranular level and corresponded to
72.5, 25.5, and 2 at 50 µM and 82.5, 15.2, and 2.3 at 6
mM, respectively, for
-ATP,
-ADP, and
-AMP. As
reported in the time function experiments, this distribution did not
change significantly with time; even after a 2-h incubation period at
25 °C, the distribution was modified only slightly. When internal
standards of
-ATP were added to the chromaffin granule pellet
after transport, and afterwards processed as indicated under
``Experimental Procedures,'' the
-ADP production as a
direct consequence of the procedure was under 0.5% and under 1% for
-AMP.
From the intragranular -nucleotide values, the
saturation curve was plotted, and a nonhyperbolic curve was observed in
our experimental conditions, as shown in Fig. 3A. The
complex dependence of transport velocity with respect to extragranular
-ATP concentration observed made it necessary to interpret the
saturation curve as the superposition of various sigmoidal kinetics.
Therefore, the processing of the experimental data was done by described under ``Experimental Procedures''
(Kurganov, 1982). This analytical procedure has been employed already
with enzymes exhibiting similar kinetic behavior (Somero and Hochachka,
1969; Irving and Williams, 1973a, 1973b; Kagan and Dorozhko, 1973). The
addition of three sigmoidal curves was necessary, in this case, to
process the experimental data, as shown in Fig. 3B. The
affinity values (K), partial V
, and the
Hill number for each curve, are summarized in Table 1.
Figure 3:
Saturation studies of -ATP transport
to chromaffin granules. A, the saturation curve was obtained
by processing the HPLC data from Fig. 2B. The
-ATP
concentration ranged from 50 µM to 6 mM.
Transport velocity is expressed as nanomoles of
-ATP transported
per min per mg of chromaffin granule proteins. B, three
sigmoidal curves the addition of which accounts for the experimentally
observed curve (A). Their affinity values (K or the
equivalent S
), together with the corresponding V
and n
values, are
summarized in Table 1. C, plot of n
against the
-ATP saturation calculated for curve A,
according to Kurganov(1982), under ``Experimental
Procedures.'' These results represent a typical experiment
performed in triplicate, which is very
reproducible.
The
true cooperativity that is stated by the transporter depends on the
saturation level, because the n number is
variable. This variation can be evaluated according to Kurganov's
differential procedure ( under ``Experimental
Procedures'') and is shown in Fig. 3C.
At the extragranular level, some small
amounts of -ATP appeared during the incubation time, reaching a
maximum of 4% of the total
-nucleotide (Fig. 4A).
The action of adenylate kinase and the improbable action of the
mitochondrial adenine nucleotide translocase were excluded, because the
experiments were carried out in the presence of Ap
A and
atractyloside.
Figure 4:
HPLC chromatographic profiles of -ADP
transport to chromaffin granules. The transport experiments were
accomplished with 0.38 mg of protein of purified chromaffin granules
and incubated with
-ADP concentrations ranging from 50 µM to 6 mM, for 10 min at 25 °C as described under
``Experimental Procedures.'' Both the extragranular and the
intragranular
-nucleotide levels were quantified after separation.
To avoid a crowded figure, only the HPLC chromatograms corresponding to
the
-ADP concentrations of 0.4, 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 4,
and 6 mM, in a consecutive order, have been represented. A, HPLC chromatograms of the extragranular
-nucleotides
after a 10-min incubation period, as a function of
-ADP
concentration, represented in consecutive chromatograms. B,
HPLC chromatograms of the intragranular levels of
-nucleotides as
a function of
-ADP concentration and represented in a consecutive
way. C, percentage distribution of the intragranular
-nucleotides as a function of the
-ADP concentration in the
incubation media. These values represent a single but very reproducible
experiment.
