Activation of Dopamine 8-Monooxygenase by External and Internal

Vol. 262, No. 4, Issue of Fehruary 5, pp. 1485-1492. 1YX7

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Printed in 1J.S.A

B 1987 by The American Society of Biological Chemists, Inc.

Activation of Dopamine 8-Monooxygenase by External and Internal Electron Donors in Resealed Chromaffin Granule Ghosts* (Received for publication, March 10, 1986)

Natalie G . Ah& and JudithP. Klinmant From the Department of Chemistry, University of California, Berkeley, California 94720

Membrane ghosts derived from chromaffin vesicles of bovine adrenal medullas have been usedto examine the mechanism of reduction of dopamine B-monooxygenase in its compartmentalized state. The rate of the dopamine 8-monooxygenase-catalyzed conversion of dopamine to norepinephrine is greatly stimulated by the presence of ATP, reflecting substrate hydroxylation on the ghost interior subsequent to the active transport of dopamine. We demonstratea 2-3-fold increase in the turnover rate forghosts resealed with 0.2-2 mM potassium ferrocyanide, conditions leading to a slight decrease in the rate of dopamine transport. These data provide the first evidence that an intravesicular pool of reductant can activate dopamine 8-monooxygenase, as required by models in which vesicular ascorbatebehaves as enzyme reductant. Although there issufficient catecholamine (endogenousplus substrate) to keep internal ferrocyanide reduced in these experiments, an additional 2-3-fold increase in turnover occurs in the presence of 0.2-2 mM ascorbate on the ghost exterior. The magnitudeof this activation is found to be constant at all concentrations of internal ferrocyanide (both below and above saturation), implying that reductantson opposite sides of the membrane behave independently. Replacement of ascorbate by potassium ferrocyanide as external reductant leads to almost identical results, andwe are able to rule out an inward transport of dehydroascorbate as the source of activation by external ascorbate. We conclude that externalreductants are capable of reducing membrane-bound dopamine 8-monooxygenase from the exterior face of the vesicle, either by direct reduction or through a membrane-bound mediator. It appears that two viable modes for reduction of dopamine B-monooxygenase may exist in vivo, involving the reduction of membrane-bound enzyme by cytosolic ascorbate as well as the reduction of soluble enzyme by the pool of intravesicular ascorbate present in chromaffin vesicles.

which is driven by a chemiosmotic proton gradient supported by ATP hydrolysis, prior to hydroxylation, Equation 1:

+Pi

’

DA+Oz 2e-,2H+ DPM NE + HZ0

I

1

where DA,’ NE, and DBM represent dopamine, norepinephrine, and dopamineP-monooxygenase, respectively. Extensive studies haveelucidated many chemical details of the dopamine P-monooxygenase mechanism (1-6). However, thesestudies employ the solubleform of DPM and basic features of the mechanismof dopamine hydroxylation within the chromaffin vesicle remain unanswered.A fundamental question concerns the mechanismwhereby DPM obtains reducing equivalents in its compartmentalized state. Ascorbic acid is the best electron donor for DPM in vitro (7). Inaddition, 10-20 mM ascorbatehas beenmeasured within bovine chromaffin vesicles (8, 9), implying that it functions as thein vivo electron donor aswell. Recent studies have shown that a membrane-bound cytochrome beGIin ghost vesicles can mediate transfer of electrons from ascorbate in the ghost interior to the electron acceptorsferricyanide and ferric cytochrome c in the external medium (10-12). Accordingly, a model for DPM reduction hasbeen proposed in which ascorbate within the vesicle is regenerated by external electrondonors via transmembraneelectrontransferthrough cytochrome bM1. In an important initial study, Grouselle and Phillips (13) reportedtheassay of DPM inchromaffin vesicle ghosts. Although this study indicated a role for external reductants in the activation of DPM, the authors failed to see a role for either ascorbate orferrocyanide located in the ghost interior. To clarify this issue, the ATP-dependent hydroxylation of dopamine hasbeen studied in ghosts which are preloaded with Chromaffin granule ghostvesicles derived from bovine ad- varying concentrationsof potassium ferrocyanide and subserenal cells contain all proteinsnecessary to convert dopamine quently exposed to either ascorbate or ferrocyanide in the to norepinephrine. Theprocess involves uptake of dopamine, assay medium. The results presented herein demonstrate first that both internally and externally localized reductants acti* This work was supported by National Institutes of Health Grant vate hydroxylation and second that they appear to act indeGM 25765. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address: Dept. of Medicine RG-26, University of Washington, Seattle, WA 98195. To whom correspondence should be addressed.

The abbreviations used are: DA, dopamine; NE, norepinephrine; DBM, dopamine P-monooxygenase; sDBM, soluble dopamine P-monooxygenase; mbDBM, membrane-bound dopamine 0-monooxygenase; acid; Mes, 4Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic morpholineethanesulfonic acid; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography.

