granule ghosts loaded with varying levels of ferrocyanide as reductant, has indicated that external ferrocyanide produces a %fold activation in norepinephrine ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 266, No. 18, Issue of June 25. pp. 11544-11550,1991 Printed in U.S.A.
(0 1991 by The American Society for Biochemistryand Molecular Biology, Inc.
Activity of Membranous Dopamine 8-Monooxygenase within Chromaffin Granule Ghosts INTERACTIONWITH
ASCORBATE* (Received for publication, August 2, 1990)
Bernard G. Huyghe and Judith Pollock KlinmanS From the University ofCalifornia, Department of Chemistry, Berkeley, California 94720
The role of intra- and extravesicular ascorbate has vitro (5,6). However, the intragranular concentration of prodbeeninvestigatedindopamine8-monooxygenase uct norepinephrine (-0.5 M), relative to the presumedi n vivo (DBM) turnoverusingadrenal medullachromaffin reductant ascorbate (10-20 mM) (7, 8) necessitates an addigranule ghosts. Resealing of vesicle ghosts with high tional source of electrons to sustainenzymatic activity. Given levels of intravesicular ascorbate leads to viablevesi- the inefficiency of catecholamines themselves aselectron cles, as evidenced from the high rates of the ATPdonors (6), the most likely source of reducing equivalents is dependent accumulation of tyramine, V,,, = 14 f 1 cytosolic ascorbate(present at -2 mM (9)). According to nmol/min*mgand K , = 2 0 f 6 PM. However, theDBM- current literature models, DPM hydroxylates dopamine to catalyzed conversionof tyramine tooctopamine occurs produce norepinephrine and semidehydroascorbateradical. slowly, V,,, = 0.50 f 0.13 nmol/min*mg andK , = 29 Rapidreduction of semidehydroascorbate by membranous f 18 mM. When ascorbate is present instead in the external buffer, theDBM rate increases 3.6-fold for a cytochrome bSfi1is proposed to prevent radical disproportionfinal V,,,,, = 1.8 f 0.2 and K , = 1.2 f 0.3 mM. This ation, thereby maintaining a constant pool of intravesicular relatively high rate of enzyme turnoveris retained in ascorbate. Cytochrome b5fi1oxidized in this cycle is subseghosts resealed with a large excess of ascorbate oxi- quently reduced by cytoplasmic ascorbate and depletion of by a mitochondrial dase, ruling out contamination by intravesicular ascor-cytoplasmic ascorbateisprevented bate as the sourceof enzyme activity. The synergistic NADH:semidehydroascorbate oxidoreductase (10-12). effect of intravesicular ascorbate was examined under Earlier work from this laboratory (13), using chromaffin granule ghosts loaded with varying levels of ferrocyanide as conditions of 2 mM external ascorbate, showing that the enzymatic rate increases 2.7-fold, from 1.2 (0 in- reductant, has indicated that externalferrocyanide produces ternal ascorbate) to3.2 f 0.4 nmol/min-mg (saturating a %fold activation in norepinephrine production in the presinternal ascorbate). This result confirms that high levence of saturating internal ferrocyanide. These results led to els of internal ascorbateare not damaging to intrave- the proposal that dopamine turnover can be supportedvia a sicular DBM. These studies demonstrate very clearly direct reduction from the exterior face of the vesicle memthat external ascorbate is the preferred reductant for brane. A new model was suggested in which intravesicular the membranous form of DBM in chromaffin granule ascorbate reduces the soluble form of DPM, with membrane ghosts. associated DPM undergoing reduction by a transmembrane electron transferprocess (13). In the present studyof intravesicular DPM, ascorbate has Dopamine P-monooxygenase (DPM)’ exists in both a solu- been used as both an internal and external electron donor, in order to approximatemore closely the physiologic conditions ble and membrane associated form within the chromaffin DPM turnover. In light of the nearly identical kinetic of granule of adrenal medulla (1-4). As summarized in Equation DPM substrates (271, properties of tyramine and dopamine as 1, the enzyme catalyzes the conversion of dopamine t o nortyramine has been used place in of the redox active dopamine epinephrine with the concomitant uptake of two electron to insure that no turnoverwill occur in the absenceof added equivalents. reductant. Initial rate parameters of DPM in granule ghosts indicate that saturating external ascorbate(2 mM) produces significantlyhigher rates of octopamineformationthan ghosts resealed with high (>20 mM) ascorbate, reinforcing the extrafacial reduction mechanism of membranous DPM proposed by Ahn and Klinman (13). When ascorbate is present to the rateof DPM turnover T h e in vivo reductant isnormally ascribed to ascorbate,which both within and external vesicles, alone. This has been shown to support high levels of DPM turnover i n is %fold greater than with external ascorbate enhancer effect of internal ascorbate upon external reductant * This work was supported by Grant GM 25765 from the National is attributed to a small residual pool of the soluble form of Institutes of Health. The costs of publication of this article were DPM, together with the binding of internal ascorbate to a defrayed in part by the payment of page charges. This article must regulatory site on the intravesicular face of DPM (5).
therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. The abbreviations used are: DPM, dopamine P-monooxygenase; MES, 2-(N-morpholino)ethanesulfonicacid; HEPES, 4-(2-hydroxyethyl-1-piperazineethanesulfonicacid; HPLC, high pressure liquid chromatography.
EXPERIMENTALPROCEDURES
Materials All materials purchased were of analytical grade or the highest purity available. Sucrose, disodium fumarate, MES, glycerol, MgS04,
11544
Dopamine 0-Monooxygenme Activity ATP, HEPES, ascorbate, tyramine hydrochloride, and octyl glucoside were from Sigma. Catalase (260,000 units/ml) and ascorbate oxidase (1700 units/ml) were from Boehringer Mannheim. Ficoll and Sephadex G-25 (medium)were purchased from Pharmacia LKB Biotechnology Inc. Dowex AG50WX8 ion exchange resin was from Bio-Rad. Potassium ferrocyanidewas fromMallinckrodtandCuS04from Baker.[I4C]Tyramine(50mCi/mmol), [14C]ascorbate (15 mCi/ mmol), and ‘H20 (5 mCi/mmol) were supplied by Amersham Corp. [I4C]Dextran (0.8mCi/mmol) was from Du-Pont-New England Nuclear. Methods Preparation of Chromaffin Granule Ghosts-One to two pounds of adrenal glands were obtained from a local slaughterhouse and were transported back to the laboratory on ice. The medullae were then dissected out and immersed in ice-cold 0.32 M sucrose, 100 pg/ml catalase. All subsequentoperations were carriedout a t 4°C. The medullae were homogenized at low speed in a Waring blender for 45 s. Cellular debris was removed by centrifugation in a Sorvall SS34 rotor for 10 min a t 2500 rpm. The supernatant was filtered through glass wool and recentrifuged in the SS34 for 30 min a t 14,000 rpm. The resulting pellet, containingthecrude vesicles, was carefully dissected with a glass rod to remove the top brown layer and a clear gelatinous band, leaving behind the pink fraction at the bottom of the tube. This was resuspendedinto 0.32 M sucrose, 100 pg/ml catalaseand layeredover 50 ml of 1.6 M sucrosein a Beckman SW25.2 centrifuge tube. The discontinuous sucrose gradient was spun at 22,000 rpm for 2 h. The tubes were decanted and immediately wiped with kimwipes to avoid anycontamination.The pellet of purified chromaffin vesicles was resuspended in 30 ml of a lysis buffer containing 5 mM sodium fumarate, 10mM MES, pH 6.5, and 100 pg/ ml catalase.To ensure complete lysis (14),1/8 volume of 30% glycerol (v/v), 10 mM MES, pH 6.5, was added to each tube after 5 min and allowed to sit at 4°C for 45 min. Lysed membranes were collected by centrifugation inan SS34 rotor(15,000 rpm, 30 min) and resuspended in about 1 ml of a column buffer comprised of 5 mM fumarate, 10mM MES, pH 6.5, 100pg/ml catalase, 3.5% glycerol. The membranes wereloadedona pre-equilibrated 2 X 40-cm G-25(m)Sephadex column (22 g of dry resin), andallowed to flow by gravity in order to purify the membranes away from endogenous catecholamines and ascorbate. The membrane fraction was concentrated again by centrifugation in an SS34 rotor (15,000 rpm, 30 min). The pellet was divided into aliquotsfor resealingwith different internal components. Resealing was accomplished by incubating the membranesa t 4°C for 1 h in a buffer containing 5 mM fumarate, 10 mM MES, pH 6.5, 100 pg/ml catalase, 150 mM KC1 (15), and varying amountsof ascorbate. The loaded ghosts were collected by spinning them down in an SS34 rotor at 14,000 rpm for 30 min over a 10% (v/v) Ficoll, D 2 0 pad. The ghosts were washed twice in a buffer containing 10 mM MES, pH 6.5, and 150 mM KC1. Ascorbate