The intragranular content of -nucleotides, when
-ADP was the substrate to be transported, is shown in Fig. 4B. In this case, the
-ADP was present, but
-ATP was the major compound, together with very small amounts of
-AMP. The nucleotide percent distributions were around 7%,
30-40%, and 55-65%, for
-AMP,
-ADP, and
-ATP, respectively, at every concentration studied and for 10-min
transport experiments. The percent distribution is shown in Fig. 4C. At longer experimental times, e.g. 30
min, no significant changes were observed in the intragranular percent
distribution among the three nucleotides. To control the
-ADP
stability, an external standard of this nucleotide was added to the
chromaffin granules pellet and processed as indicated under
``Experimental Procedures.'' No production of
-ATP was
observed to any extent, and only a minor
-AMP production, which
was under 1%.
To approach the kinetic parameters for -ADP
transport, the total granular
-nucleotide content was considered
to calculate the transport velocity at each
-ADP concentration
used, and the data obtained are represented in Fig. 5A.
A nonhyperbolic curve was obtained, very similar to that reported for
-ATP, and the data were analyzed in the same way.
Figure 5:
Saturation studies of -ADP transport
to chromaffin granules. A, the saturation curve was obtained
by processing the HPLC data from Fig. 4B. The
-ADP
concentration ranged from 50 µM to 6 mM.
Transport velocity is expressed as the total of the granular
nucleotides per min per mg of chromaffin granule proteins. B,
three sigmoidal curves the addition of which account for the
experimentally observed curve (A). The affinity values,
together with the corresponding V
and n
values, are summarized in Table 1. C, plot of n
against the
-ATP
saturation calculated for curve A, according to the Kurganov
method(1982), under ``Experimental Procedures.''
These results represent a typical experiment performed in triplicate,
which is very reproducible.
The kinetic
parameters were obtained considering three superimposed nonhyperbolic
curves (Fig. 5B) and are summarized in Table 1. Fig. 5C shows the n variability as
a function of saturation (V/V
).
Figure 6:
Saturation studies of -AMP transport
to chromaffin granules. The transport experiments were accomplished
with 0.38 mg of protein of purified chromaffin granules and incubated
with
-AMP concentrations ranging from 100 µM to 6
mM, for 10 min at 25 °C as described under
``Experimental Procedures.'' To avoid a crowded figure, only
the HPLC chromatograms corresponding to the
-AMP concentrations of
0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, and 5 mM, in a
consecutive order have been represented. The extragranular
-AMP is
not shown, because no changes have been observed. A, HPLC
chromatograms of the intragranular content of
-AMP. B,
saturation curve of
-AMP transport as a function of
-AMP
concentration in the incubation media. V means the nanomoles
of
-AMP transported per min per mg of chromaffin granule proteins.
These results represent a typical and very reproducible experiment
performed in triplicate.
The variation of n values with
respect to saturation (V/V
) is similar
to that obtained for ATP (results not shown).
Figure 7:
Effect of extragranular nucleotides on the
induced release of stored -adenine nucleotides. Samples of
purified chromaffin granules containing 0.38 mg of protein were
incubated in the presence of 4 mM
-ATP for a 10-min
period. The charged granules were isolated as described under
``Experimental Procedures.'' Samples without further
processing were employed as controls to measure the total content of
-nucleotides and their distribution (B). The other
samples were subsequently resuspended in assay buffer with or without
addition of adenine nucleotides, incubated for a 10-min period at 25
°C, and afterwards centrifuged at 100,000
g,
resulting in a supernatant and a granular pellet. A,
quantification and distribution of
-nucleotides in the above
described supernatant from incubations in assay buffer and with the
addition of 4 mM each of ATP, ADP, and AMP. B,
quantification and distribution of
-nucleotides in the granular
pellet after incubation and centrifugation under the same conditions as
above. Values are the means ± S.D. of three experiments in
duplicate.
The pattern of released nucleotides changes with
respect to the granular one, and -AMP and
-ADP appeared even
in higher concentrations than reported in the granules (Fig. 7A). One plausible explanation is the action of
membrane ATPases and other nucleotidases on the
-ATP and
-ADP
when they are not protected inside the granules. In the presence of a
large amount of ATP, this nucleotide, acting as a competitor, protected
the released
-ATP, now appearing in large quantities (Fig. 7A).
