Isolation and reconstitution of the membrane-bound form of dopamine beta-hydroxylase

Dopamine beta-hydroxylase is present in the bovine adrenal medulla in two forms, soluble and membrane bound. Previous isolation procedures for the membranous hydroxylase have resulted in a form of enzyme identical in subunit structure with the soluble type. We report here the isolation of a membrane-bound form of dopamine beta-hydroxylase which is structurally different from the soluble form. The isolated membranous enzyme has a large apparent molecular weight on gel filtration, is amphiphilic, and contains bound phospholipid which is predominantly phosphatidylserine. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows that the membranous hydroxylase contains two nonidentical subunits under both reducing and nonreducing conditions. Under reducing conditions the apparent molecular weights of the two subunits are 70,000 and 75,000 and both contain carbohydrate. The purified membranous hydroxylase binds to phospholipid vesicles and chymotryptic digestion of the bound enzyme suggests that two forms of the membranous hydroxylase exist.     

The membrane-binding segment of dopamine beta-hydroxylase is not an uncleaved signal sequence

Dopamine beta-hydroxylase exists in bovine adrenal medulla chromaffin granules in both soluble and membrane-bound forms. The mechanism by which membranous dopamine beta-hydroxylase is bound to granule membranes has been elusive. Recently, evidence that covalently attached phosphatidylinositol does not serve as an anchor for membranous dopamine beta-hydroxylase was reported (Stewart, L. C., and Klinman, J. P. (1988) J. Biol. Chem. 263, 12183-12186). It was suggested that an uncleaved signal sequence could serve as a mode of attachment for the membrane-bound hydroxylase. Amino-terminal sequence analysis of purified bovine membranous dopamine beta-hydroxylase demonstrates that this form of the enzyme possesses an amino-terminal sequence similar to the soluble enzyme. Additionally, the 75- and 72-kDa bands of membranous dopamine beta-hydroxylase were electrophoretically eluted from a preparative sodium dodecyl sulfate-polyacrylamide gel and sequenced. Both bands had the amino-terminal sequence characteristic of the soluble bovine enzyme. These sequence results eliminate the possibility that an uncleaved signal sequence serves as the membrane anchor.     

Multiple forms of human dopamine beta-hydroxylase in SH-SY5Y neuroblastoma cells

Dopamine beta-hydroxylase exists as three forms in human neuroblastoma (SH-SY5Y) cells. The membrane-bound form of the hydroxylase contains three different species with apparent relative molecular weights of 73,000, 77,000, and 82,000. The intracellular soluble form of dopamine beta-hydroxylase was present as a single species with an apparent molecular weight of 73,000. Pulse-chase experiments showed that membranous dopamine beta-hydroxylase contains two subunit forms of 73,000 and 77,000 after short chase times. The soluble hydroxylase was synthesized as a single species of 73,000 at approximately the same rate as the lower molecular weight species of the membranous enzyme. A constitutively secreted third form of the enzyme with an intermediate apparent molecular weight also incorporated [35S]sulfate, whereas no significant amount of [35S]sulfate was observed in the cellular forms of the enzyme. The [35S]sulfate was incorporated on N-linked oligosaccharides. Approximately 12% of the enzyme is released constitutively within 1 h. These results demonstrate that neuronal cells have the ability to constitutively secrete a specific form of dopamine beta-hydroxylase which may contribute to the levels of this enzyme found in plasma.     

Subunit exchange between membranous and soluble forms of bovine dopamine beta-hydroxylase

Dopamine beta-hydroxylase is present in the bovine adrenal medulla in two forms: soluble and membrane-bound. In a previous study, it was shown that the tetrameric, soluble form of the enzyme undergoes dissociation into two identical dimeric subunits and that this subunit dissociation is dependent on pH and ADP binding (Dhawan, S., Hensley, P., Osborne, J. C., Jr., and Fleming, P. J. (1986) J. Biol. Chem. 261, 7680-7684). Here we report the effect of pH and ADP on the dissociation of the membranous form of dopamine beta-hydroxylase into two nonidentical subunits. Negative stain electron microscopy of purified membranous hydroxylase showed largely tetrameric species together with occasional dimeric species. The tetrameric images of membranous hydroxylase were similar to, but clearly different from, previously published negative stain images of soluble hydroxylase (Duong, L. T., Fleming, P. J., and Ornberg, R. L. (1985) J. Biol. Chem. 260, 2393-2398). Quantitative binding of ADP to the membranous hydroxylase revealed the existence of two binding sites per dimeric subunit. ADP binding and low pH both promote dissociation of a hydrophilic, catalytically active subunit from the membranous enzyme reconstituted onto phospholipid vesicles. Kinetic analyses of reconstituted membranous hydroxylase activity were consistent with the existence of tetrameric and dimeric catalytic species in equilibrium. All of the hydrophilic subunits of the purified soluble hydroxylase bind to the hydrophobic subunits of the reconstituted membranous hydroxylase. We propose that, in the chromaffin granules, the soluble hydroxylase subunits are in equilibrium association with the membrane-bound hydroxylase subunits and that the hydrophilic subunits of both soluble and membranous hydroxylase are identical.     

