Mechanism of the insect enzyme, tyramine beta-monooxygenase, reveals differences from the mammalian enzyme, dopamine beta-monooxygenase

Tyramine beta-monooxygenase (TbetaM) catalyzes the synthesis of the neurotransmitter, octopamine, in insects. Kinetic and isotope effect studies have been carried out to determine the kinetic mechanism of TbetaM for comparison with the homologous mammalian enzymes, dopamine beta-monooxygenase and peptidylglycine alpha-hydroxylating monooxygenase. A new and distinctive feature of TbetaM is very strong substrate inhibition that is dependent on the level of the co-substrate, O(2), and reductant as well as substrate deuteration. This has led to a model in which tyramine can bind to either the Cu(I) or Cu(II) forms of TbetaM, with substrate inhibition ameliorated at very high ascorbate levels. The rate of ascorbate reduction of the E-Cu(II) form of TbetaM is also reduced at high tyramine, leading us to propose the existence of a binding site for ascorbate to this class of enzymes. These findings may be relevant to the control of octopamine production in insect cells.     

Correlation of copper valency with product formation in single turnovers of dopamine beta-monooxygenase

Chemical- and freeze-quench EPR techniques have allowed single-turnover studies of the copper-containing enzyme dopamine beta-monooxygenase. Reduction of enzyme by a stoichiometric amount of ascorbate followed by rapid mixing with tyramine leads to oxidation of bound copper and formation of hydroxylated product in the expected 2:1 ratio. The tyramine dependence of single turnovers yields a limiting rate of 82 +/- 9 s-1 and Km of 3 +/- 1 mM, in agreement with kinetic modeling based on steady-state parameters. Together these results show that the reduced enzyme is a catalytically competent species, with bound copper acting as the sole reservoir of reducing equivalents. The correlation of copper oxidation and substrate hydroxylation rules out significant antiferromagnetic spin coupling in the enzyme-product complex. Since the enzyme-product complex's Cu2+ EPR signal is absent in the transient approach to the steady state [Brenner, M. C., Murray, C. J., & Klinman, J. P. (1989) Biochemistry (preceding paper in this issue)], this result implies that ascorbate reduces copper in the enzyme-product complex. These findings have important consequences for catalysis and active site structure in dopamine beta-monooxygenase.     

Mechanism-based inhibition of dopamine beta-monooxygenase by aldehydes and amides

A mechanism for beta-chlorophenethylamine inhibition of dopamine beta-monooxygenase has been postulated in which enzyme-bound alpha-aminoacetophenone is generated, followed by an intramolecular redox reaction to yield a ketone-derived radical cation as the enzyme inhibitory species (Mangold, J. B., and Klinman, J. P. (1984) J. Biol. Chem. 259, 7772-7779). If correct, additional compounds capable of producing enzyme-bound (formula; see text) reductant should inhibit dopamine beta-monooxygenase. Phenylacetaldehyde was chosen to test this model, since beta-hydroxyphenylacetaldehyde is expected to function as a reductant in a manner analogous to alpha-aminoacetophenone. Phenylacetaldehyde exhibits the properties of a mechanism-based inhibitor. Kinetic parameters are comparable to beta-chlorophenethylamine under both initial velocity and inactivation conditions. Since phenylacetaldehyde bears little resemblance to beta-chlorophenethylamine, its analogous inhibitory action provides support for an intramolecular redox reaction (via beta-hydroxyphenylacetaldehyde oxidation to a radical cation) in dopamine beta-monooxygenase inactivation. beta-Hydroxyphenylacetaldehyde was identified as the enzymatic product of phenylacetaldehyde turnover. As predicted, this product behaves both as a time-dependent inhibitor of dopamine beta-monooxygenase and as an electron donor in enzyme-catalyzed hydroxylation of tyramine to octopamine. Phenylacetamide and p-hydroxyphenylacetamide are also found to be mechanism-based inhibitors of dopamine beta-monooxygenase. In this case the product of hydroxylation (beta-hydroxyphenylacetamide) is redox inactive and, therefore, is unable to function as either a reductant or an inhibitor. Thus, mechanism-based inhibitors are divided into two types: type I, which undergoes hydroxylation prior to inactivation, and type II, which only requires hydrogen atom abstraction. A general mechanism for dopamine beta-monooxygenase inactivation is described, in which a common mechanistic radical intermediate is formed from both pathways.     

Continuous spectrophotometric assays for dopamine beta-monooxygenase based on two novel electron donors: N,N-dimethyl-1,4-phenylenediamine and 2-aminoascorbic acid.