oxidase loaded ghosts were prepared by including ascorbate oxidase (80-100 units/ml) in the lysis, column, and resealing buffers in order to oxidize any endogenous or contaminating ascorbate,as well as toload the ghosts with ascorbate oxidase. Subsequent washing (2 X ) of resealed ghosts with the final resuspension buffer (10 mM MES, pH 6.5, and 150mM KCI) was sufficient to remove contaminating external ascorbateoxidase. Transport of Tyramine and Ascorbate-Assays for tyramine transport were performed as described previously (13), except that radiolabeled tyramine or ascorbate was used in place of [“Cldopamine. Data were fit using HYPER (29). Tyramine Turnouer Assay-Resealed vesicle ghosts (0.1-0.2 mg/ ml of protein) were preincubated for 10 min at 35°C in a reaction medium containing 50 mM HEPES, pH 7.2, 150 mM KC1, 6 mM MgS04, f 6 mM ATP. Ascorbate oxidase (-10 units/ml) was included in all samples assayedfor internalreductant effects (to remove extravesicular ascorbate lost from leaky vesicles). The reaction was initiated at t = 0 with ascorbate and [14C]tyramine. Aliquots of 100 pl were quenched into 0.4 N HClO, and frozen overnight. Tyramine and octopamine were purified away from the reaction mixture by adsorption onto 0.5 ml of AG50W-X8 H-form (100-200 mesh) cation exchange resin. The columns were washed with approximately 10bed volumes of water. Elution of tyramine plus octopamine was accomplished with 1.5 ml of 6 M NH40H in an overallyield of >go%. Ammonia was removed under vacuum ina SpeedVac for 1 h. Samples were titrated to pH 3-4 with the addition of 50% acetic acid.Tyramine and octopamine were separated by reverse-phase HPLC (AltexC-18 Ultrasphere column) witha mobile phase of methanol, 1% acetic acid
in Chromaffin Granule Ghosts
11545
(1:g) a t a flow rate of 1 ml/min. Octopamine elutes a t 7.5 min and tyramine a t 14 min. One-min fractionswere collected and counted in 10 ml of Ecolite scintillation mixture. Fractional conversion (octopamine (counts/min)/octopamine (counts/min) + tyramine (counts/ min)) for each aliquotwas plotted uersus time toyield turnover rates, which were then normalized for protein concentration. The rate of turnover reported, u, is equal to the rateof octopamine formation in the presence of ATP, u (+ATP), minus the rate without ATP, u (-ATP). The valuefor u (-ATP) was calculatedfrom asingle timepoint at 25 min. In assays involving external ascorbate asreductant, the minus ATP corrections were in the range of8-18% of observed turnoverrates,withan average correction of 12%. As describedpreviously, activation by externalascorbate is directed toward substrate in the ghost interior (13,28). Assay for Ascorbate Oxidase Activity and Znternalization-Ascorbate oxidase activity was determined by measuring the rateof oxygen consumption ona polarographic oxygen electrode. The specific activity of enzyme was determined by incubating known amounts of commercially prepared ascorbate oxidase in 50 mM HEPES, pH 7.0, 150 mM KC1, and initiating the reaction with 2 mM ascorbate. One unit is 1 pmol of O2 consumed/min. mg under these conditions. The measurements were thenrepeatedinthepresence of 0.5% octyl glucoside to check for any inhibition or activation by detergent. No effect was detected. In order to assay for the amount of internal enzyme activity in ghosts resealed with ascorbate oxidase, loaded ghosts were placed in the oxygen electrode in 50 mM HEPES,pH 7.0, 150 mM KC1. Ascorbate