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Kinetic evidence for channeling of dopamine between monoamine transporter and membranous dopamine-beta-monooxygenase in chromaffin granule ghosts.

The nature of coupling between the uptake and dopamine-beta-monooxygenase (DbetaM) catalyzed hydroxylation of dopamine (DA) was studied in bovine chromaffin granule ghosts. Initial rate and transient kinetics of DA uptake and conversion were determined under a variety of conditions. The uptake kinetics of DA, norepinephrine (NE), and epinephrine demonstrate that DA is a better substrate than NE and epinephrine under optimal uptake conditions. The transient kinetics of DA accumulation and NE production under both optimal uptake and uptake and conversion conditions were zero-order with no detectable lag or burst periods. The mathematical analyses of the data show that a normal sequential uptake followed by the conversion process could not explain the observed kinetics, under any condition. On the other hand, all experimental data are in agreement with a mechanism in which DA is efficiently channeled from the vesicular monoamine transporter to membranous DbetaM for hydroxylation, prior to the release into the bulk medium of the ghost interior. The slow accumulation of DA under optimal conversion conditions appears to be caused by the slow leakage of DA from the channeling pathway to the ghost interior. Because DbetaM activity in intact granules is equally distributed between soluble and membranous forms of DbetaM, if an efficient channeling mechanism is operative in vivo, soluble DbetaM may not have access to the substrate, making the catalytic activity of soluble DbetaM physiologically insignificant, which is consistent with the increasing experimental evidence that membranous DbetaM may be the physiologically functional form.

Dopamine-␤-monooxygenase (D␤M 1 ; EC 1.14.17.1) catalyzes the conversion of dopamine (DA) to norepinephrine (NE) within the neurosecretory vesicles of the adrenal medullae and the large dense cored synaptic vesicles of the sympathetic nervous system (1)(2)(3)(4)(5). Cytosolic DA is actively transported into the storage vesicles through relatively nonspecific vesicular monoamine transporters (VMATs (Refs. 6 -9)) prior to the D␤M reaction. VMATs are responsible for maintaining a high concentration of catecholamines in storage vesicles. The free energy for this process is provided by pH and electrochemical gradients (10,11) generated by a transmembrane proton translocating ATPase. Evidence from numerous experiments suggests that both electrochemical (⌬) and pH gradients (⌬pH) are mandatory for the intragranular accumulation of monoamines (12,13).
Various mechanistic aspects of DA uptake and DA to NE conversion have been studied using bovine chromaffin granule ghosts as a model for adrenergic neurotransmitter storage vesicles (14 -17). Large quantities of purified granule membranes can be easily isolated from bovine adrenals and stored and resealed to produce fully functional and easily characterizable granule ghosts for detailed mechanistic studies. The coordinated functions of granule membrane proteins with respect to DA uptake and conversion, including membranous D␤M (m-D␤M), ATPase, cytochrome b 561 , and VMATs, have been widely studied using chromaffin granule ghosts (14 -21). Despite these efforts, some critical aspects of the transport-coupled D␤M hydroxylation of DA are not fully understood. For example, numerous studies have shown that D␤M activity in granule ghosts, even under optimal conditions, is orders of magnitude less than the total enzyme activity released following the lyses and solubilization (16,22,23), suggesting that DA transport may be rate-limiting in granule ghosts. However, isotope effects and other studies appear to indicate that the D␤M reaction is rate-limiting in ghosts regardless of the experimental conditions, whereas kinetic studies suggest that the rate of NE production could be much slower than the net uptake of DA, especially under non-optimal conversion conditions (16). Although the D␤M activity in granules is distributed between the soluble and membranous forms (23)(24)(25)(26), the physiological significance of these two forms of enzyme is not clear, but increasing recent experimental evidence suggests that the catalytic activity of the soluble form of D␤M (s-D␤M) may not be physiologically significant (15,23).
To resolve some of these inconsistencies, we examined the nature of the coupling between VMAT-mediated DA transport and the D␤M hydroxylation reaction in chromaffin granule ghosts. Initial rate and transient kinetic studies of DA uptake and conversion have been carried out under a variety of conditions, and the experimental data were analyzed and mathematically modeled by assuming a sequential uptake followed by a conversion process. These analyses clearly demonstrate that a sequential uptake and conversion process could not explain the kinetics of DA uptake and conversion under any condition. On the other hand, all the experimental data are in excellent agreement with a channeling mechanism (for recent reviews on substrate channeling in enzymes, see Refs. [27][28][29][30] in which DA is efficiently channeled from the VMAT to m-D␤M for hydroxylation prior to the release of the ghost interior into the bulk medium.