1485

1486

Dopamine P-Monooxygenase Activation in

pendently of one another. A mechanism invoking a transfer of electrons from an external donor to an internal mediator is unnecessary to explain our data, which instead suggest that electron donors may reduce membrane-bound enzyme from the external membrane surface. EXPERIMENTALPROCEDURES

Materials Reagents were analytical grade wherever possible and were purchased from the following sources:disodium fumarate, vanadium-free dopamine disodium ATP, Hepes, Mes, n-octyl-p-D-glucopyranoside, . HC1, and (-)norepinephrine bitartrate, Sigma; ascorbic acid, British Drug House; potassium ferrocyanide, Mallinckrodt; CuSOl. 5Hz0, J. T. Baker Chemical Co.; bovine liver catalase, Boehringer Mannheim; bovine serum albumin, Behring Diagnostics; Ficoll and Sephadex G25, Pharmacia; Surfactol-100, Westchem; sodium dodecyl sulfate (SDS), P-mercaptoethanol, and polyacrylamide gel reagents, Bio-Rad. [1-“CIDopamine and 3H20 were from Amersham Corp. and [“C] methylamine, [14C]thiocyanate, and [“Cldextran were from New England Nuclear. Methods Absorbance measurements were carried out on a Cary 118 spectrophotometer and pH measurements on a Radiometer pH meter 26. HPLC separations utilized a Beckman Model 332 system and scintillation counting a Beckman LS8000 spectrometer. The scintillation mixture contained 2.85 g/liter 2,5-diphenyloxazoleand 35.7 mg/liter 1,4-bis[2-(5-phenyloxazolyl)]benzene dissolved in toluene with 30% Surfactol-100 detergent. For measurements of oxygen consumption, a Yellow Springs Instrument Model 53 biological oxygenmonitor was employed. Protein concentrations were determined by the method of Bradford (14) using the Bio-Rad protein assay with bovine serum albumin as a standard. This method has been reported to show little interference by contaminating catecholamines (15). SDS-polyacrylamide gel electrophoresis was carried out according to Laemmli (16) using a 10% running gel and a 5% stacking gel. Gels were stained overnight with a solution of 0.1% (w/v) Coomassie Blue, 25% (v/v) isopropyl alcohol, 10% (v/v) acetic acid, and 0.1% (w/v) cupric acetate. Protein samples were prepared by boiling for 3 min in 1%SDS, 10%glycerol, 2.5% p-mercaptoethanol, and 35 mM Tris, pH 6.8. Intensity of staining inselected bands was quantitated at 530 nm using a Kratos Model 5D3000 Spectrodensitometer with a Kratos SDC Density Computer and a Hewlett-Packard 3380A Integrator.

Chromaffin Granule Ghosts

tration of about 5 mg/ml with the final wash buffer. The entire isolation procedure lasts 12-14 h from the time of slaughter and yields approximately 20 mg of ghost membrane protein. Experiments with ghosts were performed immediately and generally completed within 9-15 h after isolation, during which time the membranes were stored on ice. Protein determinations were found to be artifactually low unless the membranes were solubilized hence, octyl glucoside was added to each sample and standard to3.3% (w/v), prior to addition of the dye reagent (final detergent concentration, 0.1% (w/v)). Catecholamine (norepinephrine + epinephrine) concentrations inthe resealed ghosts were estimated from the integration of absorbance peaks on HPLC traces and found to be in the range of 60-100 nmol/mg of protein. Measurement of ApH, A#, and Intravesicular Water Space Standard techniques were used for measuring ApH, A$, and intravesicular water space (21) which wereapplied to chromaffin granules by Johnson et al. (17) and Johnson and Scarpa (22). (a) Intravesicular water space was estimated using [“Cldextran as a membrane-impermeant label and 3Hz0 asa permeant radiolabel. Chromaffin granule ghosts were incubated in the presence of both labels for 10 min and concentrated by centrifugation, after which aliquots of both supernatant and pellet were counted. Internal vesicle volume per milligram of protein is given as: vesicle vol

(pl) =

Vol,

(pl)

X

1

I

’Hpllet l4cpeuet -’H,, “C,,

(b) Measurements of ApH and A$ were performed the same way, replacing [“Cldextran by [’4C]methylamine(MA) for measuring ApH ratios and by [“C]thiocyanate (SCN) for measuring A$. (“C/3H)p~~et were corrected for the presence of extravesicular water in the pellet:

= RT/F logJS

+ ( S - 1)(X/1 - X ) ]

= RT/F In

S

- X/1 - X

where X = ([“C]dextran/3H20)~~~e,/([14C]dextran~Hz0)~w~mt, R= ([“ClMA/3H~O),~~,~/([14ClMA/3H~O)~~~~~mt, and S = ([“ClSCN/

3HzO)p,~,/([14ClSCN/3Hz0)~upsmamt.

Preparation of Chromaffin Granule Ghosts

Assays for Dopamine Transport and Turnover

Our ghost preparations use techniques borrowed from many researchers in the chromaffin granule field (17-20). About one pound of bovine adrenal glands was collected from a local slaughterhouse. Medullas were dissected out within 2 h of slaughter and homogenized in 0.3 M sucrose. Fragments of medullas and intact nuclei were removed bylow speed centrifugation (620 X g for 10 min) and the supernatant was centrifuged at higher speed (10,400 X g for 30 min) to concentrate the organelles. Chromaffin granules in this fraction were purified away from mitochondria, lysosomes, and microsomes by centrifugation through a 1.6 M sucrose density step gradient (58,400 X g for 90 min). The granules were then hypoosmotically lysed by >100-fold dilution into a buffer containing 5 mM Hepes, pH 7, 5 mM fumarate, 20 pg/ml catalase, 4 p M cuso4, and varying concentrations of K,Fe(CN)+ These preparationswere allowed to sit at 4 “C for 60 min. Lysed granule membranes were collected by centrifugation (18,000 X g for 30 min) and resuspended into approximately 2 ml of the same buffer. The granules were then loaded onto a Sephadex G-25 column (1.2 X 20 cm) which was pre-equilibrated with a buffer containing 5 mM Hepes, pH 7,5mM fumarate, 150 mM KCl, 20 pg/ml catalase, 4 p M CuSO,, and varying concentrations of K4Fe(CN)6and allowed to flow by gravity. It has been reported that high salts in the medium lead to resealing of the vesicles in which the original sidedness is maintained (17). During this process, catecholamine and other low molecular weight constituents rapidly separated from the membranes, which were collected in the void volume fraction. The membranes were allowed to sit in the isoosmotic buffer for 60 min and collected by centrifugation. As a final wash, pellets were resuspended into a buffer containing 5 mM Hepes, pH 7, and 150 mM KCl. They were then collected by centrifugation onto a 10 mM Hepes, 10% (w/v) Ficoll D20pad and resuspended to a final proteinconcen-