The significant extragranular presence of
-nucleotides in buffer-resuspended granules (30% of the total
content) (Fig. 7A) could be due, almost in part, to the
breakdown of granules that occurred during their resuspension and later
centrifugation. This hypothesis was supported by the activity values of
the granular enzyme dopamine
-monooxygenase (EC 1.14.17.1). The
activity of its soluble form was 19-24% (n = 3)
of the total content in the extragranular media, independent of the
nucleotide addition (results not shown).
Figure 8:
Inhibition of -nucleotides transport
by several effectors.
-
,
-ATP transport;
-
,
-ADP transport;
-
,
-AMP transport. A, effect
of AMPPNP on
-nucleotide transport. Both
-ATP and
-AMP
are at 0.5 mM concentrations.
-ADP is at a 1 mM concentration, to observe the different behavior in inhibition. B, effect of ATP
S.
-nucleotide concentrations are as
in A. The open square symbol (
) represents
-ADP transport but submitted to the effects of variable ADP
S
concentration instead of ATP
S. C, effect of N-ethylmaleimide.
-nucleotide concentrations are as in A. D, effect of atractyloside.
-Nucleotide
concentrations are as in A. Values are the means ± S.D.
of a typical experiment performed in triplicate. 100%
-ATP
transport (0.5 mM) corresponded to 0.026 ± 0.003
nmol
min
mg of protein
.
100%
-ADP transport (1 mM) corresponded to 0.038 ±
0.004 nmol
min
mg of
protein
. 100%
-AMP transport (0.5 mM)
corresponded to 0.013 ± 0.001
nmol
min
mg of
protein
.
The nucleotide transport was
inhibited, although not completely, by atractyloside, as shown in Fig. 8D. The IC values were, respectively,
0.10 ± 0.01 mM for
-ATP, 0.033 ± 0.002
mM for
-ADP, and 0.17 ± 0.02 mM for
-AMP (n = 3).
N-Ethylmaleimide
inhibits the transport of -nucleotides at maximal levels of 41,
49, and 60% for
-ATP,
-ADP, and
-AMP, respectively (Fig. 8C). The IC
values were,
respectively, 0.094 ± 0.008, 0.80 ± 0.07, and 0.20
± 0.01 mM (n = 3).
Granule treatment
with ionophores such as the charge carrier valinomycin (10
µM), or the K/proton exchanger nigericin
(20 µM), in the presence of K
, resulted
in no inhibition of the
-ATP transport. The proton translocator
FCCP (0.25 mM) inhibited the transport of
-ATP,
-ADP, and
-AMP to an extent of 40 ± 6%, 45 ±
7%, and 53 ± 10% (n = 3), respectively.
Transport studies using the -adenine nucleotides to
characterize the vesicular nucleotide transport present the advantage
of their direct fluorescent detection, which combined with HPLC
techniques makes it possible to obtain a large set of information
necessary to understand the secretory vesicle replenishment and
circumvents the drawbacks of the traditional radioactive techniques.
The results presented here show that the nucleotide vesicular
transport, when studied in a very broad range of substrate
concentration, exhibits a saturable nonhyperbolic kinetic, for all
-adenine nucleotides, in bovine chromaffin granules. Previous
studies of nucleotide vesicular transport to chromaffin granules and
torpedo synaptic vesicles reported hyperbolic saturable kinetics for
both experimental models (Aberer et al., 1978; Luqmani, 1981;
Weber and Winkler, 1981). The K
values described
ranged between 0.9 and 1.4 mM for ATP, 1.2 and 1.4 mM for ADP, 0.7 and 2 mM for UTP, 0.3 and 1.2 mM for GTP, and, finally, 2.9 and 3.3 mM for AMP. This range
of values is nevertheless comprised in the affinities reported here for
the three-step saturation curves, for
-ATP,
-ADP, and
-AMP (Table 1).
The V values
reported for the ATP transport to chromaffin granules are about 0.4
nmol
min
mg of protein
,
that are in the same order as that reported here for
-ATP.