Materials
L-Norepinephrine hydrochloride, DL-epinephrine bitartrate, dopamine hydrochloride, MgATP, HEPES, Tris base, and ascorbate oxidase were from Sigma. Ascorbic acid (Asc) and sodium fumarate were from Aldrich. Ficoll was from Amersham Biosciences, and catalase was from Roche Molecular Biochemicals. The protein assay reagent and bovine serum albumin were from Bio-Rad. All other chemicals were of the highest grade obtainable. Membrane pellets were homogenized using glass-Teflon Potter-Elvehjem homogenizers. Centrifugations were performed using Beckman Coulter J2MC and Optima LE-80K refrigerated centrifuges. HPLC-EC analyses were performed using an ESA model 582 solvent delivery module and a Coulochem-II electrochemical detector with ESA 501 chromatographic software.

Methods
HPLC-EC Analyses-NE, E, and DA contents of granule ghosts under various incubation conditions were quantified using HPLC-EC. Acidic extracts of ghosts were applied to a C 18 reversed phase column (ESA, HR-80) equilibrated with a mobile phase composed of 90 mM NaH 2 PO 4 , 50 mM citric acid, 0.05 mM Na 2 EDTA, 1.7 mM octanesulfonic acid sodium salt, pH 3.0, with 10% CH 3 CN at a flow rate of 1 ml/min as described previously (15). All three analytes were oxidized at 300 mV. Sample peak areas were quantified by comparison to standard curves that were linear over the range of sample sizes encountered.
Preparation of Chromaffin Granule Ghosts-Chromaffin granules were prepared from fresh bovine adrenal medullae using the original methods of Kirshner (33) as modified by Njus and Radda (34) except that the granules were further purified by a discontinuous sucrose density gradient (35) as described previously (14,15,32). The granules were homogenized in 0.2 M Tris phosphate, pH 7.0, containing 100 g/ml catalase and lysed by the addition of 0.14 volume of a glycerol solution (glycerol, 0.2 M Tris phosphate, pH 7.0 (3:7, v/v)). The lysate was stored in 1.5 ml aliquots at Ϫ78°C. Granule membranes were isolated from the stores on the day of the experiment by diluting with water containing 20 mM Asc and 100 g/ml catalase, incubating for 10 min at 4°C, and centrifuging at 36,000 ϫ g for 25 min at 4°C. The membrane pellet was homogenized and resuspended in 2 ml of a solution containing 20 mM Tris phosphate, 100 mM KCl, 150 mM sucrose, 10 mM sodium fumarate, 4.0 M Cu 2ϩ , 100 g/ml catalase, and 20 mM Asc (optimal uptake and conversion conditions) or 7.5 units/ml ascorbate oxidase (optimal uptake conditions) adjusted to pH 7.0. The ghost membranes were allowed to reseal by incubation for 20 min at room temperature, diluted to 3.75 ml with the same solution as that placed inside (but without Asc, ascorbate oxidase, or catalase), and layered over 1.5 ml of 15% Ficoll, 0.3 M sucrose, 10 mM HEPES, pH 7.0, and 2.25 ml of 0.4 M sucrose, 10 mM HEPES, pH 7.0, and centrifuged for 30 min at 90,000 ϫ g at 4°C. The resealed ghosts, which separated as a band at the 0.4 M sucrose-HEPES/Ficoll interface, were drawn out, diluted with 5 ml of 0.3 M sucrose, 10 mM HEPES, pH 7.0, and homogenized and centrifuged at 36,000 ϫ g for 25 min at 4°C. The supernatant was removed, and the pellet was gently washed with the same buffer and homogenized and resuspended in 1.0 or 2.0 ml of 0.3 M sucrose, 10 mM HEPES, pH 7.0, depending on the experiment, and stored at 0°C. Protein content of ghosts was determined by the Bradford method (31) using the Bio-Rad protein assay with bovine serum albumin as the standard.