was injected into the chambera t t = 0 to obtain a baseline rate of oxygen consumption due to contaminating externallylocated enzyme.After 1-2 min, octyl glucoside (0.5% final concentration) wasinjected and the concurrent increase in oxygen consumption measured. The concentration (units/ml) of internal ascorbate oxidase was estimated from the octylglucoside-dependent rate (base linecorrected) and the internal volume of the vesicle ghosts. Typically, vesicle ghosts retained 40-60%of the available ascorbate oxidase, resulting invesicle ghosts with 5-8 units/mg intravesicular activity. Measurement of Intrauesicular Volume and Final Ascorbate Concentrations-Intravesicular water space was measured as described previously (13), leading to avalue of 3pl/mg of protein for our modified vesicle preparation. In the determination of intravesicular ascorbate concentrations, -0.5 mg of [‘4C]ascorbate containing vesicles were lysed by three freeze thaw cycles followed by a 30-s sonification. Lysed membranes were pelleted for 15 min at 14,000 rpm in a microcentrifuge. The supernatant was removed and counted. The pellet was resuspended and counted to determine background dpm due tononspecific binding. After correction for nonspecific ascorbate binding to membranes, internal ascorbate concentrations were calculated from the specific activity of [‘4C]ascorbate used in the resealing buffer and an internal vesicle volume of 3 pl/mg. Final concentrations are indicated in parentheses, relative to the ascorbate concentration in the resealing buffer: 1 mM (0.98 mM), 5 mM (5.6 k 0.8 mM), 10 mM (8.3 t 1.9 mM), 25 mM (17 & 5 mM), 50 mM (19 2 3 mM), and 100 mM (66 mM). RESULTS AND DISCUSSION
Tyramine Transport and Accumulation-The parameters characterizing tyramine transport and accumulationwere examinedinourexperimentalsystemas ameasure of the viability of our vesicle ghosts. The ability of chromaffin granule ghosts t o accumulate substrate in the presenceof an ATP-induced proton gradient is the best indicator of the integrity of themembranesafter isolation and resealing. Although tyramine transport parameters are notwell as characterized in the literature as for dopamine, values of V,,, = 1.1nmol/min. mg protein andK , = 8 PM have been reported (16). More recently, Knoth etal. (17) observed a value of 1.6 nmol/min. mg under conditions of 20 PM tyramine, 35°C and for dopamine p H 7.2; this rateis approximately one-third that transport. In the present study, we have obtained a V,,, = 14 f 1.0 nmol/min.mg and K , = 20 f 6 PM (Fig. 1)at 3 5 T ,pH 7.2. Although this rate is significantly greater than those in the literature, the value for tyramine remains at about onethird that of dopamine. Final concentration gradients obtained for tyramine (in)/tyramine (out) are typically 200-300-
Dopamine /?-Monooxygenme Activityin Chromaffin Granule Ghosts
11546 16
BOW
14
12
f
+ATP 6000
10
.-e
--E 0
E i
8
6
0
4000
4
2
0
I
20
40
60
80
100
120
2000
-ATP [Tyramine], pM FIG. 1. Tyramine transport into chromaffin granule ghosts. Chromaffin granule ghosts containing 10 mM HEPES, pH 7, 10 mM fumarate, and 150 mM KC1 were preincubated for 10 min at 35°C in 50 mM HEPES, pH 7, 150 mM KCl, and 6 mM M F / A T P . At t = 0, varying levels of ["Cltyramine were added to initiate transport. 50p1 aliquots were assayed each 20 s as described (13). Limiting kinetic parameters were obtained from fitting of the data toHYPER.
. . , .
0
. ,
. . , . . , . .
40
20
60
80
I
.
.
