(Received for publication, April 3, 1995; and in revised form, August 30, 1995)
From the
The role of internal and external reductants in the dopamine
-monooxygenase (D
M)-catalyzed conversion of dopamine to
norepinephrine has been investigated in resealed chromaffin granule
ghosts. The rate of norepinephrine production was not affected by the
exclusion of internal ascorbate. The omission of ascorbate from the
external medium drastically reduced the norepinephrine production
without affecting the net rate of dopamine uptake. In the presence of
the external reductant, the internal ascorbate levels were constant
throughout the incubation period. The rate of norepinephrine production
was not affected when ghosts were resealed to contain the D
M
reduction site inhibitor, imino-D-glucoascorbate. Ghosts
incubated with external imino-D-glucoascorbate reduced the
norepinephrine production. The weak D
M reductant,
6-amino-L-ascorbic acid, was found to be a good external
reductant for granule ghosts. The outcome of the above experiments was
not altered when dopamine was replaced with the reductively inactive
D
M substrate, tyramine. These results and the known topology of
membrane-bound D
M disfavor the direct reduction of the enzyme by
the external reductant. Our observations are consistent with the
hypothesis that external ascorbate is the sole source of reducing
equivalents for D
M monooxygenation and that internal soluble
ascorbate (or dopamine) may not directly reduce or mediate the
reduction of membrane-bound D
M in resealed granule ghosts.
Dopamine -monooxygenase (D
M;
EC 1.14.17.1)
catalyzes the conversion of dopamine (DA) to the neurotransmitter,
norepinephrine (NE), within the neurosecretory vesicles of the adrenal
medullae and the large dense-cored synaptic vesicles of the sympathetic
nervous system. D
M exists in both soluble and membrane-bound forms
in these tissues(1, 2, 3) . The reducing
equivalents required for in vivo D
M monooxygenation is
believed to be provided by ascorbic acid (AscH
) which is
present in high concentrations (20 mM) within the
D
M-containing neurosecretory vesicles(4, 5) .
Despite the fact that AscH
can function as an overall
two-electron reductant, it has been shown to function as a
single-electron reductant producing semidehydroascorbic acid as the
immediate product during the D
M
turnover(6, 7, 8) . Semidehydroascorbic acid
is a relatively nonreactive radical which spontaneously
disproportionates to produce AscH
and the oxidized form,
dehydroascorbic acid, under normal conditions. Although dehydroascorbic
acid is reduced back to AscH
by a glutathione-dependent
enzyme system in plants(9, 10) , no mechanism is
available to regenerate AscH
from dehydroascorbic acid in
neuroendocrine cells.
Numerous detailed studies have demonstrated
that neuroendocrine secretory vesicles of the adrenal medullae or other
endocrine glands do not transfer AscH across the vesicle
membrane at a detectable
rate(11, 12, 13, 14) . Based on
these observations and the fact that the concentration of AscH
in the catecholamine secretory vesicles is approximately 20
mM and the catecholamine concentration is 500
mM(4, 5) , a transmembrane electron carrier
which shuttles electrons from the cytosolic pool of AscH
(2
mM(15) ) to the intercellular matrix to regenerate
AscH
from semidehydroascorbic acid has been proposed. The
chromaffin granule-transmembrane hemoprotein, cytochrome b
(cyt b
) has been
identified as the most likely candidate to serve as an electron
carrier, since it has a favorable midpoint potential of +140
mV(16, 17, 18) . According to this model,
semidehydroascorbic acid generated by the D
M monooxygenation
reaction within the granule matrix is efficiently reduced to AscH
by the reduced cyt b
, preventing the
radical disproportionation and thereby maintaining a constant pool of
AscH
within the granule
matrix(19, 20, 21, 22) . Oxidized
cyt b
is subsequently reduced by cytoplasmic
AscH
, and depletion of cytoplasmic AscH
is
prevented by a mitochondrial NADH:semidehydroascorbic acid
oxidoreductase(19, 20, 21, 22) .
The dynamics and various mechanistic aspects of NE biosynthesis has
been mostly studied by using resealed chromaffin granule ghosts as a
model system for adrenergic neurotransmitter storage
vesicles(19, 20, 21, 22, 23, 24, 25, 26, 27) .