DA Uptake Experiments-To aliquots of ghosts prepared under optimal uptake conditions as described above were added 5 mM MgATP, 5 mM MgSO 4 , 100 g/ml catalase, 7.5 units/ml ascorbate oxidase. The aliquots were then diluted with 0.3 M sucrose, 10 mM HEPES, pH 7.0, to a final volume of 0.5 ml for initial rate kinetic experiments or 5.0 ml for transient kinetic experiments. This mixture was incubated for 10 min at 30°C, and the reactions were initiated by adding the desired concentration of DA (see corresponding figure legends) and incubated further at 30°C. At the desired time intervals, 400-l aliquots of the incubate were withdrawn and diluted into 5.0 ml of ice-cold 0.4 M sucrose and 10 mM HEPES, pH 7.0, and stored at 0°C until the incubation was completed. These samples were then centrifuged at 36,000ϫ g for 25 min at 4°C, the supernatants were removed, the pellets were gently washed three times with 0.4 M sucrose, 10 mM HEPES, pH 7.0, and the tubes were swabbed dry. The pellets were homogenized in 500 l of 0.1 M HClO 4 and allowed to extract for 20 min at room temperature. After low speed centrifugation, 20 l of the acidic extracts was analyzed for catecholamines by HPLC-EC as described above. The raw data were corrected for the loss of internal content of the ghosts by standardizing the DA and NE levels to the average indigenous E levels as reported previously (14 -15). Resealed ghost preparations contained an average of 8 -10, 6 -9, and 0 nmol/mg of protein of NE, E, and DA, respectively, depending on the preparation.
DA Uptake and Conversion Experiments-These experiments were carried out using identical protocols as described above for uptake experiments, except that the ghosts were prepared under optimal uptake and conversion conditions as described above, and ascorbate oxidase in the external incubation medium was replaced with 20 mM Asc.

RESULTS
The data in Fig. 1 demonstrate that chromaffin granule ghosts prepared according to the above procedures actively accumulated DA, E, and NE in a concentration-dependent manner. The uptake kinetics of all three catecholamines followed simple Michaelis-Menten behavior in a wide concentration range (5-300 M). The kinetic parameters determined by fitting the initial rate data to the hyperbolic form of the Michaelis-Menten equation show that DA is kinetically a better substrate for the transporter than E and NE (Table I). The depletion of the external substrate during the incubations was estimated to be less than 10% for all the concentrations tested. Therefore, the change in external substrate concentration during the incubation period (6 min) should not have significantly affected the initial rate kinetic parameters determined. In agreement, the control experiments revealed that the corrected uptake rates for all three catecholamines were linear at least up to 6 -8 min of incubation in the entire concentration range FIG. 1. Kinetics of DA, NE, and E uptake into chromaffin granule ghosts under optimal uptake conditions. Granule membranes were resealed and ghosts were incubated under optimal uptake conditions in a total volume of 0.5 ml as detailed under "Experimental Procedures." The uptake was initiated by adding desired amounts of DA (q), NE (E), or E (OE) (5-300 M final concentration), and after 6-min time intervals, 400-l aliquots of the incubate were withdrawn and rates of uptake were determined by HPLC-EC quantification of intragranular catecholamines as detailed under "Experimental Procedures." Dashed lines represent the fit of the experimental data to the hyperbolic form of the Michaelis-Menten equation. of 2-300 M. Although the uptake kinetic parameters varied slightly from one membrane preparation to another, they were highly reproducible within a single preparation; therefore, the most critical experiments were carried out using the same preparation for comparison purposes.
The uptake and conversion of DA inside the granule ghosts as a function of the external DA concentration (DA out ) under optimal uptake and conversion conditions is shown in Fig. 2. These data show that the maximum rate of NE production was about 60% of the net DA uptake (i.e. internal DA ϩ NE). As expected, net DA uptake displayed saturable Michaelis-Menten kinetics with a K m of 33.2 Ϯ 2.4 M and a V max of 1.30 Ϯ 0.03 nmol/ mg⅐min with respect to DA out . Interestingly, the initial rates of DA accumulation as well as NE production also showed saturable Michaelis-Menten kinetics with respect to DA out with K m values of 33.9 Ϯ 3.7 and 32.7 Ϯ 2.0 M and V max values of 0.51 Ϯ 0.02 and 0.79 Ϯ 0.02 nmol/mg⅐min, respectively (see below).