,
120
100
Time, s FIG. 2. Tyramine transportas a function of ascorbate. Chrofold using 30 pM external tyramine, 35"C, pH 7.2, for com- maffin granule ghosts containing 10 mM HEPES, pH 7,10 mM parison to dopamine gradients of 640-650-fold under condi- fumarate, 150 mM KCl, and either 0 or 50 mM ascorbate were ) dopamine, 35"C, pH 7.0 (13). As preincubated for 10 min at 35'C in 50 mM HEPES, pH 7, 150 mM tions of low (25 p ~ external originally noted by Knoth et al. (17), tyramineyields reduced KC1, and 0 or 2 mM ascorbate, +6 mM M F / A T P . At t = 0, 30 p~ concentration gradients relative to dopamine, with reported [14C]tyraminewas added. 50-pl aliquots were analyzed each 20 s as values of 35 f 10 and 221 f 36 for 20 pM external tyramine described (13). 0-0, 0 ascorbate (in), 0 ascorbate (out); 0-0, 0 ascorbate (in), 2 mM ascorbate (out); 0--0, 50 mM ascorbate (in), 2 and dopamine, respectively. mM ascorbate (out); m ,." 50 mM ascorbate (in), 0 mM ascorbate We suggest that the higher transport rates and concentra- (out).
tion gradients reported herein arise from our preparatory procedures which are less damaging than those in the literature. A notable difference in our method is the avoidance of dialysis of isolated vesicle ghosts, a step which we have found can reduce the viability of ghosts (as measured by substrate transport capabilities) by 30-50% within 12 h. Residual catecholamine contamination in our system has been measured to be -5 nmollmg protein. Kelly and Njus (18)report residual catecholamine levels of 2 nmol/mg after 40-48 h of dialysis. We find that rapid preparation and characterization of vesicles is essential to detection of high activity. For the experiments described in this report, all manipulations were complete within 18 h of slaughter. Effect of InternalandExternal Ascorbateon Tyramine Transport-Before investigating the activation of tyramine turnover by ascorbate in chromaffin granule ghosts, it was important to ascertain whether high concentrations of reductant would lead to damage of resealed vesicles. The rate of tyramine (30 p M ) transport by ghosts was examined under experimental conditions similar to those found in uiuo, i.e. with both external andhigh internal ascorbate, and under the limiting condition of either internal or externalascorbate. In all cases, tyramine transportwas found to be ATP dependent (Fig. 2). More importantly, rates were indistinguishable, independent of the location and level of ascorbate (Table I).
TABLE I Initial rates of transport of tyramine by chromaffinvesicle ghosts as a function of intra- and extravesicular ascorbate Resealed vesicle ghosts (0.5 mg/ml) were preincubated for 10 min at 35 "C in a buffer containing 50 mM HEPES, pH 7.0, 150 mM KCI, 6 mM M F / A T P in order to establish a proton gradient. At t = 0,30 p~ ['4C]tyramine was added to a final volume of 0.5 ml. For samples with external ascorbate, 2 mM ascorbate was added just prior to ["C] tyramine. 50.~1aliquots were removed at 20-s intervals and vesicles isolated and washed with ice-cold 0.32 M sucrose by quick filtration as described Dreviouslv (13). Ascorbate u
Intravesicular
Extravesicular nmollmin .mg
mM
0 0 50 50
0 2 0 2
+ +
8.4 1.0 8.1 + 1.0 7.8 1.0 8.7 + 1.0
We conclude that preparation of vesicle ghosts with ascorbate does not lead to oxidative damage. The difference between these results and earlier studies (13, 19) is attributed to the elimination of copper (a catalystfor ascorbate autooxidation) from the resealing buffer.
