Ascorbic acid within chromaffin granules. In situ kinetics of norepinephrine biosynthesis

Ascorbic acid requirements for norepinephrine biosynthesis were investigated in intact bovine chromaffin granules using the physiologic substrate dopamine and a novel coulometric electrochemical detection high pressure liquid chromatography system for ascorbic acid. 10 mM external dopamine, 1 mM Mg-ATP, and 1 mM ascorbic acid produced maximal norepinephrine biosynthesis without granule lysis. When external ascorbic acid was omitted, intragranular ascorbic acid was consumed in a 1:1 ratio with respect to norepinephrine biosynthesis. The initial concentration of intragranular ascorbic acid was 10.5 mM, which was depleted in stepwise fashion to 15 lower concentrations over the range of 9.2-0.2 mM. Chromaffin granules containing these varying concentrations of intragranular ascorbic acid were then incubated with 1 mM exogenous ascorbic acid, and norepinephrine biosynthesis from dopamine was determined. The apparent Km of norepinephrine biosynthesis for intragranular ascorbic acid was 0.57 mM by Eadie-Hofstee analysis and 0.68 mM by Lineweaver-Burk analysis. These data indicate that intragranular ascorbic acid is available and required for norepinephrine biosynthesis, that ascorbic acid is a true co-substrate for dopamine beta-monooxygenase, and that intragranular ascorbic acid is maintained by extragranular ascorbic acid. Continued norepinephrine biosynthesis in granules is dependent on both intragranular and extragranular concentrations of the vitamin. Furthermore, in situ kinetics of dopamine beta-monooxygenase for ascorbic acid may be most accurately determined using intact granules and the true physiologic substrate.     

Ascorbic acid and Mg-ATP co-regulate dopamine beta-monooxygenase activity in intact chromaffin granules

Ascorbic acid and Mg-ATP were found to regulate norepinephrine biosynthesis in intact secretory vesicles synergistically and specifically, using the model system of isolated bovine chromaffin granules. Dopamine uptake into chromaffin granules was shown to be unrelated to the presence of Mg-ATP and ascorbic acid at external dopamine concentrations of 7.5 and 10 mM. Under these conditions of dopamine uptake, norepinephrine biosynthesis was enhanced 5-6-fold by Mg-ATP and ascorbic acid compared to control experiments with dopamine only. Furthermore, norepinephrine formation was enhanced approximately 3-fold by ascorbic acid and Mg-ATP together compared to norepinephrine formation in granules incubated with either substance alone. The action of Mg-ATP and ascorbic acid together was synergistic and independent of dopamine content of chromaffin granules as well as of dopamine uptake. The apparent Km of norepinephrine formation for external ascorbic acid was 376 microM and for external Mg-ATP was 132 microM, consistent with the larger amounts of cytosolic ascorbic acid and ATP that are available to chromaffin granules. Other physiologic reducing agents were not able to increase norepinephrine biosynthesis in the presence or absence of Mg-ATP. In addition, maximum enhancement of norepinephrine biosynthesis occurred only with the nucleotide ATP and the cation magnesium. The mechanism of the effect of ascorbic acid and Mg-ATP on norepinephrine biosynthesis was investigated and appeared to be independent of a positive membrane potential. The effect was also not mediated by direct action of ADP, ATP, or magnesium on the activity of soluble or particulate dopamine beta-monooxygenase. These data indicate that Mg-ATP and ascorbic acid specifically and synergistically co-regulate dopamine beta-monooxygenase activity in intact chromaffin granules, independent of substrate uptake. Although the mechanism is not known, the data are consistent with the possibility that the chromaffin granule ATPase mediates these effects.     

Ascorbic acid transport and accumulation in human neutrophils

The transport, accumulation, and distribution of ascorbic acid were investigated in isolated human neutrophils utilizing a new ascorbic acid assay, which combined the techniques of high performance liquid chromatography and coulometric electrochemical detection. Freshly isolated human neutrophils contained 1.0-1.4 mM ascorbic acid, which was localized greater than or equal to 94% to the cytosol, was not protein bound, and was present only as ascorbic acid and not as dehydroascorbic acid. Upon addition of ascorbic acid to the extracellular medium in physiologic amounts, ascorbic acid was accumulated in neutrophils in millimolar concentrations. Accumulation was mediated by a high affinity and a low affinity transporter; both transporters were responsible for maintenance of concentration gradients as large as 50-fold. The high affinity transporter had an apparent Km of 2-5 microns by Lineweaver-Burk and Eadie-Hofstee analyses, and the low affinity transporter had an apparent Km of 6-7 mM by similar analyses. Each transporter was saturable and temperature dependent. In normal human blood the high affinity transporter should be saturated, whereas the low affinity transporter should be in its linear phase of uptake.     