Based on the novel chromophoric electron donors, N,N-dimethyl-1,4-phenylenediamine (DMPD) and 2-amino-2-deoxy-L-ascorbic acid (2-aminoascorbic acid), two sensitive, convenient, and continuous spectrophotometric assays for dopamine beta-monooxygenase (EC 1.14.17.1) are described. Both, DMPD and 2-aminoascorbic acid are kinetically and stoichiometrically well-behaved electron donors for dopamine beta-monooxygenase with kinetic parameters comparable to the most efficient physiological electron donor, ascorbic acid. During dopamine beta-monooxygenase turnover, DMPD is converted to its chromophoric cation radical which is stable under the standard assay conditions. The rate of the enzyme-dependent formation of DMPD cation radical under standard assay conditions could easily be followed at 515 nm with high accuracy and reproducibility. Similarly, dopamine beta-monooxygenase-mediated oxidation of 2-aminoascorbic acid results in the formation of the known, stable chromophoric product, 2,2'-nitrilodi-2(2')-deoxy-L-ascorbic acid (red pigment), which has a very strong absorption maximum at 385 nm. Both the above assays are superior to the existing assays in their convenience, reproducibility, and sensitivity for routine kinetic analysis of dopamine beta-monooxygenase and may be adopted as a simple color test for the enzyme. We propose that the above assays could also be adopted to design continuous and sensitive spectrophotometric assays for ascorbate oxidase, peptidyl alpha-amidating monooxygenase, and the chromaffin granule electron transport protein, cytochrome b561, due to their remarkable similarity to dopamine beta-monooxygenase in the chemistry of catalysis with regard to the electron donor.     

Inhibition of dopamine beta-hydroxylase by 2-mercapto-1-methylimidazole

The inhibition of bovine dopamine beta-hydroxylase (dopamine beta-monooxygenase, EC 1.14.17.1) by 2-mercapto-1-methylimidazole has been studied using a simple, 'metal-free' assay system. 2-Mercapto-1-methylimidazole is an uncompetitive inhibitor of dopamine beta-hydroxylase with respect to ascorbate and a mixed type of inhibitor with respect to tyramine. These findings are consistent with 2-mercapto-1-methylimidazole interacting exclusively with the reduced form of dopamine beta-hydroxylase.     

Activation of dopamine beta-monooxygenase by external and internal electron donors in resealed chromaffin granule ghosts

Membrane ghosts derived from chromaffin vesicles of bovine adrenal medullas have been used to examine the mechanism of reduction of dopamine beta-monooxygenase in its compartmentalized state. The rate of the dopamine beta-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 demonstrate a 2-3-fold increase in the turnover rate for ghosts 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 beta-monooxygenase, as required by models in which vesicular ascorbate behaves as enzyme reductant. Although there is sufficient catecholamine (endogenous plus 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 magnitude of this activation is found to be constant at all concentrations of internal ferrocyanide (both below and above saturation), implying that reductants on opposite sides of the membrane behave independently. Replacement of ascorbate by potassium ferrocyanide as external reductant leads to almost identical results, and we are able to rule out an inward transport of dehydroascorbate as the source of activation by external ascorbate. We conclude that external reductants are capable of reducing membrane-bound dopamine beta-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 beta-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.     

On the copper transfer between dopamine beta-monooxygenase and Cu-thionein

Metallothionein saturated with copper is able to donate copper to apodopamine beta-monooxygenase. The complete recovery of dopamine beta-monooxygenase activity is observed at the molar ratio Cu-thionein/apoenzyme of 25. On the other hand, apothionein accepts copper easily from the holoenzyme.     

Relation between the membrane and soluble forms of dopamine beta-monooxygenase

The quantitative ratio of membrane-bound and soluble forms of dopamine beta-monooxygenase from chromaffin granules obtained under different experimental conditions was determined. The amount of the membrane-bound form of dopamine beta-monooxygenase made up to no less than 60% of the total enzyme pool, when the granules were obtained and lyzed in the presence of pepstatin, phenylmethylsulfonyl fluoride, N-ethylmaleimide and catalase. In the absence of protectors practically all the enzyme can be obtained in the soluble form without detergent treatment. The effects of some ionic and nonionic detergents on the enzymatic activity of both forms of dopamine beta-monooxygenase were studied. No inhibition of dopamine beta-monooxygenase by 2% octyl glucoside or 1% Triton X-100 was observed. A comparative analysis of specific activities, subunit compositions, antigenic and physico-chemical properties of membrane-bound and soluble forms of dopamine beta-monooxygenase was carried out.     