To determine the effect of intragranular DA concentration (DA in ) on the rate of NE production, the DA in for each DA out was estimated based on the average of net DA uptake between 0 and 6 min from the data in Fig. 2. These estimates gave 1.0 -3.8 nmol/mg at 10 -300 M DA out . However, the rate of NE production was non-saturable and linearly increased with increasing DA in in the above concentration range (Fig. 3). Although accurate kinetic parameters could not be obtained, estimates showed that if the enzyme is saturable with respect to DA in , K m must be Ͼ30 nmol/mg (ϳ15-10 mM, assuming an internal volume of 2-3 l/mg for granule ghosts (36)) and V max should be Ͼ 6.5 nmol/mg⅐min (Fig. 3).
As shown in Fig. 4A, transient kinetics of DA accumulation and NE production under optimal uptake and conversion conditions were linear in the presence of saturating DA out (200 M). The net rate of DA uptake was 1.3 nmol/mg⅐min, which is in good agreement with the initial rate kinetics under similar conditions ( Fig. 2; V max ϭ 1.3 nmol/mg⅐min), and the rates of DA accumulation and NE production were 0.40 and 0.88 nmol/ mg⅐min, respectively. The data in Fig. 4B show the time courses of net DA uptake and accumulation and NE production under optimal uptake conditions. Again, whereas net uptake and accumulation of DA and NE production were linear, the rate of NE production was significantly reduced, and the rate of DA accumulation was significantly increased, as expected. Under these conditions, the rates of net DA uptake, NE production, and DA accumulation were 1.5, 0.3, and 1.2 nmol/mg⅐min, respectively, again in agreement with the initial rate kinetics under similar conditions (Table I).
The transient kinetic data presented in Fig. 5A for longer incubation times demonstrate that NE production was linear even up to ϳ40 min under optimal uptake and conversion conditions in the presence of saturating DA out (200 M). Whereas the rate of DA accumulation was linear only up to about 6 -8 min, under these conditions DA in reached an apparent steady state with ϳ7 nmol/mg. However, no lag or burst periods in either DA accumulation or NE production were observed. As shown in Fig. 5B, under optimal uptake conditions, DA accumulation increased by ϳ50 -60% in comparison to the levels determined under optimal uptake and conversion conditions above, whereas the rate of NE production decreased by ϳ50 -60%. Again the DA accumulation reached a plateau (ϳ18 nmol/mg), whereas NE production was linear with no lag or burst periods (Fig 5B). In the presence of limiting DA out (25 M ϳ K m ; data not shown), both DA accumulation and NE productions were significantly decreased in comparison to the saturating DA out experiments under uptake and conversion conditions, as expected. Whereas DA accumulation reached an apparent steady state at ϳ2 nmol/mg in ϳ15 min, NE production increased throughout the incubation period, but significantly deviated from the linearity at longer incubation times (likely because of the depletion of DA out ). Under uptake conditions, the NE production was linear throughout the time course, whereas DA accumulation exceeded the NE production and reached an apparent steady state at ϳ6 nmol/mg. No lag or burst periods were observed for DA accumulation or NE production under both sets of conditions, similar to that observed under saturating DA out conditions (data not shown). DISCUSSION The procedures that we developed earlier and used in the present study to determine the DA uptake and conversion in granule ghosts give highly reproducible results (15). A key step FIG. 2. Kinetics of DA uptake and NE production in chromaffin granule ghosts under optimal uptake and conversion conditions. Granule membranes were resealed and ghosts were incubated under optimal uptake and conversion conditions in a total volume of 0.5 ml as detailed under "Experimental Procedures." The uptake and conversion was initiated by adding desired amounts of DA (0 -300 M final concentration) and, after 6 min time intervals, 400 l aliquots of the incubate were withdrawn and rates of uptake and conversion were determined as described for Fig. 1. q, DA levels; E, NE levels; f, net DA uptake. Dashed lines represent the fit of the experimental data with respect to DA out to the hyperbolic form of the Michaelis-Menten equation.