Ascorbic acid regulation of norepinephrine biosynthesis in isolated chromaffin granules from bovine adrenal medulla

The effect of ascorbic acid on the conversion of dopamine to norepinephrine was investigated in isolated chromaffin granules from bovine adrenal medulla. Ascorbic acid was shown to double the rate of [3H]norepinephrine formation from [3H]dopamine, despite no demonstrable accumulation of ascorbic acid into chromaffin granules. The enhancement of norepinephrine biosynthesis by ascorbic acid was dependent on the external concentrations of dopamine and ascorbate. The apparent Km of the dopamine beta-hydroxylation system for external dopamine was approximately 20 microM in the presence or absence of ascorbic acid. However, the apparent maximum velocity of norepinephrine formation was nearly doubled in the presence of ascorbic acid. By contrast, the apparent Km and Vmax of dopamine uptake into chromaffin granules were not affected by ascorbic acid. Norepinephrine formation was increased by ascorbic acid when the concentration of ascorbate was 200 microM or higher; a concentration of 2 mM appeared to induce the maximal effect under the experimental conditions used here. The effect of ascorbic acid on conversion of dopamine to norepinephrine required Mg-ATP-dependent dopamine uptake into chromaffin granules. In contrast to ascorbic acid, other reducing agents such as NADH, glutathione, and homocysteine were unable to enhance norepinephrine biosynthesis. These data suggest that ascorbic acid provides reducing equivalents for hydroxylation of dopamine despite the lack of ascorbate accumulation into chromaffin granules. These findings imply the functional existence of an electron carrier system in the chromaffin granule which transfers electrons from external ascorbic acid for subsequent intragranular norepinephrine biosynthesis.     

Enhancement of norepinephrine biosynthesis by ascorbic acid in cultured bovine chromaffin cells

Ascorbic acid donates electrons to dopamine beta-monooxygenase during the hydroxylation of dopamine to norepinephrine in vitro. However, the possible role of ascorbic acid in norepinephrine biosynthesis in vivo has not been defined. We therefore investigated the effect of newly accumulated ascorbic acid on catecholamine biosynthesis in cultured bovine adrenal chromaffin cells. Cells supplemented for 3 h with ascorbic acid accumulated 9-fold more ascorbic acid than found in control cells. Under these conditions, the cells loaded with ascorbate were found to double the rate of norepinephrine biosynthesis from [14C]tyrosine compared to control. By contrast, the amounts present of [14C] 3,4-dihydroxyphenylalanine and [14C]dopamine synthesized from [14C]tyrosine were unaffected by the preloading of ascorbic acid. Ascorbate preloaded cells incubated with [3H]dopamine also showed a similar increase in the rate of norepinephrine formation, without any change in dopamine transport into the cells. Thus, these data were consistent with ascorbate action at the dopamine beta-monooxygenase step. In order to determine if ascorbate could interact directly with dopamine beta-monooxygenase localized within chromaffin granules, we studied whether isolated chromaffin granules could accumulate ascorbic acid. Ascorbic acid was not transported into chromaffin granules by an uptake or exchange process, despite coincident [3H]dopamine uptake which was Mg-ATP dependent. These data indicate that ascorbic acid does augment norepinephrine biosynthesis in intact chromaffin cells, but by a mechanism that might enhance the rate of dopamine hydroxylation indirectly.     

Improved assay of reaction products to quantitate catechol-O-methyltransferase activity by high-performance liquid chromatography with electrochemical detection

We applied coulometric detection (three electrochemical electrodes in series) to quantitate vanillic acid and isovanillic acid using reversed-phase HPLC. The formation of these reaction products from dihydroxybenzoic acid was used as a precise and reproducible measure of catechol-O-methyltransferase (COMT) activity in striatal homogenates and recombinant membrane-bound COMT protein. This detection system has a higher sensitivity (0.5 pmol per injection) than a single-cell amperometric detection. As in a previous method, the deproteinized supernatants of the COMT assay could be injected directly onto the HPLC system allowing the handling of a large number of samples in one day.     

Carbachol induced release of diadenosine polyphosphates--Ap4A and Ap5A--from perfused bovine adrenal medulla and isolated chromaffin cells.