Several granule membrane proteins, including membrane-bound DM,
ATPase, cyt b
, and monoamine transporter, have
been well characterized, especially in relation to catecholamine
neurotransmitter biosynthesis. The transmembrane proton-translocating
ATPase has been shown to be responsible for maintaining a low
intravesicular pH of 5.5 relative to the neutral cytosolic
pH(28, 29) , which is proposed to be mandatory for the
proper functioning of chromaffin granule ghosts. In accordance with the
chemiosmotic hypothesis(30) , the vectorial transport of
protons across the chromaffin granule membrane by proton-translocating
ATPase creates an electrochemical (
) and a pH gradient
(
pH) across the granule membrane. It has been hypothesized that
the electrochemical and proton gradients across the granule membrane
are important for the cyt b
-mediated electron
transport process, since the pH gradient will shift the reduction
potential of the AscH
/semidehydroascorbic acid couple from
+70 mV (outside (pH 7.0)) to +220 mV (inside (pH 5.5)),
facilitating the electron flux from cytosol to the interior of the
granule ghosts through cyt b
(19, 20, 21, 22, 31, 32) .
However, in contrast to the above generally accepted mechanism for
the reduction of DM in catecholamine secretory vesicles, several
recent reports appear to indicate that the external AscH
is
the kinetically preferred reductant for the membrane-bound D
M in
resealed chromaffin granule ghosts(14, 33) . In the
present study, we have examined the role of internal and external
electron donors in the D
M-catalyzed conversion of DA to NE and the
net rate of DA uptake in resealed chromaffin granule ghosts using three
new AscH
analogs, 6-amino-L-ascorbic acid
(6-AAscH
), 2-amino-L-ascorbic acid
(2-AAscH
), and imino-D-glucoascorbic acid (IGA) as
probes (Fig. S1). The results presented in this report suggest
that the external reductant, AscH
, is the sole source of
the reducing equivalents for the membrane-bound D
M-catalyzed
monooxygenation in resealed chromaffin granule ghosts. More
importantly, our results also suggest that internal soluble AscH
(or DA) may not directly reduce or mediate the reduction of
membrane-bound D
M in chromaffin granule ghosts. The implications
of these findings on the mechanism of the reduction of membrane-bound
and soluble D
M in relation to in vivo NE biosynthesis is
discussed.
Figure S1: Scheme 1.
Membrane-bound DM from the
above preparation was used in the estimation of the inhibition potency
of IGA and the substrate activity of 6-AAscH
using the
oxygen monitor assay as described previously(35) .
The resealed chromaffin granule ghosts prepared to contain 20
mM AscH, 20 mM Tris phosphate, 100
mM KCl, 150 mM sucrose, 10 mM sodium
fumarate, and 100 µg/ml catalase according to the above procedures
(standard resealed granule ghosts), actively accumulate DA and convert
it to NE in a time-dependent manner in the presence of 5 mM ATP, 5 mM MgSO
, and 20 mM AscH
in the external incubation medium (standard uptake and conversion
incubation conditions). Appropriate control experiments with the
standard resealed granule ghosts revealed that the exclusion of ATP or
inclusion of 2 µM reserpine in the external incubation
medium completely abolishes the DA accumulation and NE production under
the above conditions as expected (data not shown).
A series of
control experiments were carried out with standard resealed granule
ghosts in order to quantify the experimental variability of the rate of
DA uptake and its conversion to NE and to examine the viability of the
standard ghost preparations during the time course of the experiment
under the standard uptake and conversion incubation conditions. Under
these conditions, the average internal E levels of the standard
resealed granule ghosts varies from 23.0 to 15.7 nmol/mg within a
25-min incubation period (Fig. 1a) probably due to the
slow lysis or the leakage of the internal contents of the resealed
granule ghosts (see ``Discussion''). The data standardized
against average constant indigenous E levels show that the average
internal AscH (reduced) levels were 23.5 nmol/mg
(8-12 mM depending on 2- or 3-µl internal volume of
granule ghosts) under the above experimental conditions (Fig. 1b) demonstrating that the trapping efficiencies
are in the range of 50% (resealing solution contained 20 mM AscH
). Since only the levels of reduced AscH
were measured by electrochemical detection methods, and reduced
AscH
is susceptible to slow auto-oxidation during the
experiment, the trapping efficiencies of these experiments may be
higher than these values. The maximum experimental variability of the
standardized rate of NE production was less than 30% (an average of
five different experiments; see Fig. 2a, vertical
bars) for these preparations suggesting a good reproducibility of
the results regardless of the complexity of the system. However, this
experimental variability has been taken into account in the analysis of
all the rates of DA accumulation and NE production under various
experimental conditions and was assumed to be similar to the standard
resealed granule ghosts, if the rates were in the above range. The
above experiments also revealed that the rate of NE production is in
the range of 1.7-2.4 nmol/mg/min (average rate 2.0 nmol/mg/min; Fig. 1b), and the rate of DA accumulation varies in the
range of 0.2-0.7 nmol/mg/min (average rate 0.5 nmol/mg/min; Fig. 1b) for different standard resealed granule ghost
preparations under the standard uptake and conversion incubation
conditions. In addition, the time courses of both NE production and DA
accumulation are relatively linear (Fig. 2, a and b; vertical bars), and the internal AscH
levels were found to be relatively constant throughout the
incubation period in all these experiments (Fig. 1, a and b).