11547
Dopamine @-Monooxygenuse Activity in Chromaffin Granule Ghosts
0.8 Transport of ~4C]Ascorbate-Although it is generally assumed that chromaffin granules are impermeable to ascorbate, the ability of vesicle ghosts to transport and accumulate radiolabeled ascorbate, either by active transport or passive diffusion, was re-examined (data not shown). Ghosts were 0.6 preincubated for 10 min in 6 mM M$’/ATP, 35”C, prior to addition of 2 mM [’4C]ascorbic acid. The resultsshow no ATP dependence on uptake of radioactivity and, hence, no active .-t transport of ascorbate. The lack of a time dependence also E L indicates that passive diffusion of ascorbate is not occurring 0.4 at a measurablerateduringthetimeframe of tyramine E turnover experiments. Using the specific activity of the [“C] s ascorbic acid used in these studies, together with an internal volume of 3 pl/mg (cf. “Methods”), ghostscould be calculated 0.2 to contain internal ascorbate a t 3-9 mM on the assumption that all of the radioactivity is internal. This calculatedvalue exceeds the theoretical maximum allowed by equilibration with 2 mM external ascorbate. A more likely explanation for the detection of vesicle associated [’4C]a~c~rbate is a nonspe0.0 20 40 60 80 100 120 cific binding to the outerface of the membrane, in agreement with conclusions reached by others (20-22). Activation of Tyramine, Turnover by Internal Ascorbate[Ascorbate], mM The ability of an internal pool of reductant to support turnFIG. 3. Activation of tyramine turnover by internal ascorover was examined by resealing chromaffin vesicle ghosts in the presence of ascorbate concentrations ranging from 0 to bate. Vesicle ghosts containing 10 mM MES, pH 6, 10 mM fumarate, 100 mM. Quantitation of ascorbate trapped within the vesicle 150 mM KC1, and varying amounts of ascorbate were preincubated for 10 min,35°C in50 mM HEPES, pH 7,150 mM KCI, +6 mM Mg”/ ghosts indicated that40-100% of the ascorbate in thereseal- ATP and10 units/ml ascorbate oxidase. Att = 0,30 p M [14C]tyramine ing buffer could be incorporated into the isolated ghosts (cf. was added (final volume 0.8 ml). 100-pl aliquots were removed every “Methods”). Loaded ghosts were preincubated at 35°C for 10 3 min and quenched into 2 ml of 0.4 N HC1O4.The rateof octopamine formation was analyzed byHPLC asdescribed under “Methods.” The minin 6 mM M$’/ATP to establish the proton gradient neededfor substrate transport. Ascorbateoxidase was in- data reflectfour separate vesicle preparations overaperiod of 6 cluded in the preincubation medium to oxidize any contami- months. Data points which show error bars are the average of three to five data points. Velocity has been plotted as a function of the nating ascorbate and to ensure that measured octopamine ascorbateconcentration in the resealingbuffer. Limiting kinetic formation would be due toDPM reduction by an internalpool parameters were obtained by nonlinear least squares fitting of the of ascorbate. data to HYPER. Reanalysisof the velocity data as a function of the Rates at each ascorbate concentration were calculated from estimated intravesicular ascorbate concentrations(cj. “Methods”) led to V,,, and K,,, parameters which were essentially identical to those plots of percent tyramine conversion versus time using the linear portion of the curve to determine the initial rates of obtained from Fig. 4. formation. Time frames varied from 5 to 40% conversion as presence ofCu’+ and a function of internal ascorbate levels, to minimize deviations oxidative Fenton’s chemistry in the ascorbate. In order to assess the requirement for copper in from linearity arising from depletion of ascorbate. The V,,, the present studies, ghostswere resealed with 4 PM Cu2+and for octopamine formation with internal ascorbate as reductant K,,, is 29 then exposed to 2 mM external ascorbate as reductant (see is 0.5 f 0.13 nmol/min- mg protein and the apparent f 18 mM (Fig. 3). The observed value of K,,, is within experi- below); under this conditiona relatively small activation was mental error of a previously published value of 15 f 2 mM seen by Cuz+ (-1.4-fold), suggesting that loss of copper was (23), although itis significantly elevated relative to a value of not a major concern. This result is consistent with previous 0.6 mM reported by Dhariwal etal. (26) for intact chromaffin studies, which found addition of copper to resealing buffer granules. Inlight of the presenceof two K,,, values for purified containing ferrocyanide as reductant to havea very small forms of DPM ( 5 ) ,it is likely the first K,,, value for DPM (3000fold excess)), no inhibition of DBM turnover was observed (Fig. 5). The data inFigs. 3-5 demonstrate first that internal ascorbate is not requiredfor octopamine formation and second that there must bea mechanism for the transferof reducing equivalents from an extravesicular source to intravesicular DPM. In lightof the recentevidence formembrane anchoring of DPM by an uncleaved signal peptide (24), a direct interaction of externalreductantwith enzyme appears highly unlikely. A direct interaction between DPM and cytochrome bsGlalso appears to beruled out from studies of Fleming and co-workers (25) on artificial lipid vesicles reconstituted with
-
2.0
1.5
-
1.0-
-
0.5
t
0.00
1
2
3
4
5
[Ascorbate], mM
FIG. 4. Activation of tyramine turnover by external ascorbate. Vesicle ghosts containing 10 mM MES, pH 6, 10 mM fumarate and 150 mM KC1 were preincubated for 10 min at 35°C in 50 mM HEPES, pH 7, 150 mM KC1, +6 mM Mg2+/ATP. External ascorbate was added at varying concentrations just prior to[14C]tyramine (final volume 0.8 ml). 100-pl aliquots were removedevery 3 minand quenched into2 ml of 0.4 N HC10,. The rateof octopamine formation was analyzed by HPLC as described under “Methods.” Data points reflect three separatevesicle preparations prepared and analyzed over a n eight month period. Limiting kinetic parameterswere obtained by least squares fittingof the data to HYPER.