In all assays using radiolabeled dopamine, chromaffin granule ghosts were incubated for approximately 10 min at 35 “C ina medium containing 50 mM Hepes, pH 7, 150 mMKC1, 6 mM MgS04, +6 mM ATP, and varying concentrations of external reductant. Final membrane protein concentration was about 0.5 mg/ml. At t = 0, [“C] dopamine was added to a final concentrationof 100 p~ (-10 Ci/mol) and transportor turnover assayed in thefollowing ways. Dopamine Uptake-At various times, 50-111 aliquots (-25pg of protein) were removed,added to 0.45-pm cellulosenitrate filters over a vacuum manifold, and washed with 4-6 ml of cold 0.3 M sucrose buffered with 10 mM Hepes, pH 7. Filters were dried and counted in 5 ml of scintillation fluid. Standards of each reaction mixture were added onto filters inscintillation vials (without filtration orwashing) and counted to measure the apparent specific activity of [“Cldopamine. In measurements of substrate accumulation a t equilibrium, time points were taken from 0 to 60 min. Net counts incorporated into ghosts typically reached a maximum value at 10-20 min (Fig. U).In measuring initial rates of transport, aliquots were sampled within 100 s, after which the curves became nonlinear (Figure 1B). To obtain these time points, multiple 0.12-ml reaction mixture aliquots were used to sample measurements at 10 and 60 s, 20 and 80 s, and 40 and 100 s. Turnover of Labeled Dopamine-After initiating the reaction, 100p1 aliquots were quenched at 1-6 min, +ATP, and 6 min, -ATP into a mixture containing 2 ml of 0.33 M HClO,, 81 mM Tris, pH 8.4, 10.6 mM EDTA, and 3.25 mgof sodium bisulfite (23). Catecholamines were extracted from the quenched mixture by adsorption onto basic alumina at pH 8.4 and elution in 0.2 N acetic acid, which proceeds with about 90% recovery of counts. Catecholamines were separated by reversed-phase HPLC (Altex C-18 Ultrasphere column) with a

Dopamine /3-Monooxygenase Activation

in Chromaffin

Granule Ghosts

Frc. 1. [“C]Dopamine accumulation into chromaffin granule ghosts verslls time (O-60 min) is shown in A. Ghosts were resealed with 2.0 mM K,Fe(CN), and 4 PM CuSOa, and uptake assays were performed in the presence of 2.0 mM ascorbate and 100 pM [Wldopamine, + 6 mM ATP (0) or in its absence (A). [‘“C]Dopamine accumulation into chromaffin granule ghosts versus time (O-100 s) is shown in B. Preparation of ghosts and assay conditions are as described for A.

TimeCmin)

mobile phase of methanol, 1% acetic acid (15:85, v/v). One-minute (1 ml) fractions were collected and counted in 5 ml of the scintillation fluid described above. Fractional turnover of dopamine is calculated as: fractional turnover = counts/minute (NE)/counts/minute (NE) + counts/minute (DA). This assay is sensitive to a fractional turnover of 0.01, which typically ranged from 0.01 to 0.4. ATP-dependent norepinephrine formation (Fig. 2) shows a small lag due to substrate transport prior to hydroxylation, and rate measurements generally utilize data taken after 1 min. For the purposes of this experiment, ATP-dependent hydroxylation was calculated as Au = v(+ATP) u(-ATP), where v(-ATP) is derived from a single time point at 6 min. Magnitudes of transport and turnover rates typically vary between ghost preparations even when prepared under identical conditions. In order to compare data derived from various preparations, the following correction procedure was used. Turnover rates were measured at both 0.2 mM KdFe(CNJG(in) and 2.0 mM K,Fe(CN)a(in) with 2 mM ascorbate in the external medium, conditions common to every preparation. The observed rates under these conditions were then compared to standard values from a single representative preparation of 10.5 nmol/min.mg for 0.2 mM &Fe(CN)G(in) and 9.3 nmol/min mg for 2.0 m&t KIFe(CN)&n). The ratio of standard to observed values was then averaged and yielded a correction factor for each preparation by which all other rates (both transport and turnover) were multiplied.

Time

dopamine to a final concentration of 10 mM. Solubilization of membrane ghosts by octyl glucoside results in expression of latent activity, representing DBM located in correctly sealed vesicles. This activity is 60-70 % of the total and is maximally expressed with 0.5% octyl glucoside. In the comparison of kinetic parameters and deuterium isotope effects with ascorbate versus KIFe(CN)G, initial velocities were measured at a fixed oxygen concentration (0.2 mM) and varying concentrations of either [2-‘Hz]- or [2-‘H.Jdopamine as described by Ahn and Klinman (2). RESULTS

Chromaffin granules were lysed and resealed as described under “Methods.” Although ascorbate is believed to be the redox mediator in uiuo, we found that resealing granule membranes in the presence of ascorbate results in reduced and irreproducible rates of dopamine transport and that this destructive effect is accentuated by the addition of copper (Table I). Copper was added in accordance with previous findings that micromolar concentrations of the metal are required for optimal soluble dopamine &monooxygenase (sDBM) activity (3, 4). Samuni et ul. (24) have described the damaging effects of ascorbate and Cu2+ on T7 and bacteriophage through

Assay of Solubilized, Membrane-bound Dopamine &Morwoxygenme (mbD,YM)-The assay of detergent-solubilized mbD@M activity

involved the continuous monitoring of oxygen consumption using a polarographic oxygen electrode. In the study of the additivity of ascorbate and K9e(CN)e as reductants, ghosts (-80 &g/ml) were incubated in media containing 50 mM Mes, pH 6.0, 150 rnM KCl, 5 mM fumarate, 4 PM CuSO,, 0.5% octyl glucoside, and varying concentrations of reductant. Reactions were initiated with the addition of

Measurements

TABLE I of [‘Qdopamine transport varying conditions

nmol/min

28.1 6.9

5 mM ascorbate -Cu’+, pH 5.5” 3 PM cu*+, pH 5.5 10 Pi

CL?+, pH

2 rnM K,Fe(CN), -Cd+, pH 7 4 /LM CL?+, pH 7

Time

2. [“ClNorepinephrine

Preparation for Fig. 1.