Figure 1:
The time courses of
NE production and DA uptake in standard resealed chromaffin granule
ghosts under standard uptake and conversion conditions. a,
ghosts were resealed to contain 20 mM Tris phosphate, 100
mM KCl, 150 mM sucrose, 10 mM sodium
fumarate, 100 µg/ml catalase, and 20 mM AscH (pH 7.0) and incubated in a medium (2.5 ml total volume)
containing 0.3 M sucrose, 10 mM HEPES, 5 mM Mg-ATP, 5 mM MgSO
, 20 mM
AscH
, and 100 µg/ml catalase (pH 7.0) at 30 °C for
10 min. At zero time, the uptake and conversion reaction was initiated
by adding DA to a final concentration of 200 µM, and, at
5-min time intervals, 400-µl aliquots of the incubate were
withdrawn, and HPLC-EC quantitation was performed as noted under
``Experimental Procedures.'' The data presented are the
averages of five different experiments.
, NE levels;
, E
levels;
, AscH
levels;
, DA levels. b, same as above, but the catecholamine and AscH
levels are standardized with respect to the average indigenous E
levels.
Figure 2:
The
effect of internal and/or external AscH on the time courses
of NE production (a) and DA accumulation (b) in
resealed chromaffin granule ghosts. Ghosts were resealed to contain 20
mM Tris phosphate, 100 mM KCl, 150 mM sucrose, 10 mM fumarate, and 100 µg/ml catalase with
AscH
, without AscH
, or with ascorbate oxidase
(pH 7.0) and incubated in a medium (2.5 ml total volume) containing 0.3 M sucrose, 10 mM HEPES, 5 mM Mg-ATP, 5
mM MgSO
, and 100 µg/ml catalase with
AscH
, without AscH
, or with ascorbate oxidase
(pH 7.0) at 30 °C for 10 min. At zero time, the uptake and
conversion reaction was initiated by adding DA to a final concentration
of 200 µM, and, at 5-min time intervals, 400-µl
aliquots of the incubate were withdrawn, and HPLC-EC quantitation was
performed as noted under ``Experimental Procedures.''
,
, no AscH
inside, 20 mM AscH
outside;
, 25 units of ascorbate oxidase inside, 20
mM AscH
outside;
,
, and
, 20
mM AscH
inside, 25 units of ascorbate oxidase
outside (multiple experiments under the same conditions were carried
out in some cases to examine the reproducibility). Note: the
experimental variability of the rate of NE production and DA
accumulation under standard uptake and conversion incubation conditions
(20 mM AscH
outside) with standard resealed ghosts
(20 mM AscH
inside) for 5 separate experiments are
shown as vertical bars.
The rates of NE production or DA uptake under
standard uptake and conversion incubation conditions were not affected
when the granule ghosts were resealed to contain no added
AscH and a high concentration of active ascorbate oxidase (Fig. 2, a and b). The rate of NE production
in this preparation was in the normal range in comparison with that of
the control standard resealed granule ghosts suggesting a high rate of
DA to NE conversion in the absence of internal AscH
. On the
other hand, when standard resealed granule ghosts were incubated in a
medium in the absence of external AscH
, but in the presence
of a high concentration of external ascorbate oxidase, the rate of DA
accumulation was increased by about 3-fold, and NE production was
decreased by about 3-fold in comparison with the standard resealed
granule ghosts (Fig. 2, a and b). Furthermore,
when AscH
was excluded from the interior and the external
incubation medium (by the addition of ascorbate oxidase into both
media), the rate of NE production was again reduced by about 5-fold
without significantly altering the net rate of DA uptake (data not
shown).