The diadenosine polyphosphates--Ap4A and Ap5A--were released from perfused bovine adrenal glands and recently isolated chromaffin cells by the action of carbachol. The H.P.L.C. technique reported here allowed the quantification of pmol amounts of these compounds present in biological samples from the perfusion media after stimulation. Both compounds (Ap4A and Ap5A) were identified by the retention time in H.P.L.C. chromatography, co-elution with standards, re-chromatography and destruction by the phosphodiesterase action. Bovine adrenal glands stimulated with 100 microM carbachol released 0.47 +/- 0.12 nmol/gland of Ap4A and 1.11 +/- 0.26 nmol/gland of Ap5A. Isolated bovine chromaffin cells after 100 microM carbachol, as secretagogue, released 11.1 +/- 0.8 pmol/10(6) cells of Ap4A and 15.8 +/- 1.1 pmol/10(6) cells of Ap5A. The ratio of these compounds with respect to the exocytotically released ATP and catecholamines was in the same order as that found in isolated chromaffin granules.     

Stimulatory Effect of Ascorbic Acid on Norepinephrine Biosynthesis in Digitonin-Permeabilized Adrenal Medullary Chromaffin Cells

The regulatory role of ascorbic acid in norepinephrine biosynthesis was studied using digitonin-permeabilized chromaffin cells. When permeabilized chromaffin cells were incubated with [3H]3,4-dihydroxyphenylethylamine ([3H]dopamine) in calcium-free medium, the amounts of radioactive dopamine and norepinephrine measured in the cell fraction were increased as a function of incubation time and dopamine concentration. Both the accumulation of dopamine and the formation of norepinephrine were shown to require the presence of Mg-ATP in the medium. These results indicate that the permeabilization of chromaffin cells by digitonin treatment does not disrupt the functions of chromaffin granules, including dopamine uptake, norepinephrine formation, and storage of these amines. Using this permeabilized cell system, the effect of ascorbic acid on the rates of dopamine uptake and hydroxylation was investigated. The formation of norepinephrine was stimulated by ascorbic acid at concentrations of 0.5-2 mM in the presence of Mg-ATP. By contrast, dopamine uptake was not affected by the presence or absence of ascorbic acid in the medium. These findings provide evidence that ascorbic acid may stimulate the conversion of dopamine to norepinephrine by increasing dopamine beta monooxygenase activity rather than by increasing the substrate supply of dopamine. These observations also suggest that the rate of norepinephrine biosynthesis in adrenal medullary cells may be regulated by the concentration of ascorbic acid within the cell cytoplasm.     

A specific high-performance liquid chromatography assay for dehydroascorbic acid shows an increased content in CLL lymphocytes

A method for the assay of dehydroascorbic acid using high-performance liquid chromatography with uv detection is described. The dehydroascorbic acid is separated from ascorbic acid and reduced with dithiothreitol, and is then quantitated as ascorbic acid following rechromatography. Since as little as 22 pmol can be detected, sensitivity is at least 40-fold greater than that of other currently available procedures. This method was used to measure the level of dehydroascorbic acid in normal and chronic lymphocytic leukemia lymphocytes. A significantly higher concentration of dehydroascorbic acid was found in leukemic (21.80 +/- 3.55 nmol/10(8) cells, mean +/- SE) than in normal lymphocytes (9.32 +/- 1.15 nmol/10(8) cells) (P less than 0.03). Analysis of extracts from normal B cell lymphocytes revealed comparable dehydroascorbic acid levels to unfractionated lymphocytes, indicating that the elevated level in chronic lymphocytic leukemia was not simply a reflection of the increased percentage of B lymphocytes in this disorder. These studies illustrate that the technique can be used to measure the dehydroascorbic acid content from sources where only scanty material is available or low levels are found.     

Simultaneous determination of uric and ascorbic acids in human serum by reversed-phase high-performance liquid chromatography with electrochemical detection

A rapid, easy, and accurate method for the determination of uric acid and ascorbic acid in human serum by reversed-phase high-performance liquid chromatography with electrochemical detection has been developed. Human serum (0.5 ml) was mixed with 1.5 ml of an aqueous solution containing 2.0% metaphosphoric acid and the mixture was centrifuged at 3000g for 30 min. The supernatant was passed through a membrane filter to remove the particulate matter. Ten microliters of the filtrate was injected into the chromatographic system employed in this study. Complete separation of uric acid and ascorbic acid was achieved in about 2 min. The assay limit for quantitation was about 10 pg for uric acid and ascorbic acid under the present chromatographic conditions. The analytical recoveries of uric acid and ascorbic acid in human serum samples were found to be almost 100%.     