Time, min
FIG. 5. Octopamine formation in the presence and absence of intravesicular ascorbate oxidase. Vesicle ghosts containing 10 mM MES, pH 6, 10 mM fumarate, 150 mM KC1, and +20 units/ml of ascorbate oxidase were preincubated for 10 min, 35°C in 50 mM HEPES, pH7, 150 mM KCI, 6 mM M?/ATP. External ascorbate a t 2 mM was added just prior to30 mM [14C]tyramine (final volume 0.8 ml). 100-ml aliquots were analyzed by HPLC at the times indicated. The solid circles are plus ascorbate oxidase, and the open circles are minus ascorbate oxidase.
Dopamine P-Monooxygenase
Activity Chromaffin in
Granule Ghosts
11549
purified cytochrome and DPM. Additionally, Wakefield and a model was advanced in which soluble and membrane assoRadda ( l l ) , using lysed chromaffin granules and selective ciated DPMwere reduced by internal and external ascorbate, reduction of cytochrome b561by NADH and a mitochondrial respectively. It was suggested that the 3-foldincrease in vesicles reductase, failed to see electron transferbetween cytochrome activity following the additionof external reductant to ferrocyanidereflected the relative b5,j,and DPM. It is apparent that further studies areneeded, saturatedwithinternal both to identify and characterize the redox component(s) levels of membrane associatedversus soluble DPM in resealed catalyzing membranousDPM reduction invesicle membranes. vesicles. In this study (Fig. 6), a larger discrepancy in rates Activation of Tyramine Turnover by Internal and External has been observed using internal and internal plus external Ascorbate-In a final set of experiments, we investigated the reductant, 6-7-fold. Although vesicle ghost preparations were combined effect of ascorbate present on both sides of the modified in the present study to lead to a greater lossof vesicle membrane. As shown in Fig. 6, activation of substrate soluble components (cf. “Experimental Procedures”), we still turnover by external ascorbate is observed at all levels of estimate -30% of total DPM as the soluble form. Thus, the internal ascorbate. The presence of saturating levels of inter- relative levels of soluble uersus membranous enzyme no longer nal ascorbate together with2 mM external ascorbate leads to correlate well with relative activities using internal and extera rate of product formation of 3.2 f 0.4 nmol/min.mg, with nal reductant. K , = 5.7 k 1.9 mM. This rate is -3-fold greater than that Interpretation of the behavior seen in Fig. 6 is aided by our observed with external ascorbate alone (Fig. 4), proving that recent studies on ascorbate activationusing isolated forms of the resealing of vesicles with high internal ascorbate is not DPM (5). Examination of enzyme activity over a wide range damaging to intravesicularDPM activity. of ascorbate concentrations indicates cooperative behavior, The demonstration that both internal and external ascor-such that ascorbate concentrations above 1 mM lead to a 3batesupportintravesicular DPM turnoverissimilarto a 4-fold increase in V,,,,, with apparent K, values of 16-50 mM previous observation using ferrocyanideas the internalreduc- forsolubleDPM and 10-40 mM for membranous enzyme. tant and dopamine as substrate (13). In the earlier study Analysis (13), of this activation has indicated that it is independent of enzyme form and concentration, as well as tyramine and 5 O2 concentrations leading to the proposal of a regulatory site for ascorbate in DPM ( 5 ) .Comparison of Figs. 4 and 6 in the present study indicates that turnover inpresence the of 2 mM external ascorbate yields values of 1.2 (0 internal ascorbate) and 3.2 nmol/min.mg(saturatinginternalascorbate). We 4propose that the 