The effect of external and/or internal IGA, which is a
potent inhibitor for the AscH site of both soluble (K
= 26.4 ± 5.2 µM(36))
and membrane-bound D
M, (
)on the rate of DA accumulation
and NE production in resealed chromaffin granule ghosts was examined in
a series of experiments. When granule ghosts were resealed to contain
10 mM IGA together with 20 mM AscH
and
incubated under standard uptake and conversion conditions, the rates of
either the DA accumulation or NE production were not significantly
altered compared to identical controls without IGA (data not shown). In
fact, the rate of NE production under these conditions was in the high
normal range in comparison to the control standard resealed granule
ghosts. In addition, the rates of both DA accumulation and NE
production in ghosts resealed to contain either 1 or 10 mM IGA
in the presence (5 mM or 20 mM) or absence of added
AscH
were also in the normal range under standard uptake
and turnover conditions (Fig. 3, a and b). On
the other hand, the rate of NE production was significantly decreased
when standard resealed granule ghosts were incubated under uptake and
conversion conditions in the presence of 1 mM IGA and 5 mM AscH
with respect to a parallel identical control
without IGA. The rate of DA accumulation in the IGA-containing
incubates was significantly higher than the rate of the controls
suggesting a slow rate of DA to NE conversion without changing the net
rate of DA uptake as expected (Fig. 3b). Furthermore,
when ghosts were prepared to contain either AscH
, IGA, or
ascorbate oxidase and incubated in the presence of external IGA (note
that IGA is not a substrate or inhibitor for ascorbate
oxidase(42) ) and ascorbate oxidase, the rate of NE production
dropped to the background level while the rate of DA accumulation
increased to a very high level confirming that external IGA does not
support the D
M monooxygenation reaction in resealed granule
ghosts. Control experiments revealed that IGA is not actively or
passively accumulated into granule ghosts under the experimental
conditions (data not shown).
Figure 3:
The effect of internal and/or external
imino-D-glucoascorbic acid (IGA) on the time courses of NE
production (a) and DA accumulation (b) in resealed
chromaffin granule ghosts. Ghosts were resealed to contain 20 mM Tris phosphate, 100 mM KCl, 150 mM sucrose, 10
mM fumarate, and 100 µg/ml catalase with AscH,
IGA, ascorbate oxidase, or a combination of the above (pH 7.0) and
incubated in a medium (2.5 ml total volume) containing 0.3 M sucrose, 10 mM HEPES, 5 mM Mg-ATP, 5 mM MgSO
, and 100 µg/ml catalase with
AscH
, IGA, ascorbate oxidase, or a combination of the above
(pH 7.0) at 30 °C for 10 min. At zero time, the uptake and
conversion reaction was initiated by adding DA to a final concentration
of 200 µM, and, at 5-min time intervals, 400-µl
aliquots of the incubate were withdrawn, and HPLC-EC quantitation was
performed as noted under ``Experimental Procedures.''
,
5 mM AsCH
inside, 20 mM AscH
outside;
, 20 mM AscH
inside, 5
mM AscH
outside;
, 5 mM AscH
and 1 mM IGA inside, 20 mM AscH
outside;
, 20 mM AscH
inside, 5
mM AscH
and 1 mM IGA outside;
, 10
mM IGA and 25 units of ascorbate oxidase inside, 20 mM AscH
outside;
, 20 mM AscH
inside, 10 mM IGA and 25 units of ascorbate oxidase
outside;
, 25 units of ascorbate oxidase inside, 10 mM
IGA outside; X, 10 mM IGA and 25 units of ascorbate oxidase
inside, 10 mM IGA outside. Note: vertical bars are
the same as in Fig. 2.
The specificity of the extragranular
reduction site of the chromaffin granule membrane was examined by using
the AscH analog, 6-AAscH
, which is a very weak
reductant for purified soluble (36) as well as membrane-bound
D
M (less than 10% activity at 20 mM concentration in
comparison to AscH
). The data presented in Fig. 4a clearly demonstrate that in contrast to its
inability to reduce soluble or membrane-bound purified D
M
efficiently, under standard experimental conditions, external
6-AAscH
supports the NE production in standard
resealed granule ghost with the efficiency similar to that of
AscH
itself. The data also show that external AscH
supports NE production in 6-AAscH
-loaded ghosts with
the efficiency similar to that of AscH
-loaded ghosts.
Furthermore, the rate of DA accumulation is in the normal range for
both of these sets of experiments suggesting that external or internal
6-AAscH
has no significant effect on the net rate of DA
uptake (Fig. 4, a and b). Although
6-AAscH
has a remarkable structural similarity to DA,
control experiments revealed that it is not accumulated into granule
ghosts through the DA uptake mechanism in an ATP- and time-dependent
manner, under standard uptake and conversion incubation conditions.
Other control experiments indicated that 6-AAscH
had no
significant effect on the viability of resealed ghosts under the
experimental conditions.