Effects of dopamine beta-monooxygenase substrate analogs on ascorbate levels and norepinephrine synthesis in adrenal chromaffin granule ghosts

Chromaffin granule ghosts from bovine adrenal medullae have been used to investigate the effects of prototypic dopamine beta-monooxygenase substrate analogs of two distinct classes on intravesicular reduced ascorbic acid (AscH2) levels and on norepinephrine synthesis. Phenyl-2-aminoethyl sulfide (PAES), a sulfur-containing substrate, was shown to concentrate within ghosts, a process that was time and ATP dependent, but reserpine insensitive. Dopamine beta-monooxygenase oxygenation of PAES resulted in accumulation of the oxygenation product, PAESO, without affecting intravesicular levels of AscH2. Similarly, incubations of ghosts with phenyl-2-aminoethyl selenide (PAESe) also resulted in rapid, time- and ATP-dependent, but reserpine-insensitive uptake. However, oxygenation of PAESe by dopamine beta-monooxygenase within ghosts was found to cause a marked decrease in intravesicular AscH2, without buildup of the oxygenated product, phenyl 2-aminoethyl selenoxide. These results illustrate two basic differences between the consequences of PAES and PAESe turnover: while PAES accumulation proceeds concomitant with PAESO production and without AscH2 depletion, PAESe accumulation proceeds with a marked lowering of internal AscH2 but without observable product formation. Both PAES and PAESe were capable of competing with dopamine, the physiological substrate, for enzymatic oxygenation and/or vesicular uptake, and were capable of significantly reducing norepinephrine synthesis. In experiments where ghosts were preincubated with either PAES or PAESe with delayed addition of dopamine, it was clear that neither compound nor their oxygenated products interfered with electron transport via cytochrome b561. These results are consistent with the hypothesis that the physiological activity observed with both PAES and PAESe may be related to their ability to gain entrance to adrenergic neurons and decrease norepinephrine synthesis within neurotransmitter storage vesicles.     

Use of isotope effects to characterize intermediates in mechanism-based inactivation of dopamine beta-monooxygenase by beta-chlorophenethylamine

A mechanism for beta-chlorophenethylamine inhibition of dopamine beta-monooxygenase has been postulated in which bound alpha-aminoacetophenone is generated followed by an intramolecular redox reaction to yield a ketone-derived radical cation as the inhibitory species (Mangold, J.B., and Klinman, J.P. (1984) J. Biol. Chem. 259, 7772-7779). Based on the assumption that the ketone radical is the inhibitory intermediate, an analogous system was predicted and verified (Bossard, M.J., and Klinman, J.P. (1986) J. Biol. Chem. 261, 16421-16427). In the present study, the role of alpha-aminoacetophenone as the proposed intermediate in the inactivation by beta-chlorophenethylamine was examined in greater detail. From the interdependence of tyramine and alpha-aminoacetophenone concentrations, ketone inactivation is concluded to occur at the substrate site as opposed to potential binding at the reductant-binding site. Using beta-[2-1H]- and beta-[2-2H]chlorophenethylamine, the magnitude of the deuterium isotope effect on inactivation under second-order conditions has been found to be identical to that observed under catalytic turnover, D(kappa inact/Ki) = D(kappa cat/Km) = 6-7. By contrast, the isotope effect on inactivation under conditions of substrate and oxygen saturation, D kappa inact = 2, is 3-fold smaller than that seen on catalytic turnover, D kappa cat = 6. This reduced isotope effect for inactivation is attributed to a normal isotope effect on substrate hydroxylation followed by an inverse isotope effect on the partitioning of the enol of alpha-aminoacetophenone between oxidation to a radical cation versus protonation to regenerate ketone. These findings are unusual in that two isotopically sensitive steps are present in the inactivation pathway whereas only one is observable in turnover.     

Cloning and characterization of insect arylalkylamine N-acetyltransferase from Bombyx mori

Arylalkylamine N-acetyltransferase (AANAT) catalyzes N-acetylation of arylarkylamines. A cDNA of Bombyx mori insect AANAT (Bm-iAANAT) was found by searching an expressed-sequence tag (EST) database of B. mori (SilkBase). The cDNA encoded a 261 amino acid protein. The mRNA of Bm-iAANAT was expressed in eggs, larvae, adults and various tissues. Recombinant Bm-iAANAT protein was expressed in Sf9 cells by a baculovirus expression system. The AANAT activity of Bm-iAANAT was inhibited by high concentrations (over 0.01 mM) of tryptamine used as a substrate. The Bm-iAANAT acetylated tryptamine, serotonin, dopamine, octopamine, tyramine and norepinephrine. This is the first report of a cloned AANAT that acetylated norepinephrine. These results suggest that Bm-iAANAT is a novel member of insect AANAT family with unique kinetic properties and a broad substrate range.     