FIG. 3. Kinetics of NE production with respect to DA in in chromaffin granule ghosts under optimal uptake and conversion conditions. The internal concentrations of [DA in ] were estimated as detailed under "Results" from the net DA uptake in Fig. 2. q, experimentally determined rates of NE production with respect to estimated DA in ; dashed lines, the simulated curve for Michaelis-Menten behavior, assuming a K m of 30 nmol/mg and V max of 6.5 nmol/mg⅐min, respectively.
in this procedure was the use of cold dilution to separate cleanly the internal contents of the ghost incubates from the external medium and to minimize the back transport of the internal contents. The control experiments have shown that granule ghosts prepared and processed according to these procedures contained relatively constant levels of internal E (6 -9 nmol/mg), which slowly declined during longer incubation times, probably because of slow lyses, back transport, nonspecific leakage, etc. Therefore, similar losses of DA and NE in ghost incubates could accurately be corrected by standardizing the DA and NE levels to the average indigenous E levels as reported previously (15), because NE is not converted to E within granule ghosts. We have also shown that ghosts resealed to contain 10 -20 mM Asc could be used to examine the DA uptake and conversion in the presence of 10 -20 mM extragranular Asc (under optimal uptake and conversion conditions). Ghosts prepared to contain excess ascorbate oxidase with no added Asc could be used to examine essentially the DA uptake in the absence of extragranular Asc (optimal uptake conditions) because intragranular DA to NE conversion is significantly slower under these conditions (15).
The uptake kinetics of DA, NE, and E demonstrate that they all are good substrates for bovine adrenal VMAT and obey Michaelis-Menten kinetics in a wide concentration range under optimal uptake conditions (Fig. 1). The kinetic data in Table I show that DA is a better substrate than NE and E under the same experimental conditions. The V max and K m parameters determined for DA and E uptake were in good agreement with the previously reported values under similar conditions (14,32,38,39). Although recent studies suggest that bovine adrenal VMAT contains a mixture of VMAT2 (ϳ85%) and VMAT1 (ϳ15%) (40), our studies could not identify two kinetically distinguishable transport processes for any of the three catecholamines. This may be for two reasons: either the two trans-  Fig. 4A except that 400-l aliquots of the incubate were withdrawn at 2-5-min time intervals, and intragranular catecholamine concentrations were determined as described for Fig. 1. q, DA levels; E, NE levels; f, net DA uptake. Dashed lines, the fit of the experimental data to the equations 6 and 7 and 6 ϩ 7, respectively, in the text. B, transient kinetics of DA uptake into chromaffin granule ghosts under optimal uptake conditions in a long time frame. The conditions are the same as in Fig. 4B except that 400-l aliquots of the incubate were withdrawn at 2-5-min time intervals, and intragranular catecholamine concentrations were determined as described for Fig. 1. q, DA levels; E, NE levels; f, net DA uptake. Dashed lines, the fit of the experimental data to the equations 6 and 7 and 6 ϩ 7, respectively, in the text.
porters may be kinetically indistinguishable under the experimental conditions, or the ghost preparation procedures may have enriched a specific population of ghosts with predominantly one of the transporters.
The net DA uptake (DA ϩ NE) into ghosts under optimal uptake and conversion conditions displayed saturable initial rate kinetics with respect to DA out (Fig. 2), and the kinetic parameters determined were similar to previously reported values under comparable experimental conditions (14,15,24). The uptake kinetics with respect to estimated DA in (see "Results") showed that the rate of NE production was apparently non-saturable within the experimental concentration range and linearly increased with increasing DA in (Fig. 3). In addition, kinetic parameters estimated for DA in to NE conversion, assuming saturating kinetics with respect to DA in , are highly inconsistent with the kinetic parameters of the purified m-D␤M (15). On the other hand, both NE production and DA accumulation displayed saturable initial rate kinetics with respect to DA out (Fig. 2) with similar K m parameters that were very comparable with that of net DA uptake, suggesting that DA out could be the kinetically relevant substrate for all three processes (see below).