Figure 4:
The effect of internal or external
6-aminoascorbic acid (6-AAscH) on the time courses of NE
production (a) and DA accumulation (b) in resealed
chromaffin granule ghosts. Ghosts were resealed to contain 20 mM Tris phosphate, 100 mM KCl, 150 mM sucrose, 10
mM fumarate, and 100 µg/ml catalase with AscH
and 6-AAscH
(pH 7.0) and incubated in a medium (2.5
ml total volume) containing 0.3 M sucrose, 10 mM HEPES, 5 mM Mg-ATP, 5 mM MgSO
, and
100 µg/ml catalase (pH 7.0) at 30 °C for 10 min. At zero time,
the uptake and conversion reaction was initiated by adding DA to a
final concentration of 200 µM, and, at 5-min time
intervals, 400-µl aliquots of the incubate were withdrawn, and
HPLC-EC quantitation was performed as noted under ``Experimental
Procedures.''
, 20 mM AscH
inside, 20
mM 6-AAscH
outside;
, 20 mM 6-AAscH
inside, 20 mM AscH
outside;
, no added AscH
inside, 20 mM
AscH
outside. Note: vertical bars are the same as
in Fig. 2.
The data presented in Fig. 5demonstrate that the chromophoric DM reductant,
2-AAscH
(35) , also supports the conversion of DA to
NE in standard resealed granule ghosts with an efficiency similar to
that of AscH
(control experiments indicate that
2-AAscH
does not permeate the granule membrane at a
detectable rate although it is somewhat less polar than AscH
due to the alteration of the pK
of the 3`-OH
group). Although quantitative studies have not been completed yet, we
have observed the formation of the chromophoric oxidized product of
2-AAscH
, the red pigment, in a time-dependent manner in
these experiments when 2-AAscH
is the external electron
donor (we believe this reaction could be adopted to measure the rate of
electron flux into granule ghosts; detailed experiments are in
progress). On the other hand, the chromophoric non-ascorbate D
M
reductant, N,N-dimethyl-1,4-phenylenediamine(35) , does
not appear to be an external reductant for membrane-bound D
M in
chromaffin granule ghosts (data not shown).
Figure 5:
The effect of external 2-aminoascorbic
acid (2-AAsCH) on the time courses of NE production and DA
accumulation in resealed chromaffin granule ghosts. Ghosts were
resealed to contain 20 mM Tris phosphate, 100 mM KCl,
150 mM sucrose, 10 mM fumarate, 100 µg/ml
catalase, and 20 mM AscH
(pH 7.0) and incubated in
a medium (2.5 ml total volume) containing 0.3 M sucrose, 10
mM HEPES, 5 mM Mg-ATP, 5 mM
MgSO
, 100 µg/ml catalase, and either 20 mM AscH
or 20 mM 2-AAscH
(pH 7.0) at
30 °C for 10 min. At zero time, the uptake and conversion reaction
was initiated by adding DA to a final concentration of 200
µM, and, at 5-min time intervals, 400-µl aliquots of
the incubate were withdrawn, and HPLC-EC quantitation was performed as
noted under ``Experimental Procedures.''
, NE levels;
, DA levels with 20 mM AscH
outside;
,
NE levels;
, DA levels with 20 mM 2-AAscH
outside.
The possibility that DA
may function as a redox mediator between the extragranular reduction
site and membrane-bound DM was investigated by using reductively
inactive tyramine as the D
M substrate. In these experiments, the
rates of intragranular octopamine formation were accurately quantified
by reversed phase HPLC-EC as described under ``Experimental
Procedures.'' The data presented in Fig. 6clearly
demonstrate that standard resealed granule ghosts uptake tyramine and
convert it to the D
M hydroxylation product, octopamine, in a
time-dependent manner with a rate somewhat slower than that of the DA
to NE conversion under standard uptake and turnover conditions (see Fig. 6, vertical bars; however, note that the external
tyramine concentrations in these experiments were 30 µM,
whereas DA concentrations in analogous experiments were 200
µM). The exclusion of internal AscH
by
resealing the granule ghosts to contain a high level of highly active
ascorbate oxidase (
)does not significantly change the rate
of octopamine production in comparison to the standard controls under
identical conditions (Fig. 6). The rate of octopamine production
was not inhibited when ghosts were resealed to contain 5 mM IGA and no added AscH
and incubated under standard
uptake and turnover conditions (Fig. 6). In contrast, a slight
increase in the rate of octopamine production was observed compared to
the standard controls when internal IGA was present, similar to that
observed for the DA to NE conversion. In addition, external 20
mM 6-AAscH
supports the tyramine to octopamine
conversion in 20 mM AscH
-loaded granule ghosts
with a rate slightly lower than that of the standard controls.