Rapid freeze- and chemical-quench studies of dopamine beta-monooxygenase: comparison of pre-steady-state and steady-state parameters

The copper-containing enzyme dopamine beta-monooxygenase has been studied with regard to pre-steady-state kinetics of tyramine hydroxylation and reduction of enzyme-bound Cu2+ by chemical- and freeze-quench EPR techniques. The bulk of the enzyme-bound copper (approximately 70%) is reduced in a single-exponential process with a limiting rate constant of 250 s-1, Km = 0.9 mM, consistent with participation of both copper ions in the redox events of catalysis. The remaining copper is reduced much more slowly (k approximately 2 s-1) or not at all, attributed to a distribution of copper into inhibitory binding sites and the presence of some inactive enzyme. Knowledge of the Cu2+ reduction rate, together with rate constants calculated from steady-state isotope effects [Miller, S. M., & Klinman, J. P. (1985) Biochemistry 24, 2114-2127], has allowed prediction of pre-steady-state product formation transients. Measurement of these transients under conditions of excess ascorbate shows close agreement with prediction, supporting the validity of individual rate constants obtained from steady-state data. Kinetic modeling shows further that the predominant steady-state enzyme form is the enzyme-product complex (E-P), which is expected to show a correspondingly large (approximately 70% of total copper) EPR signal for bound Cu2+. Surprisingly, the steady state is characterized by a low (19% of total copper) EPR signal. This lack of correlation between the anticipated and observed steady-state EPR signal suggests either antiferromagnetic coupling in binuclear copper centers or reduction of Cu2+ in this enzyme form by ascorbic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Semidehydroascorbic acid as an intermediate in norepinephrine biosynthesis in chromaffin granules.

We investigated whether semidehydroascorbic acid was an intermediate in norepinephrine synthesis in chromaffin granules and in electron transfer across the chromaffin granule membrane. Semidehydroascorbic acid was measured in intact granules by electron spin resonance. In the presence of intragranular but not extragranular ascorbic acid, semidehydroascorbic acid was formed within granules in direct relationship to dopamine beta-monooxygenase activity. However, semidehydroascorbic acid was not generated when granules were incubated with epinephrine instead of the substrate dopamine, with dopamine beta-monooxygenase inhibitors, without oxygen, and when intragranular ascorbic acid was depleted. Experiments using the impermeant paramagnetic broadening agents [K3 [Cr(C2O4)3].3H2O] and Ni(en)3(NO3)2 provided further evidence that semidehydroascorbic acid was generated only within granules. We also investigated semidehydroascorbic acid formation in the presence of intragranular and extragranular ascorbic acid. Under these conditions, semidehydroascorbic acid was formed on both sides of the granule membrane, and formation was coupled to dopamine beta-monooxygenase activity. These data indicate that dopamine beta-monooxygenase is reduced by single electron transfer from intragranular ascorbic acid, that transmembrane electron transfer occurs by single electron transfer, and that transmembrane electron transfer is directly coupled to formation of intragranular semidehydroascorbic acid via dopamine beta-monooxygenase activity.     

Ascorbic acid specifically enhances dopamine beta-monooxygenase activity in resting and stimulated chromaffin cells

Ascorbic acid enhancement of norepinephrine formation from tyrosine in cultured bovine chromaffin cells was characterized in detail as a model system for determining ascorbate requirements. In resting cells, ascorbic acid increased dopamine beta-monooxygenase activity without changing tyrosine 3-monooxygenase activity. [14C]Norepinephrine specific activity was increased by ascorbic acid, while [14C]dopamine specific activity was unchanged. Dopamine content, dopamine biosynthesis, tyrosine content, and tyrosine uptake were also unaffected by ascorbic acid. Furthermore, increased norepinephrine formation could not be attributed to changes in norepinephrine catabolism. Enhancement of dopamine beta-monooxygenase activity was specific for ascorbic acid, since other reducing agents with higher redox potentials were unable to increase norepinephrine formation. The specific effect of ascorbic acid on enhancement of norepinephrine formation was also observed in chromaffin cells stimulated to secrete with carbachol, acetylcholine, veratridine, and potassium chloride. In stimulated cells with and without ascorbate, there were no differences in dopamine content, tyrosine uptake, dopamine specific activity, and norepinephrine catabolism. These data indicate that, under a wide variety of conditions, only one catecholamine biosynthetic enzyme activity, dopamine beta-monooxygenase, is specifically stimulated by ascorbic acid alone in cultured chromaffin cells. This model system exemplifies a new approach for determining ascorbic acid requirements in cells and animals.     