The transient kinetics of DA uptake, NE production, and DA accumulation were zero-order under optimal uptake and conversion conditions in the presence of saturating DA out (Fig. 4A), and the corresponding rate constants were in good agreement with the initial rate kinetics under similar experimental conditions (see Fig. 2). The apparent zero-order kinetics for DA accumulation and NE production indicate that the rates of back transport of DA in or NE (if any) are insignificant. Therefore, the kinetics of DA uptake followed by m-D␤M-catalyzed sequential conversion to NE could be analyzed by a simplified kinetic scheme (Scheme 1) with the following assumptions. (a) DA uptake and conversion are sequential processes. (b) The rate of DA uptake is zero-order (k in ) and irreversible. Because DA out is saturating (ϳ7 ϫ K m ) and the maximum depletion of DA out because of uptake was estimated to be less than 1-2% under these conditions, the rate of DA uptake should not be dependent on DA out . As mentioned above, the rate of back transport of DA in is insignificant, and DA uptake could be considered irreversible under these conditions. (c) The conversion of DA in to NE is DA in -dependent and first-order (k NE ) with respect to DA in (i.e. all the substrates are saturating or should remain constant) and irreversible. These assumptions are also reasonable, because the extra-and intragranular reductant, Asc, is saturating (20 mM), and the m-D␤M reaction is macroscopically irreversible. The above assumption also requires that DA in Ͻ Ͻ K m for DA in , which is also reasonable, because the estimates above indicate K m for DA in Ͼ 30 nmol/mg and the maximum DA in is less than 2 nmol/mg under the same experimental conditions. The above system could be described by the following equations.
Although the experimental data in Fig. 4A Fig. 6, DA accumulation should reach an apparent steady state in ϳ3 min, and NE production should lag behind with a non-zero transient time in the above system (27)(28)(29)(30)41). These analyses demonstrate that a sequential process of uptake followed by conversion is inconsistent with the experimental data. Although DA accumulation is zero-order at short incubation times (Fig. 4, A and B), it reached an apparent steady state at longer times under both optimum uptake and uptake and conversion conditions in the presence of saturating DA out (Fig. 5, A  and B). However, NE productions were again zero-order, suggesting that the rate of DA in to NE conversion could not be dependent on DA in . The approach of DA in to an apparent steady state could be due to the competing re-entry of DA in into the transport-coupled conversion process from the interior of the granule (k 2 in Scheme 2), which may be significant under longer incubation conditions. Thus, assuming the rate of DA in to NE conversion (k NE ) is DA in -independent, and rate of reentry of DA to the conversion process (k 2 ) is DA in -dependent and significant, the kinetics of the system could be analyzed by a simplified scheme (Scheme 2) with the following assumptions. (a) As argued above, the net rates of DA uptake (k in ) and leakage (k 1 ) are zero-order (maximum depletion of DA out due to uptake is ϳ5-6% under these conditions), and re-entry of DA in to the kinetic scheme is first order with respect to DA in . (b) The rate of conversion of DA in to NE is zero-order (k NE ) and independent on DA in under both sets of conditions. 2 (c) The rate of back transport of NE is not significant under the experimental conditions because NE production was linear throughout the time course of the incubation. This system could be described by the following equations: The fit of the data in Fig. 5A to equation 6 gave k 1 ϭ 0.52 Ϯ 0.01 nmol/mg⅐min and k 2 ϭ 0.072 Ϯ 0.001 min Ϫ1 under optimal uptake and conversion conditions. Similar fits under optimal 2 D. S. Wimalasena and K. Wimalasena, unpublished observations. SCHEME 1. Sequential model. uptake conditions (Fig. 5B) gave k 1 ϭ 1.00 Ϯ 0.05 nmol/mg⅐min and k 2 ϭ 0.06 Ϯ 0.01 min Ϫ1 . The fit of NE production data to equation 7 resulted in zero-order rate constants (k NE ) of 0.55 and 0.21 nmol/mg⅐min, yielding rates of net DA uptake (k in ) of 1.1 and 1.2 nmol/mg⅐min under optimal uptake and conversion and uptake conditions (because k 2 is small k in ϳ (k NE ϩ k 1 ) at low DA in conditions), respectively. These results are in excellent agreement with the initial rate kinetic data of net DA uptake (see Fig. 2). The steady state concentrations of DA in (DA ss ϭ (k 1 )/k 2 )) were determined to be 7.2 and 16.7 nmol/ mg⅐min under optimal uptake and conversion and uptake conditions, respectively, and are also in excellent agreement with the experimental results (Fig. 5, A and B). Furthermore, the similar and relatively small magnitudes of k 2 determined under both sets of conditions (k in Ͼ Ͼ k 2 ) are consistent with the apparent linear transient kinetics observed for net DA uptake and DA accumulation at short incubation times (Fig. 4, A and  B). Therefore, all the experimental results are fully consistent with the above model (Scheme 2).