(m1n1

production

versus

time

(O-6

of ghosts and assay conditions were as described

in ghosts

prepared

under

Assay conditions were as described under “Methods,” with 2 mM ascorbate, 6 mM MgATP, and 100 FM (‘4C]dopamine in the external medium. Transport of [“Cldopamine Components of lysis and resealing buffers Initial velocity Net uptake (6 min) -Reductant -CL?+, pH 7 4 /JM cu’+, pH 7

FIG. min).

(s)

5.5

mg

nmol/mg 110

30

5.3

36

1.4

13

0.5 21.1 16.3

6.2

90 68 10 FM CL?+, pH 7 11.5 55 4 PM cu2+, DH 5.5 7.6 49 “Preparations with ascorbate were conducted at pH 5.5 using nitrogen-bubbled buffers in an effort to minimize ascorbate oxidation.

Dopamine &Monooxygenme

1488

Activation in Chromaffin Granule Ghosts TABLEI1

Kinetic parameters and deuterium isotope effects withdetergent-solubilized mbDpM in the presence of saturating ascorbate or varying concentrations of K a e ( c N ) ~ Reductant DV-b

VW: nmol/min.rng

VIKDA." nrnollmin.mg mM 562 +- 20

D(~ / K D A ) ~

I

Ascorbate, 5 mM 1030 -C 20 1.82 ? 0.09 3.02 Ifr 0.24 132 2 9 ND' 106 k 18 ND K4Fe(CN)6,0.02 mM K4Fe(CN)G,0.2 mM 379 2 20 1.54 0.10 254 +- 13 2.77 If: 0.38 362 k 18 1.79 0.18 315 39 5.91 -+ 1.06 K4Fe(CN)6,2.0 mM Vmaris the velocity in the limit of saturating substrates and V / K D Athe velocity in the limit of zero substrate concentration. These values are apparent, having been measured at a single 0 2 concentration, as described under "Methods." * "Vmarrratio of V,,, for [l-'H?]- and [l-2H2]dopamine,respectively; "( V/KDA),ratio of V / K D Afor [l-'Hz]- and Il-'H,ldopamine. ND, not determined.

*

formation of oxygen-derived radical species. Since several hours elapse between the exposure of ghosts to ascorbate/ Cu2+/02 and transportassays, we attribute the observed inhibition to a nonspecific damage of membrane protein and ghost integrity. It is most likely that the magnitude of initial transport reflects the fitness of all membrane proteins present in theghost, rather than thecarrier alone, providing an assay for vesicle viability. As result a of the destructive effects of ascorbate, K.,Fe(CNI6,an alternate electron donor for DPM, wasused as the internal reductant. As shown in Table I, ferrocyanide has a minor effect on transport and furthermore appearsto protect against the inactivation seen by copper in the absence of reductant. The use of K4Fe(CN), in place of ascorbate in these studies appearswell justified by the similar magnitudes of the , isotope limiting parameter, VmaX,V/Ko,, and V / K D ~and effects on these parameters for sDPM.2 In addition, the use of K4Fe(CN)6with solubilized mbDPM leads to respectable rates and deuterium isotope effects analogous to ascorbate, under conditions of atmospheric oxygen (0.2 mM) and 4 pM Cu2+(Table 11). The effect of internal K4Fe(CN16on the magnitudes of the ApH and A$ generated by ATP hydrolysis in the presence of C1- is given inTable 111. As predicted from the work of Johnson et al. (17), dopamine uptakein the presence of permeant anions is driven primarily by the transmembrane pH gradient, about -1.0 f 0.2 (120 mV), with a negligible contribution from the transmembrane potential (910 mV). Accordingly, the observed ratios of [DA]i,/[DA],ut at steady state are predicted from the ApH within the range of error. Although there is a small variation in ApH and [DA]i,/ [DAlOutat varying concentrations of K,Fe(CN),(in) C ascorbate(out), these parameters are significantly reduced in the absence of ferrocyanide. This is similar to the data in Table I, which indicate a reduction in theinitial rate of transport in the presence of Cuz+upon omission of internal reductantfrom the lysis buffer. Since a direct comparison of the rates of transport and turnover +. reductant would require a correction for the fraction of viablevesicles (Table 111), the effect of internal K4Fe(CP& on DPM activity was examined by varying its concentration in the lysis medium from 0.02 t o 2.0 mM. As shown in Fig. 3A, K4Pe(CN)&n) clearly activates hydroxylation 2-3-fold over the base-line rate. Activation appears saturabie with an apparent K,,, cz 0.1 mM. Because the rate of dopamine hydroxylation has been corrected by the (small) rate measured in the absence of ATP, these data reflect L. C. Stewart and J. P. Klinman, manuscript submitted for publication.