Sulfation and constitutive secretion of dopamine beta-hydroxylase from rat pheochromocytoma (PC12) cells

The biosynthesis and secretion of dopamine beta-hydroxylase were investigated by radiolabeling rat pheochromocytoma (PC12) cells in culture. Intracellular dopamine beta-hydroxylase from a crude chromaffin vesicle fraction and secreted dopamine beta-hydroxylase from culture medium were immunoprecipitated using antiserum made against purified bovine soluble dopamine beta-hydroxylase. Analysis of the immunoprecipitated enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that: 1) the membrane-bound form of the hydroxylase from crude secretory vesicle membrane extracts contained two nonidentical subunits in approximately stoichiometric amounts (Mr = 77,000 and 73,000); 2) the soluble hydroxylase from the lysate of these secretory vesicles was composed predominantly of a single subunit (Mr = 73,000); and 3) the hydroxylase secreted into the medium under resting conditions was also composed of a single subunit (approximate Mr = 73,000). All subunits of the multiple forms of hydroxylase were glycoproteins. Under resting conditions, the rate of secretion of hydroxylase was approximately 6% of total cellular enzyme/15 min. The secreted form of the hydroxylase incorporated [35S]sulfate, whereas no significant [35S]sulfate was incorporated into the cellular forms of enzyme. We propose that in addition to the dopamine beta-hydroxylase which is found in catecholamine storage vesicles and released during stimulus-coupled exocytosis, PC12 cells also have a constitutive secretory pathway for dopamine beta-hydroxylase and that the enzyme released by this second pathway is sulfated.     

The pH-dependent subunit dissociation and catalytic activity of bovine dopamine beta-hydroxylase

The soluble form of dopamine beta-hydroxylase from bovine adrenal medulla has previously been shown to exist as a tetrameric species of Mr = 290,000 composed of two disulfide-linked dimers. Here we report that this enzyme can also undergo a reversible tetramerdimer dissociation which is dependent on pH. Gel permeation chromatography of dopamine beta-hydroxylase at pH 5.0 demonstrates a Stokes radius of 5.8 nm. When the pH is shifted to 5.7, the Stokes radius changes to 6.9 nm. Sedimentation equilibrium analysis of the purified enzyme demonstrates that this change in molecular size is due to a change in molecular weight. At low protein concentration, the estimated Mr of the enzyme is 145,000 at pH 5.0 and at high protein concentration approaches 290,000 at pH 5.7. This change in Mr is consistent with the existence of a tetramer-dimer dissociation and a change in the equilibrium constant from 1.8 X 10(-6) M to 1.16 X 10(-9) M when the pH is increased from 5.0 to 5.7. This pH-dependent subunit dissociation is correlated with pH-dependent changes in enzyme activity. Purified bovine-soluble dopamine beta-hydroxylase activity is a hyperbolic function of tyramine concentration at pH 5.0. However, the hydroxylase activity displays non-hyperbolic kinetics at pH 6.0. The kinetic data obtained at pH 6.0 can be accounted for by fitting to a model containing two nonidentical catalytic forms of enzyme generated by the pH-dependent partial dissociation of tetrameric enzyme to dimeric subunits. The two catalytic forms have apparently identical maximal velocities; however, they differ in their Michaelis constants for the substrate; the dimeric form having a low Km and the tetrameric form having a high Km. Since the pH inside bovine adrenal medullary chromaffin granules is approximately 5.5, we conclude that the subunits of dopamine beta-hydroxylase are in dynamic dissociation in a physiologically important pH range.     

Purified cytochrome b561 catalyzes transmembrane electron transfer for dopamine beta-hydroxylase and peptidyl glycine alpha-amidating monooxygenase activities in reconstituted systems

Cytochrome b561 from bovine adrenal medulla chromaffin granules has been purified by fast protein liquid chromatography chromatofocusing. The purified cytochrome was reconstituted into ascorbate-loaded phosphatidylcholine vesicles. With this reconstituted system transmembrane electron transfer for extravesicular soluble dopamine beta-hydroxylase activity was demonstrated. In accordance with the model proposed by Njus et al. (Njus, D., Knoth, J., Cook, C., and Kelley, P. M. (1983) J. Biol. Chem. 258, 27-30), catalytic amounts of a redox mediator were necessary to achieve electron transfer between cytochrome and soluble dopamine beta-hydroxylase. Our observations also showed that when membranous dopamine beta-hydroxylase was reconstituted on cytochrome containing vesicles, electron transfer occurred only in the presence of a redox mediator. Since cytochrome b561 has been found in secretory vesicles associated with peptidyl glycine alpha-amidating monooxygenase, electron transfer to this enzyme was also examined. Analogous to the results obtained for dopamine beta-hydroxylase, transmembrane electron transfer to peptidyl glycine alpha-amidating monooxygenase appears to require a redox mediator between cytochrome and this monooxygenase. These observations indicate that purified cytochrome b561 is capable of providing a transmembrane supply of electrons for both monooxygenases. Since no direct protein to protein electron transfer occurs, the results support the hypothesis that the ascorbate/semidehydroascorbate redox pair serves as a mediator for these enzymes in vivo.     