Uptake of Nucleotides and Catecholamines by Chromaffin Granules from Pig and Horse Adrenal Medulla

Abstract: The uptake of nucleotides and Catecholamines into chromaffin granules from adrenals of pigs and horses is similar to that previously seen in bovine chromaffin granules. The rate of [³H]ATP uptake at 2 mM-ATP concentration was 0.42 ± 0.06 and 0.15 ± 0.02 nmol/mg protein/min for pig and horse granules, respectively. The apparent K_m's were 1.37 mM for pig granules, 0.89 mM for horse granules, and 1.2 mM for ox granules. The sensitivity of the uptake for nucleotides and catecholamine to specific inhibitors was found to be similar in granules from pig and ox, indicating that the same mechanisms of uptake are involved in both species.     

IMMUNOLOGICAL STUDIES ON A MEMBRANE PROTEIN (CHROMOMEMBRIN B) OF CATECHOLAMINE-STORING VESICLES

Abstract— Rabbits were immunized with chromomembrin B, i.e. a membrane protein isolated from chromaffin granules of bovine adrenal medulla. When the rabbit sera were tested by immunodiffusion in the presence of various detergents, only negative results were obtained, whereas with complement fixation antibodies could be demonstrated. With this method the subcellular distribution of chromomembrin B in bovine adrenal medulla was determined. The results demonstrate that this protein is specifically localized in the membranes of chromaffin granules. In the mitochondrial and microsomal fractions it is present only in small amounts which are attributable to a contamination of these fractions with chromaffin granules. The subcellular distribution of chromomembrin R in bovine splenic nerves indicates that this antigen is also found in the membranes of noradrenalinestoring vesicles of sympathetic nerve. Chromomembrin B or a related antigen was detected in chromaffin grades isolated from pig and rat adrenal and in those isolated from a human phaeochromocytoma. It is also present in total membranes obtained from posterior and anterior hypophysis, but it is absent from membranes isolated from parotid gland, liver and adrenal cortex. This paper illustrates how a membrane protein which requires detergents for its solubilization can be characterized and measured by immunological methods.     

The in situ kinetics of dopamine beta-hydroxylase in bovine adrenomedullary chromaffin cells. Intravesicular compartmentation reduces apparent affinity for the cofactor ascorbate

The Km of dopamine beta-hydroxylase for its cofactor, ascorbic acid, was determined in situ in primary cultures of bovine adrenomedullary chromaffin cells and in isolated chromaffin vesicles. A range of intravesicular ascorbate concentrations in chromaffin cell cultures (1.1-31.2 mM) was achieved by varying the number and concentration of ascorbate additions to the culture media. The rate of octopamine synthesis from tyramine displayed a Michaelis-Menten relationship with respect to ascorbate concentration and an apparent Km of dopamine beta-hydroxylase for ascorbate of 15.0 +/- 2.0 mM was determined. In isolated chromaffin vesicles, with an initial intravesicular ascorbate concentration of approximately 10 mM, ascorbate consumption during beta-hydroxylation occurred as a first order process. This indicated that dopamine beta-hydroxylase was not saturated at this initial ascorbate concentration. When isolated chromaffin vesicles were prepared with different intravesicular ascorbate concentrations, the rate of octopamine synthesis displayed a Michaelis-Menten relationship with respect to ascorbate with an apparent Km of 17.0 +/- 5.0 mM. Ascorbate consumption also occurred as a first order process in ascorbate-loaded chromaffin-vesicle ghosts which had initial ascorbate concentrations of approximately 30 mM but which were depleted of other small molecules such as catecholamines. These results indicate that the in situ Km of dopamine beta-hydroxylase for ascorbate (approximately 15 mM) is 25-fold higher than it is for the purified or partially purified enzyme assayed under optimal conditions in vitro (0.6 mM). The factor(s) which decreases the enzyme affinity for ascorbate, relative to in vitro, resides in the chromaffin vesicle interior and is also retained in chromaffin-vesicle ghosts. The mechanism of this effect remains to be determined. The Km value determined in these experiments is close to the estimated intravesicular ascorbate concentration of bovine chromaffin granules in vivo (4), suggesting that the availability of ascorbate could become a factor in regulating the rate of dopamine beta-hydroxylation.     