*

dopamine turnover occurring exclusively in the vesicle ghost interior! Upon addition of 2 mM ascorbate to the external medium, a similar 2-3-fold activation by internal K4Fe(CNj6 is observed(Fig. 3B). Although thedata in Fig. 3 show experimental scatter, a statistical comparison (cf. Ref. 32) of the points at 0.02 to 2.0 mM internal ferrocyanide indicates a very significant increase, with the probability that these Values are thesame, being t0.01 without external reductant(Fig. 3A) and t0.005with 2.0 mM ascorbate(out) (Fig. 3B). Turning to a comparison of the data in Fig. 3A to 3B, it can be seen that external ascorbate increases the rate of hydroxylation to a similar extent at all internal K4Fe(CN)6 concentrations. Importantly, DPM activity continues to undergo an -%fold activation by externalreductant in the region of internal reductant Concentration which is saturating. This behavior is not observed in the corresponding transport measurements (Fig. 4), where both increasing concentrations of K,Fe(CN)&n) and ascorbate(out) decrease initial transport velocities and, to a small extent, the steady state levels of uptake (Table 111).The reason for this rate inhibition is not well understood, but may be related to theslight inhibition in turnover observed at high internal ferrocyanide in the presence of 2 mM external ascorbate? It is known that high copper concentrations (>2 mol of Cu2+/mo1of enzyme subunit) lead to reversible inhibition of sDPM activity in the presence of either ascorbate (3) or ferrocyanide (25). Since copper andK4Fe(CN)6 appear to interact in a competitive manner (25), we wished to demonstrate thatactivation by K4Fe(cN)6(in) in ghosts is not caused by removal of copper inhibition. Therefore, measurements were made at varying concentrations of added CuS04, 0-10 p ~Fig. . 5, A and B, shows that increasing copper across this range activates turnover in both the absence and presence of external reductant, despite a small inhibitory effect on transport (Table I). The greater activation by copper with ascorbate(out) may be related to the different pools of enzyme undergoing reduction, as described later. Significantly, the data in Fig. 5 indicate that K,Fe(CN), activation is not due to a competitive interaction with inhibitory copper. In an effort to examine moreclosely whether internal K4Fe(CN), is required for activation of turnover by external ascorbate, the concentration of ascorbate(out) was varied simultaneously with K4Fe(CN)6(in).Analogous to Fig. 3, we Reports of reversible ADP activation of purified sDBM and mbDPM suggest the possibility that ATP hydrolysis might lead to Dj3M activation (31). However, under our assay conditions, up to 6 mM ADP does not appear to activate purified sDPM from 0.1-10 mM dopamine. Since the extent of inhibition by external reductant increases as the vesicle ghost preparations age, we believe this inhibition results from free radical damage byactive oxygen species.

Dopamine P-Monooxygenase Activation in Chromaffin GranuleGhosts

1489

TABLE111 Measurements of 4pH, A$ and [DA]i,,/[DA]oucratios in ghosts prepared with vatying amounts of Kze(CN), Ghosts were prepared as described under “Methods” with5 mM Hepes, pH 7 , 5 mM fumarate, 150 mM KC], 20 pg/mI catalase, 4 p~ CuS04.The number of determinations aregiven in parentheses. Measurementswere conducted using the assay procedures described under “Methods”in the presence of 6 mM ATP. For ApH and A$, dopamine was omitted from the external buffer. Predicted

Observed

18.5 108 95 166 129

71 -+ 8 (2) 195 f 60 (2) 206 k 46 (5) 153 -+ 62 (6) 180

mV

-Reductant(out) -0.61 -0.96 -0.90 -1.07

0 mM 0.02 mM 0.2 mM 2.0 mM

Averagesb

f 0.32 (2)

(1) f 0.15 (2) f 0.26 (3) -1.00

2.9 f 1.6 (2) 7.0 (1) 10.8 f 3.9 (2) 4.9 f 4.1 (3) 6.8

+2 mM ascorbate(out) 36 69 _t 55 (3) 14.0 (1) 26 136 -+ 5 (3) 6.5 (1) 11.1 k 3.0 (2) 105 181 f 104 (8) 12.8 k 5.9 (2) 371 129 f 51 (10) 10.8 131 150 Averagesb ’[DA]i. is calculated using the average vesicular volume of 9.31 pl/mg (n = 12). Predicted ratios of [DAIi,/ [DA], are calculated accordingto Johnson et al. (17): 0 mM 0.02 mM 0.2 mM 2.0 mM

-0.66 (1) -0.67 (1) -0.92 -+ 0.08 (2) -1.18 f 0.35 (2) -0.97

Averages of measurements using ghosts prepared with 0.02, 0.2, and 2.0 mM K9e(CN)6. I

I

I

8

FIG. 3. ATP-dependent rate of hydroxylation in resealed ghosts uersus internal K4Fe(CN)G concentration is shown in A . Ghosts were resealed with 4 ~ L MCuS04and varying concentrationsof K,Fe(CN),, and assayswere performed inthe absence of reductant in the external medium. ATP-dependent rate of hydroxylationinresealedghosts versus internal K4Fe(Cb06concentration is shown in B . Ghosts were resealed in 4 ~ L MCuS04 and varying concentrations of K4Fe(CN), and assays were performed with 2.0 mM ascorbate in the external medium.

FIG. 4. Initial rateof [“Cldopamine transport into resealed ghosts versus external ascorbate concentration. Ghostswere resealed with 4 p~ CuSO, and 0.05 (O),0.2 (V),and 2.0 mM (m) K4Fe(CN)6.Assays were performed with 6 mM ATP in the external medium.

observe that ascorbate(out) in the range of 0.05-1 mM activates turnover at all concentrations of K4Fe(CN)Jin), witha maximal effect at 0.2 mM ascorbate(out) (data not shown). We also observe an inhibition by external ascorbate at concentrations greater than 2 mM. Because of the complication of inhibition by 4 ~ L MCu2+ (Table I), externalascorbate activation is difficult to demonstrate in thecomplete absence of internal K4Fe(CN)+Although omission of copper from the lysis media leads to somewhatreduced turnover rates (Fig. 5), transport rates remainhigh (Table I). Therefore, we examined D@Mturnover in the absenceof both K,Fe(CN)6 and copper, observing the same 3-fold activation of enzyme by external ascorbate as in the presence of internal reductant (Fig. 6).