Fluorescence studies on dopamine beta-monooxygenase: effects of salts, pH changes, metal-chelating agents and Cu2+

The intrinsic protein fluorescence of dopamine beta-monooxygenase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) has been characterized. The fluorescence is dominated by emission from tryptophans in a hydrophobic environment. Changes in the conformation of the enzyme induced by anions, pH changes, metal-chelating agents and Cu2+ have been determined. Conformational transitions induced by anions take place at concentrations between 0.05 and 0.2 M. Most anions give rise to a blue-shift, while ClO4- induces a red-shift of the emission spectrum. pH dependence of the protein fluorescence revealed a conformational change between pH 6.0 and 5.0. The interactions between dopamine beta-monooxygenase and seven different metal-chelating agents have been investigated using protein fluorescence, heat inactivation, and inhibition measurements. All the metal-chelating agents are able to remove the active-site copper as demonstrated by complete inhibition of enzyme activity, restoration of activity by the addition of copper, and the observation that the enzyme becomes more sensitive to heat inactivation in the presence of chelating agents, thus behaving similarly to the copper-free apoenzyme. The charge and size of the chelating agents are of importance for the reaction with the active-site copper, which is consistent with a mechanism for removal of the copper, including a ternary enzyme-copper chelating agent complex. By contrast, under turnover conditions in the presence of substrates, dissociation of the active-site copper and chelation of the free copper is a dominating mechanism. Three distinct conformations were characterized on the basis of the fluorescence spectra and the degree of quenching by Cu2+ and I-. For the copper-free apoenzyme a unique binding site for binding of the first copper was demonstrated by larger quenching of the protein fluorescence than for binding of additional copper.     

Characterization of alternate reductant binding and electron transfer in the dopamine beta-monooxygenase reaction

The steady-state limiting kinetic parameters Vmax, V/KDA, and V/KO2, together with deuterium isotope effects on these parameters, have been determined for the dopamine beta-monooxygenase (D beta M) reaction in the presence of structurally distinct reductants. The results show the one-electron reductant ferrocyanide to be nearly as kinetically competent as the presumed in vivo reductant ascorbate. Further, a reductant system of ferricyanide plus substrate dopamine yields steady-state kinetic parameters and isotope effects very similar to those measured solely in the presence of ferrocyanide, indicating a role for catecholamine in the rapid recycling of oxidized ferrocyanide. Use of substrate dopamine as the sole reductant is found to lead to a highly unusual kinetic independence of oxygen concentration, as well as significantly reduced values of Vmax and V/KDA, and we conclude that dopamine reduces enzymic copper in a rate-limiting step that is 40-fold slower than with ascorbate. The near-identical kinetic parameters measured in the presence of either ascorbate or ferrocyanide, together with markedly reduced rates with dopamine, are interpreted in terms of a binding site for reductant that is physically distinct from the substrate binding site. This view is supported by molecular modeling, which reveals ascorbate and ferrocyanide to possess an unexpected similarity in potential sites for interaction with enzymic residues. With regard to electron flux, identical values of V/KO2 have been measured with [2,2-2H2]dopamine as substrate both in the presence and in the absence of added ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of alternate substrates to probe the order of substrate addition to dopamine beta-hydroxylase

In order to determine the order of substrate binding to dopamine beta-hydroxylase during catalysis, the effect of alternate substrates upon kinetic parameters was examined. The V/K value for ascorbate was unchanged when tyramine, phenylpropylamine, p-Cl-phenethylamine, p-CH3O-phenethylamine, or phenethylamine was the hydroxylated substrate. The V/K values for tyramine and oxygen were similarly unchanged when ferrocyanide was used as the reductant in place of ascorbate. In order to use ferrocyanide as reductant it was necessary to include copper to alleviate the substrate inhibition seen with this substrate. The pattern of substrate inhibition observed with ferrocyanide was consistent with a small amount of free cyanide present in the ferrocyanide. With ferrocyanide as reductant and [2,2-2H2]tyramine as substrate, there was a measurable isotope effect on the V/K value for oxygen, but none on the values of Vmax or V/K for tyramine. These results are consistent with a ping-pong mechanism in which tyramine binds to the enzyme after the release of oxidized ascorbate. Subsequently, oxygen binds to form a ternary complex.     