Figure 6:
The
time courses of DM-catalyzed octopamine production under various
conditions when tyramine was used as the substrate. Ghosts were
resealed to contain 20 mM Tris phosphate, 100 mM KCl,
150 mM sucrose, 10 mM fumarate, 100 µg/ml
catalase with AscH
, without AscH
, and with
ascorbate oxidase or with IGA (pH 7.0) and incubated in a medium (2.5
ml total volume) containing 0.3 M sucrose, 10 mM HEPES, 5 mM Mg-ATP, 5 mM MgSO
, 100
µg/ml catalase either with AscH
, without AscH
and with ascorbate oxidase, or with 6-AAscH
(pH 7.0)
at 30 °C for 10 min. At zero time, the uptake and conversion
reaction was initiated by adding tyramine to a final concentration of
30 µM, and, at 4-min time intervals, 400-µl aliquots
of the incubate were withdrawn, and HPLC-EC quantitation was performed
as noted under ``Experimental Procedures.''
, 25 units
of ascorbate oxidase inside, 20 mM AscH
outside;
, 20 mM AscH
inside, 25 units of ascorbate
oxidase outside;
, 5 mM IGA and 25 units of ascorbate
oxidase inside, 20 mM AscH
outside;
, 20
mM AscH
inside, 20 mM 6-AAscH
outside. Note: the experimental variability of the rate of octopamine
production under standard uptake and conversion conditions (20 mM AscH
outside) with standard resealed ghosts (20
mM AscH
inside) for four separate experiments are
shown as vertical bars.
The optimum conditions and procedures to examine the DA
uptake and NE production in resealed chromaffin granule ghosts under
various experimental conditions with non-radiolabeled substrate have
been reported previously(26, 27) . The key steps in
this procedure were the resealing of the granule membranes under
optimum conditions necessary for maximum DM turnover, the
termination of the uptake of DA and its conversion to NE at a given
period of time by diluting the incubates with an ice-cold osmotically
balanced solution, the separation and reisolation of the intact granule
ghosts from the external incubation medium without contamination, and
the quantification of the internal contents of the granule ghost
incubates by HPLC-EC. The ghosts prepared, incubated, and analyzed
according to the above procedures were found to actively uptake DA and
convert it to NE in an ATP- and time-dependent and reserpine-sensitive
manner giving consistent and reproducible results. Therefore, we have
used the same protocols and procedures in the present study without
significant alterations or modifications.
The resealed granule
ghosts prepared according to the above procedures contained relatively
constant levels of internal E which were found to slowly decline during
the incubation time period (Fig. 1a). Since the
depletion of E was observed even when excess AscH was
present in both the internal matrix and in the external incubation
medium, this could not be due to the auto-oxidation of the catechol
moiety of the molecule and must be due to the slow lysis or the leakage
of the internal contents of the ghosts during the incubation period or
reisolation procedure. Therefore, any loss or leakage of internal
contents from resealed granule ghosts during the experiments could
accurately be corrected (Fig. 1b) by standardizing
against the average indigenous E levels since NE is not converted to E
within resealed granule ghosts due to the lack of the essential enzyme,
phenethylamine N-methyltransferase(5) . Furthermore,
external contamination or incomplete resealing of ghost preparations
prior to the incubation could also be estimated by measuring the DA
content at the time 0 point which is usually 0 under the above
experimental conditions (Fig. 1, a and b). The
normal rates of NE production and DA accumulation in standard resealed
granule ghosts ( Fig. 1and Fig. 2) were in good agreement
with the rates previously reported under similar experimental
conditions(26, 27, 43) . The observed low
ratio of the net rate of DA uptake to NE production (in the range of
1.1-1.3) together with the lack of lag or burst periods in both
of these time courses under the standard experimental conditions (Fig. 2, a and b), suggest that both the rate
of DA uptake and D
M turnover contribute to the net rate of NE
production in resealed granule ghosts which is also consistent with the
previous observations(44) .
In the experiments where removal
of internal AscH contaminants was necessary, ghosts were
resealed to contain high concentrations of active ascorbate oxidase.
The interior contents of the ghosts reisolated from these preparations,
after incubation under standard uptake and conversion conditions, were
found to consistently contain an average of 6-7 nmol/mg of
AscH
regardless of the presence of high levels of
intragranular ascorbate oxidase (similar observations have been
reported previously(14) ). However, these AscH
levels were not increased in a time- and/or ATP-dependent manner during
the incubation period suggesting that external AscH
was not
actively taken up into the ghosts (data not shown) and were much less
than the external AscH
concentration of the incubation
medium, ruling out the possibility that external AscH
was
passively transported and equilibrated during the incubation period.