3-fold activationwithinternalascorbate (Fig. 6 versus Fig. 4) reflects a small amount of soluble DPM, together with binding of internal ascorbate to a regulatory site on the intravesicular face of DPM. In support of this model, the K, value observed for activation by internal ascorbate is large (5.7 f 1.9 DIM),’ and in the range of the K,, values observed for the activation of isolated DPM at ascorbate concentrations inexcess of 1 mM ( 5 ) . Concluding Remarks-Through the use of the redox inactive substratetyramine,togetherwithchromaffin vesicle ghosts which have been depleted of contaminating catecholamines and ascorbate, it hasbeen possible to compare initial 1 rates for catalytic turnoverof intravesicular DPM using either internalorexternalascorbateas enzyme reductant.This e comparison indicates very clearly that external reductant is e the kinetically preferred electron donor for the membranous . , . ’ , . ‘ , form of DPM in chromaffin vesicle ghosts. Despite the low 0 20 40 60 80 100 120 activity seenwith intravesicularascorbate alone, catalytic turnover numbers with external ascorbate undergo a %fold [Ascorbate], rnM activation by internal ascorbate. The K,,, magnitude and of this activation are in the range of parameters ascribed to a FIG. 6. Activation of tyramine turnover by internal and external ascorbate. Vesicle ghosts containing 10 mM MES, pH 6, cooperative activation of DPM by ascorbate (5). We propose mM fumarate, 150 mM KC1, and varying amounts of ascorbate that one important role of intravesicular ascorbate in chrowere preincubated for 10 min a t 35°C in 50 mM HEPES, pH 7, 150 maffin granules is as a modulator of DPM activity. mM KC1, f G mM Mi”/ATP. Vesicles without external ascorbate had 10 unitsjml ascorbite’oxidase in the preincubation buffer. Samples with external ascorbate had 2 mM ascorbate added just prior t o 30 PM [I4C]tyramine addition. 100-ml aliquots were removed every minute, quenched with 2 ml of 0.4 N HC104, and analyzed by HPLC as described under “Methods.” The closed circles are without external ascorbate, and theopen circles are in the presenceof 2 mM ascorbate. The data points represent separate vesicle preparations prepared and bars analyzedover a period of 9 months. Data points with error represent theaverage of three tofive determinations. Limiting kinetic parameters were obtained by nonlinear least squares fitting of the data to HYPER. As noted under Fig. 3, the data have been fit using the concentrationof ascorbate in the resealing buffer, sincecorrection for final intravesicular ascorbate concentrations did not effect the final fitted parameters.
’
The observation that the K , for intravesicular ascorbate is reduced from 29 rt 18 (0 external ascorbate, Fig. 4) to 5.7 1.9 mM ( 2 mM externalascorbate, Fig. 6) is of possiblesignificance. Inthe present study, fumarate has been present at 5 mM in all resealing buffers. As discussed in the preceeding paper (5),the K,, for DBM activation by ascorbate is highly dependent on the presence of fumarateand on the pH of assay solutions. With decreasing pH, inhibition by fumarate increases, leading to less apparent cooperativity and lower apparent K,, values for ascorbate. Ahn and Klinman (3) have measured ApH in vesicle ghosts relative to resealing buffer showing that the pH a s a function of internal and external reductant, is reduced by an additional 0.5 pH unit when reductant is present in the intravesicular space. Thus, a reduced pH in Fig. 6, relative t o Fig. 4, may be the source of the smaller apparent K,, for ascorbate.
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11550
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