As shown (Figs. 3 and 61, the maximal degree of enzyme activation by ascorbate appears constant as the internal concentration of ferrocyanide is varied. This behavior is not predicted by a model in which K4Fe(CN)&n) is the sole reductant for DPM. If ascorbate(out) were behaving only as an electron source for the reduction of internal ferrocyanide, activation by ascorbate(out) would not be observed either in the absence of K,Fe(CN),(in) or a t saturating levels of internal reductant. Instead, K,Fe(CN),(in) does not affect activation of dopamine turnover by ascorbate(out), andconversely, ascorbate(out) does not affect activation by KBe(CN)e(in). These data lead us to suggest that ascorbate(out) reduces DPM from the external membraneface. Alternatively, ascorbatemightactasanelectrondonor

OO

5

IO

Ascorbote ( o u t ) ( m M )

Dopamine P-Monooxygenme

1490

ActivaItion i n Chromaffin Granule Ghosts I

IO

Cu+‘ ( p M )

-

-

I

0

p : ‘

Cu+‘ (pM)

FIG. 5. ATP-dependent rate of hydroxylation in resealed ghosts uersus internal CuS04.Ghosts were resealed with 0.02 (A), 0.05 (e),0.2 (V),and 2.0 mM (W) K,Fe(CN),,andassayswere performed in the absence ( A ) or presence ( B ) of 2 mM ascorbate in the externalmedium.

I

I

A

.-C

0

E

\

I

I

1.0

2.0

K,Fe(CN)e(in)(mM)

I

I

I

1.0

2.0

I

K 4 Fe (CN)6(in) (mM)

FIG. 7. ATP-dependent rate of hydroxylation in resealed Ghosts were ghosts uersus internal K4Fe(CN)B concentration. resealed in 4 p~ CuSO4and varying concentrations of K4Fe(CN)6 and assays were performed as a function of &Fe(CN)6in the external medium: A, 0 K4Fe(CN)6(out); B, 0.05 mM IdFe(CN)&(out); c, 0.2 mM K4Fe(CN)6(out); and D,2.0 mM K4Fe(CN)6(out).