Concerning the V
for the ADP, the bibliographic
data report lower values than for ATP (Aberer et al., 1978;
Winkler and Carmichael, 1982); in our conditions, the
-ADP
transport exhibits a higher V
than that of
-ATP. This result can be explained on the basis of the
intragranular modifications of the
-ADP that experimentally can be
quasi-assimilated to a Zero-trans transport conditions. In fact, when
-ADP was the extragranular nucleotide to be transported, the
intragranular
-ATP accounted for more than 55% of the total
-nucleotides. A transphosphorylation or interchange reaction of
-ADP with the adenine nucleotides already present in the granule
can be suggested. This type of reaction has previously been described
for labeled nucleotides (Aberer et al., 1978; Roisin and
Henry, 1982; Winkler and Carmichael, 1982; Taugner et al.,
1988). The enzyme responsible for this reaction still needs to be
identified, and its physiological relevance needs to be analyzed, but
from the transport studies with
-AMP, it is certain that the
enzyme cannot use
-AMP, or to a very limited extent, as a
substrate for the phosphate interchange reaction.
The
-nucleotide transport was inhibited by the same compounds
described for ATP, such as atractyloside, N-ethylmaleimide (a
good inhibitor of V-ATPases), and the proton translocator FCCP, at
similar concentrations (Aberer et al., 1978; Luqmani, 1981;
Weber and Winkler, 1981). None of these compounds was able to inhibit
the nucleotide transport completely as previously reported both in
bovine and torpedo vesicles (Luqmani, 1981).
The efflux or ``exchange'' of granular stored compounds is still an unresolved question. In the case of monoamines, both processes are independent of the transport inhibitor reserpine and ``exchange'' has been observed only when an excess of substrate was added (Schuldiner et al., 1995). In the case of granular nucleotides, their fluorescent labeling allowed a first experimental approach to the problem and, as in the previous case, a high extragranular ATP or ADP concentration is necessary, with the peculiarity that AMP was ineffective. The physiological relevance of these data still needs to be established.
Concerning the transport
saturation studies, the three-step curve that was obtained for each of
the -nucleotides studied needs to be analyzed and interpreted in
several ways. First, the chromaffin granule model itself needs some
discussion. It is well known that adrenergic and noradrenergic cells
exist in adrenal medulla, storing the specific catecholamines in their
granules (Moro et al., 1991). The noradrenergic granules
appear to be more dense with a higher proportion of nucleotides than
the adrenergic granules (Terland et al., 1979). Nothing is yet
known about the different transport properties of both granules.
Nevertheless, the superposition of two different transporters with
their respective affinities is not compatible with the positive kinetic
cooperativity exhibited by the saturation curve. Recently, the presence
of different vesicular monoamine transporters has been reported in the
large secretory chromaffin granules (VMAT1) and in the small synaptic
vesicles (VMAT2), both present in bovine adrenal glands (Henry et
al., 1994). Thus, the existence of different vesicular nucleotide
transporters, related to the secretory organella size in the same cell,
cannot be ruled out. As in the previous hypothetical situation, the
existence of two different transporters cannot explain the positive
cooperativity in the transport saturation studies.