Furthermore, since ascorbate oxidase is a very efficient enzyme (k
= 3,000-11,000
s
(46) ) with very high affinity toward
AscH
(K
is in the µM range(46) ), the observed levels of AscH
could
not possibly be in the interior soluble matrix of the resealed granule
ghosts and not be accessible to ascorbate oxidase. Therefore, a
possible simple explanation for the above observation is that
AscH
nonspecifically and tightly interacts with the
external phase of the resealed granule ghost membrane. Since the amino
acid sequence and tertiary structure model proposed for cyt b
contains clusters of positively charged amino
acid side chains in the cytoplasmic domain of the protein(47) ,
it is possible that negatively charged AscH
molecules may
specifically and strongly interact with these regions of the protein in
order to facilitate the subsequent reaction with the buried heme.
Alternatively, it is possible that a small pool of permanently inner or
outer membrane-associated externally reducible AscH
exists
in resealed chromaffin granule ghosts (for example, see (45) )
which may not be accessible to the intragranular ascorbate oxidase.
However, the present results do not distinguish between these
possibilities.
The internal AscH levels in standard
resealed granule ghosts remain relatively constant (average of 23.5
nmol/mg of protein of detectable reduced AscH
) throughout
the incubation period (25-30 min) under standard uptake and
conversion incubation conditions (Fig. 1, a and b) suggesting that there is no net consumption of internal
AscH
by the D
M monoxygenation reaction under these
conditions(44) . Since it is generally accepted (our results
also confirm) that AscH
is not actively or passively
transported into the interior of the granule ghosts at a detectable
rate(11, 12, 13, 14) , the reducing
equivalents essential for the D
M reaction must be provided from
the cytosolic pool of the reductant. Alternatively, although the
contaminating intragranular catecholamines present in the system could
provide necessary reducing equivalents for the D
M-catalyzed DA to
NE conversion in theory (for example, see (45) ), the rate of
depletion of the catecholamine concentration during the incubation
period was much slower (about 0.2 to 0.3 nmol/mg/min) and was not
sufficient (Fig. 1, a and b) to account for
the observed rate of NE production. On the other hand, the data
presented in Fig. 2a clearly demonstrate that external
AscH
is essential for the efficient conversion of DA to NE
in standard resealed granule ghosts. (
)However, the internal
or external AscH
has no significant effect on the net rate
of DA uptake into resealed granule ghosts (i.e. the combined
rates of DA accumulation and NE production; Fig. 2b)
under the same experimental conditions. Therefore, it can be concluded
that while external AscH
is essential for the efficient
conversion of DA to NE in resealed chromaffin granule ghosts under
standard uptake and conversion incubation conditions, internal
AscH
has no significant effect.
Internal IGA (an
efficient reductant site-competitive inhibitor of DM)
concentrations of up to 10 mM has no significant effect on
either the rate of NE production or the rate of DA accumulation under
standard uptake and conversion incubation conditions in the presence or
even in the absence of externally added AscH
in the
internal matrix of resealed granule ghosts (Fig. 3, a and b). In fact, close examination of the results
indicate that the rate of NE production in the presence of internal IGA
was slightly higher in comparison with identical controls without IGA.
Therefore, it is clear that although IGA is an excellent inhibitor for
both soluble and membrane-bound forms of D
M and effectively
interacts with their reduction sites (AscH
sites), it does
not effectively interact with the reduction site of membrane-bound
D
M in resealed granule ghosts from the internal phase of the
granule membrane. In addition, the lack of expected competition between
internal IGA and AscH
for the reduction site of the enzyme
strongly suggests that internal AscH
also may not directly
interact with the reduction site of D
M in the resealed chromaffin
granule ghost membrane. On the other hand, externally applied IGA was
found to inhibit the intragranular DA to NE conversion efficiently (Fig. 3, a and b) without significantly
affecting the net rate of DA uptake (Fig. 3, a and b) suggesting that IGA effectively interacts with the external
reduction site of the granule membrane. These results support the
proposal that, while intragranular AscH
may not directly
reduce or mediate the reduction of membrane-bound D
M in resealed
chromaffin granule ghosts, the external reductant may be the exclusive
reductant for the enzyme under these experimental conditions.
In our
previous studies (36) we have shown that 6-AAscH was not an efficient reductant for purified soluble D
M. We
have now shown that 6-AAscH
is a very weak substrate for
membrane-bound D
M as well (see ``Results''). However,
6-AAscH
was found to be a good external reductant for
membrane-bound D
M in standard resealed granule ghosts and supports
the DA to NE conversion with an efficiency only slightly lower than
that of AscH
(Fig. 4). In addition, although
6-AAscH
is structurally remarkably similar to DA, these
results clearly demonstrate that it is not actively or passively taken
up into resealed granule ghosts. These results strongly suggest that
the specificity of the reduction site of purified D
M must be
significantly different from the specificity of the external reduction
site of the resealed granule ghost membrane. Therefore, it could be
concluded that although membrane-bound D
M in resealed granule
ghosts appear to be exclusively reduced by the external reductant, the
external reductant may not directly interact with the reduction site of
the enzyme from the cytosolic phase of the granule. This conclusion is
further substantiated by our preliminary observation that the efficient
artificial chromophoric D
M reductant, N,N-dimethyl-1,4-phenylenediamine(35) , does
not support the DA to NE conversion in standard resealed granule ghosts
from the external phase.