As noted under “Methods,” lysis of chromaffin vesicles reduces endogenous catecholamines from -0.5 M to 5 mM. A corresponding decrease in endogenous ascorbate is expected I I to lead to -0.1 mM in resealed ghosts. It is therefore conceiv00 5 IO able that activation by both external ascorbate and ferrocyaAscorbate(out) (mM1 nide is the result of electron transfer to a small endogenous FIG. 6 . ATP-dependent rate of hydroxylation in resealed pool of ascorbate. To explore this possibility, the extent of ghosts uersus external ascorbate as reductant. Ghostswere enzyme activation by ascorbate was examined at varying resealed in the absenceof any added CuSO,or reductant. levels of K4Fe(CN), using detergent-solubilized mbDPM (Fig. from the membrane interior. Although results of previous 8). Under these conditions, enzyme is subject to competitive investigations eliminate the possibility that ascorbate is trans- activation by K4Fe(CN)fi andascorbate, such that the actiported in its reduced form (10, 26, 27) membrane-permeable vation by ascorbate decreases with increasing ferrocyanide dehydroascorbate might diffuse into the ghost and become concentrations. Thus, if the concentration of internal ascorreduced by way of the hypothesized transmembrane electron bate falls to zero in theabsence of external reductants but is transfer cytochrome. To clarify this point, K4Fe(CN), was maintained at itsinitial level, -0.1 mM, in their presence, we used in place of ascorbate as theexternal electron donor, since would expect activation by external reductants to decrease it ishighly unlikely that K4Fe(CN), or K3Fe(CN), could from 200-300% at 0.02 mM K,Fe(CN),(in) to 9.5 s-'). Based on these results, a model for DPM reduction has been proposed in which a soluble redox mediator, presumed to be ascorbate in vivo, interacts directly with mbDBM and sDPM within the chromaffin granule interior. Support of hydroxylation by an external reductantoccurs through transmembrane electron transport via cytochrome b561 to the internal mediator, which must be regenerated with each turnover. Although the experimentally measured electron flow has been in theopposite direction from that hypothesized in uivo, the dataprovide a reasonable mechanism for the regeneration of compartmentalized ascorbate. The model also explains the fact that no enzymatic oxidoreductase activity has yet been clearly demonstrated in chromaffin granules. A fundamental feature of the above model is the requirement for intravesicular reductants which support DPM-catalyzed hydroxylation. In earlier experiments, Grouselle and Phillips (13) demonstrated stimulationof an ATP-dependent ' OO .Ascorbate I. 0 (mM1 I 2.o 1 turnover of tyramine by external reductants, but failed to observe a role for internally localized K4Fe(CNI6.This result FIG. 8. Activity of detergent-solubilizedmbD@Hversus re- contrasts with the data presented herein, where ductant. Activities were measured by monitoring oxygen uptake at K4Fe(CN)&n) clearly activates DBM 2-3-fold at saturating varying concentrationsof ascorbate in the presence of 0 (O),0.02 (A), conditions of reductant (Figs. 3 and 7). In all studies of 0.1 (m), and 0.2 m M (V)K4Fe(CN)6. ferrocyanide activation, we have observed the initial rate of product formation to be linear after a short lag period. As 75,000 polypeptide into the supernatant. No further extraction of protein was observed upon incubating ghost mem- seen in Fig. 2, product synthesis reached 40% in 6 min, branes in 250 mM NaC1, 5 mM EDTA, whereas the bulk of corresponding to 80 nmol of norepinephrine/mg of protein. Since the vesicle volumeis small relative to thetotal (-0.5%), the M, 75,000 polypeptide extractedintoSDS(datanot shown). These results suggest that ghost preparations may the amount of internal K4Fe(CN)6(-20 nmol/mg of protein) indeed be contaminated by a DPM form which is not tightly is insufficient to account for the product formed and should membrane-associated, although we cannot be certain to what not yield linear rates of turnover. However, as noted under extent this putative sDPM originates from a small pool of "Methods," in the absence of substrate uptake, our vesicle intact chromaffin granules which fail to lyse in the course of preparations already contain -5 mM catecholamines. Furthe ghost preparation verus residual sDpM which does not thermore, Stewart and Klinman' have shown that catecholseparate from the lysed membranes before resealing occurs. amines rapidly reduce 2 mM K,Fe(CN), leading to initial Quantitation of hydroxylase activity in the sDpM uersus rates with sDPM which are comparable to those measured the reduction of mbDPM pools by gel densitometer scanning through the M, with 2 mM K.,Fe(CN),. Inallinstances 75,000 band indicated 30% sDPM, whereas activity measure- enzyme by catecholamine plus ferricyanide is much faster ments in 1%octyl glucoside lead to a value of 15%. Since than catecholamine alone, implicating ferrocyanide as the chromogranin A may co-migrate with sDPM (28), the activity enzymatic mediator. Thus we conclude that resealed vesicles estimates areconsidered more reliable and lead to anestimate contain sufficient catecholamine to maintain ferrocyanide in of sDPM to solubilized mbDBMof0.15:0.85. Comparative the reduced form. Overall, there aresufficient reducing equivstudies of octyl glucoside solubilized mbDPM versus mbDPM alents in our ghost preparations to support complete converin vesicle ghosts generally indicate an increase in latent sion of substrate to product and hence, no participation by an activity: so that following correction for differing activities external reductant need be invoked under our experimental in solubilized versus vesicular mbDpM we estimate conditions. sD@M:mbDpMas 0.3~0.7.Although these considerations supHowever, our results clearly implicate a role for external port a role for sDPM, we note that theestimated contribution electron donors in turnover, demonstrated by an activation of contaminating sDBM is -30% and as such is unlikely to of enzyme which is independent of the presence of internal be the exclusive source of internal K4Fe(CN)6activation. reductant (Figs. 3, 6, and 7). Since the same pattern is observed with either external K,Fe(CN), or ascorbate, it is DISCUSSION not caused by dehydroascorbate uptake and subsequent reEvidence to date strongly suggests that chromaffin granules duction. Furthermore, the behavior of competing reductants have the capability to transfer electrons from one side of the for DPM (Fig. 8) makes it unlikely that low levels of oxidized, membrane tothe other, mediated by a membrane-bound endogenous ascorbate are undergoing reduction by the exterprotein which can interact directly with soluble redox com- nal pool of reductant. We conclude that reductants on oppoponents on both sides. Using chromaffin granule ghosts pre- site sides of the membrane act independently of one another loaded with 100 mM ascorbate, pH 7, Njus et al. (10) and and contribute additively to the total rate. This behavior is Harnadek et al. (12) demonstrated the reduction of both not predicted by a model involving transmembrane electron Fe(CN)z3 (-+Fe(CN)g4) and Fe3' cytochrome c (-+Fe2+cyto- flow to a soluble internal mediator as the exclusive source of chrome c), concomitant with the appearance of a transmem- reducing equivalents. It is proposed that mbDPM may accept brane potential A$ > 0. Strivastava et al. (11)repeated this electrons from the membrane exterior without the participation of an internal mediator. This behavior might occur either N. Ahn, B. Huyghe, and J. P. Klinman, unpublished results. by direct interaction of the external reductant with the en-

1492

Dopamine P-Monooxygenase Activation

zyme or, alternatively, through the intervention of another mediator, e.g. the membrane-localized cytochrome bSG1. Finally, we note that the rate of ATP-dependent hydroxylation (1-2 nmol/min. mg) is non-zero in the compIete absence of added reductant. Eitherdopamine added as substrate or endogenous norepinephrine and epinephrine could function as electron donors. We were unable to detect labeled dopaquinone by HPLC in [I4C]dopamineturnover assay eliminating a major contribution by dopamine. Furthermore, Grouselle and Phillips (13) observed atime-dependent oxidation of endogenous catecholamine concomitant with the hydroxylation of tyramine. Using [I4C]tyramine as substrate, we also observe base-line rates of turnover in the absence of added reductant (data notshown). Therefore, it is likely that endogenous catecholamines representathird pool of reductant capable of supporting hydroxylation. However, it is unlikeIy that this pool contributes significantly to turnover rates in the presence of added internal or external reductants, since catecholamines are very poor reductants for DPM.* Given the demonstrationof transmembrane electron transfer with ascorbate loadedvesicles (10-12) anda role for internal reductant in DPM activity (this study), it appears reasonable that vesicular ascorbate should function as an in uiuo reductant. In addition, the data reported herein indicate a pathway for enzyme reduction from the exterior face of the membrane. This important resultimplies either that mbDPM is reduced more rapidly from the outer than the inner face of 2AL+2Ht