Complex Carbohydrate Composition of Large Dense-Cored Vesicles from Sympathetic Nerve

Highly purified noradrenergic, large, dense-cored vesicles were isolated from bovine sympathetic nerve endings by sucrose-D2O density gradient centrifugation. Their concentration of glycoprotein hexosamine and sialic acid was 6.6 and 3.9 mumol/100 mg lipid-free dry weight, respectively, values which are similar to those previously found in bovine chromaffin granules. However, whereas chromaffin granule glycoproteins are characterized by their high proportion of N-acetylgalactosamine-containing O-glycosidically-linked oligosaccharides (present in the chromogranins), such oligosaccharides accounted for only 17% of those in noradrenergic synaptic vesicle glycoproteins. Fractionation of N-3H-acetylated glycopeptides by sequential lectin affinity chromatography demonstrated that approximately two-thirds of the oligosaccharides were of the tri- and tetraantennary complex type, accompanied by 14% biantennary oligosaccharides and 3% high-mannose oligosaccharides. The vesicles had a relatively low concentration of chondroitin sulfate (less than 5% of that in chromaffin granules) but significant amounts of heparan sulfate (0.4 mumol N-acetylglucosamine/100 mg lipid-free dry weight). No hyaluronic acid was detected. The concentration of ganglioside sialic acid in the noradrenergic vesicles was approximately 1 mumol/100 mg lipid-free dry weight, which is significantly higher than that of a crude membrane mixture from which the vesicles were prepared; the ratio of N-acetyl- to N-glycolylneuraminic acid was 0.8. Several molecular species of gangliosides were detected by thin-layer chromatography, but most of these did not exactly comigrate with bovine brain gangliosides. Cholera toxin binding indicated that approximately half or less of the gangliosides belong to the gangliotetraose series.     

Inhibition of ascorbic acid transport in human neutrophils by glucose.

Because of the structural similarity between glucose and ascorbic acid, we investigated the effect of glucose on uptake and accumulation of ascorbic acid in isolated normal human neutrophils. Ascorbic acid accumulation was determined using high-performance liquid chromatography with coulometric electrochemical detection, in conjunction with liquid scintillation spectrometry. Ascorbic acid accumulation in neutrophils is mediated by a high and a low affinity transport activity. In neutrophils from different volunteers, glucose inhibited uptake and accumulation of ascorbic acid by both transport activities 3-9-fold. The mechanism of inhibition was different for each transport activity: inhibition of the high affinity transport activity was noncompetitive, while inhibition of the low affinity activity was competitive. Glucose-induced inhibition of both ascorbic acid transport activities occurred in neutrophils of all donors tested and was fully reversible. Although the mechanism of ascorbic acid accumulation appeared to be different than that for glucose transport, other monosaccharides and glucose transport inhibitors also inhibited ascorbic acid accumulation. These are the first data to suggest that ascorbic acid accumulation in neutrophils can be regulated by compounds of similar structure.     

Bombesin-like immunoreactivity in bovine adrenal medulla

The presence of immunoreactive (ir)-bombesin in bovine adrenal medulla, isolated adrenal chromaffin cells and subcellular fractions of the adrenal medulla was demonstrated using a specific antibody to the synthetic peptide. High levels of ir-bombesin were detected in acid (HCl) extracts of the adrenal tissue (27 pmol/g) and isolated cells (0.35 pmol per 10(6) cells). Subpopulations of adrenal chromaffin cells were also obtained by centrifugation of the original cell preparation through a stepwise bovine serum albumin gradient (cell layers I, II and III). The highest concentration of ir-bombesin (0.77 pmol/10(6) cells) was found in a cell population (cell layer I) enriched in noradrenaline (adrenaline/noradrenaline ratio of 0.6). At the subcellular level, ir-bombesin was mainly concentrated in the secretory granules (0.61 pmol/mg protein) along with catecholamines (1097 nmol/mg protein), but a relatively high concentration of ir-bombesin (0.26 pmol/mg protein) was also found in the microsomal fraction. Isolation and high performance liquid chromatography (HPLC) analysis of adrenomedullary ir-bombesin revealed the presence of four molecular forms, one of them corresponding to gastrin releasing peptide (GRP), another one (major peak) eluting closely to synthetic neuromedin B and another one coeluting with GRP-(18-27). HPLC analysis of the molecular forms of ir-bombesin in the microsomes and secretory granules indicated that GRP- and neuromedin B-like materials can be generated between the two fractions.     

Sensitive electrochemical detection method for alpha-acids, beta-acids and xanthohumol in hops (Humulus lupulus L.)