N,N,N',N'-tetramethyl-1,4-phenylenediamine: a facile electron donor and chromophoric substrate for dopamine beta-monooxygenase

Dopamine beta-monooxygenase is shown to catalyze the oxidation of N,N,N',N'-tetramethyl-1,4-phenylenediamine (TMPD) to its cation radical in the presence of a regular substrate and molecular oxygen. The enzyme-mediated oxidation of TMPD is stoichiometrically coupled with the hydoxylation of the substrate to the corresponding enzymatic product. TMPD is kinetically well behaved as an alternate electron donor for the enzyme with a potency comparable to that of the most efficient electron donor, ascorbate. Dopamine beta-monooxygenase mediated oxidation of TMPD has been employed to design a convenient and sensitive spectrophotometric assay for the enzyme. The finding that TMPD is a well behaved facile alternate electron donor for dopamine beta-monooxygenase raises some interesting novel questions regarding the specificity and chemistry of the reduction site, which may have important implications on the reduction of active site coppers of the enzyme.     

Catecholamine sulfates: end products or metabolic intermediates?

Dopamine-beta-hydroxylase catalyzes the beta-oxidation of dopamine to noradrenaline while phenylethanolamine-N-methyltransferase converts noradrenaline to adrenaline. Since catecholamine sulfates represent the predominant form of catecholamines in human tissues, we have studied the role of dopamine sulfate and noradrenaline sulfate as alternate substrates for dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase, respectively. Dopamine 3-sulfate, dopamine 4-sulfate and noradrenaline 3-sulfate were chemically synthesized and exhaustively purified by ion-exchange chromatography. Dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase were partially purified from human adrenals. Using tyramine as substrate, dopamine-beta-hydroxylase is slightly inhibited by dopamine 3-sulfate according to some irreversible or mixed mechanisms. When dopamine-beta-hydroxylase was incubated with dopamine 3-sulfate or dopamine 4-sulfate, we were not able to find any synthesis of either noradrenaline sulfate or free noradrenaline. Using phenylethanolamine as substrate, the enzymatic activity of phenylethanolamine-N-methyltransferase remains unchanged with addition of dopamine 3-sulfate, dopamine 4-sulfate or noradrenaline 3-sulfate. It was concluded that dopamine sulfate is not an alternate substrate for either dopamine-beta-hydroxylase or phenylethanolamine-N-methyltransferase nor is noradrenaline 3-sulfate an alternate substrate for phenylethanolamine-N-methyltransferase.     

Demonstration of the ascorbate dependence of membrane-bound dopamine beta-monooxygenase in adrenal chromaffin granule ghosts

Chromaffin granule ghosts from bovine adrenal medullae have been used to examine the ability of membrane-bound dopamine beta-monooxygenase to interact directly with intravesicular ascorbate and to investigate vectorial electron transfer from external ascorbate across the ghost membrane. Ghosts prepared by a modification of published procedures were shown to be fully active in both dopamine uptake and norepinephrine production. Dopamine uptake is dependent on the presence of a magnesium and ATP ionic complex, is abolished by reserpine, and reaches a steady-state level in the presence of dopamine beta-monooxygenase, ascorbate, catalase, and fumarate. Omission of ascorbate either inside or outside the ghosts greatly enhances dopamine accumulation, which reaches levels of approximately 30 nmol/mg under these conditions. Correspondingly, in the presence of all components, norepinephrine production reached approximately 100 nmol/mg in 30 min of incubation. Norepinephrine production was strictly magnesium-ATP-dependent, inhibited by either reserpine or dopamine beta-monooxygenase inactivation, and was markedly reduced when ascorbate was omitted from either inside or outside the ghosts. In the presence of limiting amounts of internal ascorbate, rapid norepinephrine production occurred which corresponded to the amount of initial ascorbate present, followed by a much slower endogenous norepinephrine production observable after complete depletion of internal ascorbate. The endogenous rate of norepinephrine production likely represents epinephrine-supported dopamine beta-monooxygenase turnover. Taken together, the data demonstrate that facile norepinephrine production by membrane-bound dopamine beta-monooxygenase occurs only when internal ascorbate is present, terminates upon depletion of internal ascorbate, and can only be sustained at a significant rate when reducing equivalents from external ascorbate are available.