2AH'

in Chromaffin Granule Ghosts the vesicle membrane, or that different forms of DPM are kinetically apparent in this system. With regard to the latter possibility, gel electrophoresis and activity analyses of ghost preparations do support the presence of -30% sDPM, suggesting that the observed kinetic patterns may reflect different mechanisms for the reduction of sDPM and mbDPM. We therefore propose a model for DPM in intact vesicles (Scheme I). As illustrated, cytoplasmic ascorbic acid (-1 mM (29)) serves as the ultimate reductant pool for both sDPM and mbDPM (30). Inthe case of sDpM, reduction occurs by intravesicular ascorbate, which is subsequently recycled by cytoplasmic ascorbate. Since the active sites of both sDPM and mbDPMface the vesicle interior, mbDPMmay also undergo some reduction by this mode. In addition, however, mbDPM can undergo efficient reduction from the exterior face of the vesicle. As noted above and in the legend to Scheme I, the precise mechanism of this newmode of reduction is currently unclear. It will be of particular interest to ascertain whether mbDpM can interact directly with ascorbate in the absence of a mediator, since such a mechanism would imply a long-range intramolecular electron transfer from the site of enzyme reduction (cytosolic) to that of substrate hydroxylation (vesicular). In closing, we also note that the demonstration of two viable modes for reduction of DPM in vivo may provide a functional explanation for the presence of multiple forms of DPM in chromaffin vesicles. REFERENCES 1. Miller, S. M., and Klinman, J. P.(1983)Biochemistry 22,3091-3096 2. Ahn, N., and Klinman, J. P. (1983)Biochemist 22,3096-3106 3. Klinman, J. P., Krueger, M., Brenner, M., and gknondson,D. E. (1984)J . Bwl. Chem. 259,3399-3402 4. Ash, D. E., Papadopoulus, N. J., Colombo, G., and Villafranca, J. J. (1984) J. Biol. Chem. 259,3395-3398 5. Miller, S. M., and Klinman, J. P. (1985)Biochemistry 24,2114-2127 6. Fitzpatrick, P. F., Flory, D. R., Jr., and Villafranca, J. J. (1985)Biochemistry 24, 2108-2114 7 . Levin, E. Y., Levenberg, B., and Kaufman, S. (1960)J. Biol. Chem. 235,

-"-" -"-"

vnQn-vnw

8. Ingebretsen, 0. C., Terland, O., and Flatmark, T. (1980)Biochim. Biophys. Acta 628,182-189 9. Terland, 0.. and Flatmark, T. (1975)FEBS Lett. 59,52-56 10. NJUS,D., Knoth, J., Cook, C., and Kelly, P. M. (1983)J. Biol. Chern. 258,

ADP

27-21)

11. St&a&va, M., Duong, L. T., and Fleming, P. J. (1984)J. Biol. Chem. 259,8072-8075 12. Harnadek, G. J., Ries, E. A., and Njus, D. (1985)Biochemisty 24, 2640?Add

13. Gri&ke, M., and Phillips, J. H.(1982)Biochem. J. 202, 759-770 14. Bradford, M. M. (1976)Anal. Biochem. 72,248-254 15. Pollard, H.B., Menard, R.,,Brandt,H. A., Pazoles, C. J., Creutz, C. E., and Ramu, A. (1978)Anal. Blochem. 86,761-763 16. Laemmli, U. K. (1970)Nature 227,680-685 17. Johnson, R. G., Pfister, D., Carty, S. E., and Scarpa, A. (1979)J. Biol. Ch.mn -. .-. .. 254. - - -,in~m-10972 . .. - ... -

mb DBM

2 AH' 2A"+ 2H' SCHEME I. Proposed model for DBM reduction in intact vesicles, where AH- and A" are the ionized forms of ascorbate and semidehydryascorbate, respectively. As shown, sDpM reduction occurs by intravesicular ascorbate, which i s recycled from cytoplasmic ascorbate. In addition, mbDBM can undergo reduction from the exterior face of the vesicle. Although direct reduction of mbDOM is drawn for illustrative purposes, this reduction may be mediated by a membrane-bound protein such as cytochrome b5G1.

18. Phillips J. H. (1974)Biochem J. 144,311-318 19. Bartletc S. F., and Smith, A. D. (1974)Methods Enzymol. 31,379-389 20. Helle. K.. Flatmark. T.. Serck-Hansen,. G... andLonnina. -. S. (1971)Bimhirn. Bidphys. Acta 226,l-8 21. Rottenber H (1979)Methods Enzymol. 5 5 , 547-572 22. Johnson G:, and Scarpa, A. (1976)J. Gen. Pbysiol. 68 601-631 23. Weil-Milherbe, H. (1971)Methods Biochem. Anal. P. Suipl., 119-152 24. Samuni, A., Aronovitch, J., Godinger, D., Chevion, M., and Czapski, G. (1983)Eur. J. Biochem. 137,119-124 25. Rosenherg, R. C., Gimble, J. C., and Lovenberg, W. (1980) Biochim. Bio h s Acta 613,62-72 26. Tirreif, f.,'and Westhead, E.(1979)Neuroscience 4,181-186 27. Levine, M.,Morita, K.. and Pollard, H. B. (1985)J. Biof. Chem. 260, 12942-12947 28. Winkler, H.(1976)Neuroscience 1,65-80 29. Daniels, A. J., Dean, G., Viveros, 0. H., and Diliberto, E. J., Jr. (1982) Science 216, 737-779 30. Levine, M., Morita, K., Heldman, E., and Pollard, H. B. (1985)J. Bid. Chem. 260,15598-15603 31. Ohuchi, T., Tachikawa, E., Morita, K., Ishimura, Y., and Oka, M. (1982) Adu. Biosci. 36,201-208 32. Snedecor, G., and Cochran, W. (1980)Statistical Methods, 7th Ed,, pp. 8393,Iowa University Press, Ames, IA

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