A previous study (16) reported that transient DA accumulation displays an initial burst followed by a sharp decline, whereas NE production displays saturating (or exponential growth) kinetics, using granule ghosts under similar experimental conditions. The authors have used a complex kinetic simulation to explain the results assuming a sequential model. Although we could not reproduce these results, the lack of initial burst or lag periods in either DA accumulation or NE production observed in the present study is consistent with all our previous studies (14,15,32). Thus, to explain the observed apparent zero-order transient kinetics of DA uptake and NE production, we propose a novel channeling model (27)(28)(29)(30) in which DA is efficiently channeled from bovine adrenal VMAT to m-D␤M for hydroxylation prior to the release into the bulk medium of the ghost interior (Schemes 2 and 3). The slow accumulation of DA, depending on the experimental conditions, must be caused by the leakage of DA from the transporter (or m-D␤M) into the interior of the ghosts, and the approach of an apparent steady state of DA in at longer incubation conditions could be caused by the slow concentration-dependent re-entry of DA in into the transport-coupled hydroxylation process from the interior of the granule (Schemes 2 and 3). The rate of NE production is independent of DA in but dependent on DA out , and DA out is the kinetically relevant substrate for DA uptake and accumulation and NE production, which is consistent with the similar K m parameters determined under initial rate conditions for these three processes with respect to DA out .
The transport-coupled m-D␤M activity is usually orders of magnitude less than the total enzyme activity released following the lyses and solubilization of ghosts (16,22,23). Because VMATmediated transport is directly coupled with the m-D␤M activity in ghosts according to the proposed channeling model, m-D␤M may not express its optimal activity but efficiently catalyzes the DA to NE conversion at low DA out concentrations. The kinetic isotope effect of the m-D␤M reaction in granule ghosts has been determined to be ϳ2 (similar to the purified enzyme under steady state conditions), suggesting that the m-D␤M reaction is ratelimiting relative to the transport under optimal uptake and conversion conditions (16). Although the exclusion of external Asc was expected to reduce the isotope effect caused by the decreased rate of hydroxylation relative to the transport (normally by about 50 -60%), the isotope effect was not affected (16). These inconsistencies have been attributed to the non-steady state conditions of the ghost interior. According to the proposed model, removal of external Asc significantly decreases the channeling efficiency because the oxidized enzyme does not interact with the amine substrate efficiently, leading to the increase of DA accumulation through the leakage pathway without affecting the isotope effect. Under both conditions the net uptake of DA remains relatively constant (within 10 -20%) regardless of the magnitude of the partition ratio between the DA accumulation and hydroxylation pathways. Therefore, the channeling model provides satisfactory explanations for some of the critical questions regarding the dynamics of DA uptake and conversion in chromaffin granule ghosts.
The concentration of VMAT in chromaffin granule membrane was estimated to be 40 -50 pmol/mg (42), and m-D␤M was about 7% of the total membrane proteins (37). Assuming a subunit molecular weight of 76,000 for m-D␤M (1-3), the molar ratio of m-D␤M to VMAT in the membrane is in the range of 1 to 5. Therefore, VMAT molecules in the membrane could theoretically be effectively coupled to m-D␤M, providing an efficient channeling probability in vivo (Scheme 3). Because D␤M activity in intact granules is equally distributed between s-D␤M and m-D␤M (23)(24)(25)(26), operation of a tightly coupled channeling mechanism in vivo may not give s-D␤M access to the substrate, making the catalytic activity of s-D␤M to be physiologically insignificant. Interestingly, increasing experimental evidence suggests (15,23) that m-D␤M may be the only functional form and that s-D␤M is not functional under physiological conditions and is designated for disposal through exocytosis. We note, however, that the above results do not provide any information about s-D␤M in intact granules because s-D␤M is lost during the preparation of ghosts. Therefore, a SCHEME 2. Channeling model. better understanding of the topological arrangements of VMAT and m-D␤M and the physiological role of s-D␤M in intact granules is of prime importance for further confirmation of the proposed channeling mechanism for the transport-coupled hydroxylation of DA in catecholamine storage vesicles.