A new HPLC method with coulometric detection for the quantification of xanthohumol, alpha-acids and beta-acids in hops was developed. The separation of compounds was accomplished with a C18 column and isocratic elution with methanol: 50 mM potassium phosphate: ortho-phosphoric acid=80:20:0.25 (v/v/v). The method was validated and UV and electrochemical detectors (ECD) were compared. The HPLC method with ECD was precise, accurate and very sensitive for detection of xanthohumol and alpha- and beta-acids. The detection limits of analytes were at least 8.8 to 24 times lower with ECD than those of the UV detector. The ECD method was successfully applied for quantification of studied compounds in hop pellets. The concentrations of all compounds obtained with ECD and UV were found to be equivalent. This is the first study demonstrating a very sensitive and validated method for the quantification of xanthohumol, alpha- or beta-acids in hop samples with the use of the electrochemical detector.     

Electrochemical characterization of repaglinide and its determination in human plasma using liquid chromatography with dual-channel coulometric detection

A simple, fast and sensitive HPLC method employing dual-channel coulometric detection for the determination of repaglinide in human plasma is presented. The assay involved extraction of repaglinide by ethyl acetate and isocratic reversed-phase liquid chromatography with dual-channel coulometric detection. The mobile phase composition was 50mM disodium hydrogen phosphate/acetonitrile (60:40, v/v), pH of the mobile phase 7.5 set up with phosphoric acid. For all analyses, the first cell working potential was +380mV, the second was +750mV (vs. Pd/H(2)). Calibration curve was linear over the concentration range of 5-500nmolL(-1). Rosiglitazone was used as an internal standard. The limit of detection (LOD) was established at 2.8nmolL(-1), and the lower limit of quantification (LLOQ) at 8.5nmolL(-1). The developed method was applied to human plasma samples spiked with repaglinide at therapeutical concentrations. It was confirmed that the method is suitable for pharmacokinetic studies or therapeutic monitoring.     

Specificity and properties of the nucleotide carrier in chromaffin granules from bovine adrenal medulla.

1. The influence of various substances on the uptake of [3H]ATP and [14C]-noradrenaline into isolated bovine chromaffin granules was investigated. The carrier-mediated [3H]ATP uptake is specifically inhibited by SO42-, PO43- and phosphoenolpyruvate. Compounds with carboxylic acid or sulphonic acid groups had no significant inhibitory effects on either uptake. 2. 35SO42-, 32PO43- and phosphoenol[14C]pyruvate are taken up into chromaffin granules by a temperature-dependent process that is inhibited by atractyloside, uncouplers of oxidative phosphorylation and lipid-permeant anions. The apparent Km of 35SO42- uptake is 0.4 mM. 3. These results indicate that the nucleotide carrier in chromaffin granules has a broad specificity, transporting compounds with two strong negative charges. 4. Amino acid probes influence the uptake of ATP and catecholamines differently. Pyridoxal phosphate inhibits both uptake processes, 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid preferentially blocks ATP uptake, whereas phenylglyoxal blocks only ATP transport. It is suggested that the nucleotide carrier possesses arginine residues in a functionally important position. 5. The significance of these results obtained on isolated granules for the function of chromaffin granules within the cell is discussed.     

Role of Mg-ATP in Norepinephrine Biosynthesis in Intact Chromaffin Granules

Abstract: Dopamine β-mdriooxygenase converts dopamine to norepinephrine in intact chromaffin granules using intragranular ascorbic acid as a cosubstrate. Mg-ATP with external ascorbic acid is required for maximal norepinephrine biosynthesis. Mechanisms to explain these requirements were investigated specifically using intact granules. The effect of Mg-ATP was independent of membrane potential (ΔΨ) because norepinephrine biosynthesis was unchanged whether ΔΨ was positive or collapsed. Furthermore, the effect of Mg-ATP was independent of absolute intragranular and extragranular pH as well as the pH difference across the chromaffin granule membrane (ΔpH). Nevertheless, norepinephrine biosynthesis was inhibited by N -ethylmaleimide, 4-chloro-7-nitrobenzofurazane, and N , N -dicyclohexylcarbodiimide, specific inhibitors of the secretory vesicle ATPase that may directly affect proton pumping. Biosynthesis occurred normally with other ATPase inhibitors that do not inhibit the ATPase in secretory vesicles. The data indicate that the effect of Mg-ATP with ascorbic acid is mediated by the granule membrane ATPase but independent of maintaining ΔΨ and ApH. An explanation of these findings is that Mg-ATP, via the granule ATPase, may change the rate at which protons or dopamine are made available to dopamine β-monooxygenase.