Since DA is a known weak reductant for
purified DM(48) , it is possible that the high rate of DA
to NE conversion in the absence of internal AscH
may simply
be due to the ability of internal DA to act as a redox mediator between
the external reduction site and membrane-bound D
M. On the other
hand, our observation that internal IGA does not competitively inhibit
the conversion of DA to NE in resealed granule ghosts in the presence
or absence of internal AscH
appears to rule out this
possibility because if DA is an effective redox mediator between the
external reduction site and membrane-bound D
M, this process must
also be competitively inhibited by IGA, since IGA has been shown to
compete with DA for the reduction site of membrane-bound D
M (see
``Results''). However, this possibility was further examined
by replacing DA in several key experiments with the reductively
inactive well characterized alternate D
M substrate, tyramine. The
remarkable similar outcome of these key experiments to the
corresponding experiments with DA clearly rules out the possibility
that under internal AscH
-depleted conditions DA acts as a
redox mediator between the external reduction site and membrane-bound
D
M in resealed granule ghosts.
Taken together, the above
results clearly demonstrate that the reducing equivalents for the
membrane-bound DM monooxygenation reaction in resealed chromaffin
granule ghosts are exclusively provided by the external reductant.
These results are in general agreement with the previous proposal of
Huyghe and Klinman(14) . More interestingly, in contrast to the
generally accepted notion, the results presented here also strongly
suggest that the intravesicular solution AscH
(or DA) may
not directly reduce or mediate the reduction of membrane-bound D
M
in resealed chromaffin granule ghosts. Since the specificity of the
reduction site of membrane-bound D
M appears to be different from
the specificity of the extragranular reduction site in the membrane and
the fact that membrane-bound D
M is anchored to the internal
surface of the granule membrane through a short signal
sequence(49) , it is clear that membrane-bound D
M could
not be directly reduced from the external phase. Therefore, we conclude
that there must be an electron acceptor site in the external phase of
the granule membrane which is capable of accepting and transferring the
external reducing equivalents directly or indirectly to the reduction
site of the membrane-bound D
M without using intragranular
AscH
(or DA) as an intermediate.
Numerous previous
studies have demonstrated that the transmembrane hemoprotein, cyt b, is capable of transferring external reducing
equivalents through the membrane to the interior of the
granule(19, 20, 21, 22) .
Furthermore, it has been proposed that the cyt b
-mediated reduction of D
M in chromaffin
granules requires intragranular AscH
as a
mediator(50, 51) . However, the above results strongly
suggest that internal solution AscH
(or DA) is not involved
in the mediation or the direct reduction of membrane-bound D
M in
resealed chromaffin granule ghosts. Therefore, if cyt b
is the external reducing equivalent acceptor,
then the reducing equivalents must be transferred to the reduction site
of membrane-bound D
M either directly or through an unidentified
intermediate electron carrier without using intragranular solution
AscH
(or DA) as an intermediate. Since this electron
transfer process appears to be highly efficient and requires tight
functional coupling between cyt b
and the
reduction site of membrane-bound D
M, it is very unlikely that the
soluble D
M in the granule is also reduced by this same cyt b
-mediated electron transport system. Therefore,
it is possible that while membrane-bound D
M in chromaffin granules
may be reduced through an unidentified external reduction site in the
membrane, the soluble enzyme is reduced by intragranular
AscH
, and constant internal AscH
levels are
maintained through the cyt b
-mediated
AscH
regenerating system as previously
proposed(19, 20, 21, 22) . An
attractive alternate possibility would be that the membrane-bound
D
M is the only physiologically functional form which is reduced
through cyt b
directly or using a mediator other
than solution AscH
(or DA), and the soluble form is only
designated as a disposal form of the enzyme (not recovered and disposed
through exocytosis) which might not be functional under physiological
conditions. (
)The physiological function of intragranular
AscH
may be to provide a reducing environment inside the
chromaffin granules to protect catecholamines from auto-oxidation.
While further experimental evidence is certainly necessary to
distinguish between these possibilities, the understanding of the
topological arrangement of the various proteins and co-factors in the
granule membrane, physiological function(s) of other redox-active
proteins or small molecules in the granule matrix, and the role of
soluble D
M in intact chromaffin granules are of prime importance.