The second aspect
under analysis and discussion is the cooperative phenomenon. -ATP,
-ADP, and
-AMP exhibit similar shapes in their saturation
curves. In allosteric enzymes, the occurrence of various intermediate
plateaus on V versus [S]
plots
has been explained by the presence in the system of forms of the enzyme
which exhibit differing degrees of kinetic cooperativity toward the
substrate, due to the comparatively slow isomerization induced by the
substrate. The enzymes with such a kinetic behavior are known as
hysteretic or mnemonical (Neet and Ainslie, 1976; Banks et
al., 1979; Kurganov, 1982; Nari et al., 1984; Ricard and
Cornish-Bowden, 1987; Valero and Garcia-Carmona, 1992). Enzymes with
similar kinetic complexities and saturation curves exhibiting various
intermediate plateaus have already been reported, as in the case of L-threonine dehydratase from Escherichia coli,
lactate dehydrogenase from rainbow trout muscle, and pyruvate kinase
from rabbit liver (Somero and Hochachka, 1969; Irving and Williams,
1973a, 1973b; Kagan and Dorozhko, 1973). Due to the difficulty of the
experimental approaches to study the membrane transport of metabolites,
the existence of mnemonic kinetic behaviors has been reported only for
the facilitated diffusion of adenosine in neural preparations (Casillas et al., 1993). The Hill number close to 5 in that case could
be explained only by the existence of a multimeric form, minimum
dimeric, and slow transitions between conformational states of the
protein. The existence of multimeric forms for transporters has been
demonstrated, being dimeric or tetrameric in the case of the glucose
and adenosine facilitated diffusion (Hebert and Carruthers, 1991; Kwong et al., 1992). The results reported here for the nucleotide
vesicular transport can also be explained on the basis of a dimeric
protein and slow transitions between the forms, and they adapt well to
a transporter model as has already been described for neural adenosine
transporter (Casillas et al., 1993). As there is no
information about the protein structure of the nucleotide transporter
that has not already been cloned successfully, it is difficult to make
an exact and accurate transport model. The analysis of the saturation
curve required a new approach to the n
concept and
obtention (Kurganov, 1982). The idea of three superimposed curves with
cooperativity was necessary to analyze and obtain the kinetic
parameters (Silonova et al., 1969; Kurganov, 1982).
The
transport activation by ATP-nonhydrolyzable analogues at low
concentrations, together with their poor inhibitory effect at the
highest concentrations (Aberer et al., 1978), also confirm the
hypothesis of conformational cooperative transitions. Similar behavior
has already been reported for some inhibitors of L-threonine
dehydratase at low substrate concentrations (Kagan and Dorozhko, 1973).
Based on the previous discussion, the complexity of the saturation
kinetics for -nucleotides can be interpreted by postulating the
existence of a mnemonic mechanism. Nevertheless, the heterogeneity of
the chromaffin granule model, on account of their aminergic content,
size, and the breakdown and subsequent vesicle ghost formation, could
be at the root of complex additive kinetic behaviors, except for the
cooperativity phenomena that cannot be explained in this way.
The third aspect to be considered concerns the physiological relevance and the advantages of such kinetic behavior. In our opinion and in agreement with the results reported here, the mnemonic behavior is essential to establish the priority order on the remaining cellular ATP after physiological situations demanding large amounts of energy, as neurosecretion or in anomalous situations as anoxic episodes. The rational is as follows.
(a) It is well known that glucose
is the main energetic substrate in neurosecretory cells, and the
remaining ATP in the situations previously described is necessary for
the phosphorylation of glucose; the K value for
the neural type I hexokinase for ATP is close to 0.1 mM. This
isoenzyme accounts for 90% of the total hexokinase activity in the
adrenomedullary tissue; the remaining 10% corresponds to hexokinase II
(Millaruelo et al., 1986). Thus, the priming of glycolysis as
the main neural catabolic energetic pathway is assured because the K (S
) for the first intermediate
plateau of transport is about 0.25 mM for ATP (it is assumed
that
-ATP and the natural substrate ATP have a similar kinetic
behavior). Only when the glycolysis goes on and the ATP level recovers,
does the nucleotide vesicular transporter increase its capacity in a
highly cooperative way. As the affinities for all nucleotides are very
similar, this kinetic behavior presents the advantage of preventing the
massive entrance to the granule of lower phosphorylated metabolites.
(b) The presence for the last plateau with K (S) values for the ATP transport (about 3
mM) is in our opinion related to the phosphofructokinase
reaction. This enzyme needs ATP as a substrate with a K
value in the range of 10 to 20 µM, but ATP also
regulates the glycolytic flux by its inhibitory action on this enzyme.
The K
values for ATP reported for the brain
isoenzyme are in the 2-3 mM range (Vora et al.,
1985). Thus, when the high ATP levels are on the edge of inhibition of
the glycolytic flux, they are in the best situation to be transported.
In the absence of a full theoretical development of the kinetic mechanism operating in membrane transport, the possibility of a mnemonic behavior opens new perspectives in this field. Moreover, the mnemonic behavior in the specific case of the nucleotide transporter allows a security threshold in the cellular energetic metabolism and sheds some light on the harmony existing in cellular functioning.