Adenosine 5'-diphosphate-dependent subunit dissociation of bovine dopamine beta-hydroxylase

In a previous study, it was shown that purified soluble bovine dopamine beta-hydroxylase exhibits pH-dependent reversible tetramer-dimer dissociation (Saxena, A., Hensley, P., Osborne, J. C., Jr., and Fleming, P. J. (1985) J. Biol. Chem. 260, 3386-3392). Here we report evidence for the dissociation of this enzyme by magnesium-adenosine diphosphate independent of pH in the pH range 5-7. Quantitative binding of ADP to dopamine beta-hydroxylase revealed that there are two binding sites/dimeric species of hydroxylase and that ADP is tightly bound with a KD less than 10(-8) M. Kinetic data obtained at pH 5.5, the pH inside the chromaffin granule, shows that the apparent Km values for both the substrates tyramine and ascorbate are lowered by the presence of ADP without affecting the Vmax of the enzyme. The ADP-dependent lowering of apparent Km values results from a dissociation of the enzyme to the dimeric species which has inherently lower apparent Km values for substrates.     

Soluble and membrane-bound forms of dopamine beta-hydroxylase are encoded by the same mRNA.

A full length cDNA clone for bovine dopamine beta-hydroxylase was expressed in rat pheochromocytoma PC12 cells by stable transformation of this cell line with a plasmid expression vector. The recombinant protein exhibited dopamine beta-hydroxylase enzyme activity and was found in both the soluble and membrane fractions of the secretory vesicle. Immunoprecipitation of cell extracts from recombinant cell lines with dopamine beta-hydroxylase antisera followed by fractionation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two subunits, which migrated to relative molecular masses of 76 and 78 kDa. The recombinant protein co-fractionated with neurotransmitter when subcellular structures were separated by sucrose gradient density centrifugation, suggesting that the protein was routed to the secretory vesicles. Dopamine beta-hydroxylase immunoreactivity in those sucrose gradient fractions presumed to contain secretory vesicles was resistant to treatment with trypsin unless the nonionic detergent Triton X-100 was also present to disrupt membrane structure. The 76- and 78-kDa isoform were each found in both the membrane and soluble fractions of the secretory vesicle. Treatment of cultured cells with nerve growth factor or 8-(4-chlorophenylthio)-cyclic AMP alters the relative distribution of the subunits such that the 76-kDa form predominates. The subcellular distribution of a dopamine beta-hydroxylase cDNA clone lacking the first 16 nucleotide residues was also determined. The predicted amino acid sequence of the protein encoded by this cDNA would be deleted of the first 13 residues of the signal sequence, which were reported to be present in the membrane-bound form, but not the soluble form, of native dopamine beta-hydroxylase (Taljanidisz, J., Stewart, L., Smith, A. J., and Klinman, J. P. (1989) Biochemistry 28, 10054-10061). Immunoprecipitable dopamine beta-hydroxylase derived from expression of the deleted cDNA was found in both the membrane-bound and soluble fractions of the secretory vesicle. These experiments demonstrate that the membrane-bound and soluble forms of dopamine beta-hydroxylase are derived from one primary translation product, which is also sufficient to produce enzyme activity. In addition, the amino-terminal amino acids encoding residues 1-13, which compose the hydrophilic region of the signal sequence, are not necessary for the biogenesis of membrane-bound dopamine beta-hydroxylase.     

Hydrophilic form of dopamine β-hydroxylase from purified noradrenergic vesicles

Bovine splenic nerve and adrenal medulla were used as homologous sources of dopamine beta-hydroxylase permitting the isolation of enzyme specific to a purified fraction of large dense cored noradrenergic vesicles and chromaffin granules, respectively. The hydrophilic (water soluble) form of the enzyme was purified to homogeneity on the bases of gel electrophoresis, isoelectric focusing, and double immunodiffusion tests from the physical lysates of the vesicles and granules. Amino acid analyses suggest that the hydrophilic dopamine beta-hydroxylase is the predominant form in the nerve vesicles. It has higher neutral and lower hydrophobic amino acid group residues when compared to the adrenomedullary enzyme prepared in this and most other laboratories. Among the neutral amino acids, this difference appears to reflect approximately 40% higher serine and glycine contents, and among the hydrophobic amino acids it may reflect in part approximately 25% lower leucine content. Although the terms hydrophilic and amphiphilic can be properly applied to certain chemical properties of the D beta H forms, it is not at all certain that these terms can be used quantitatively to describe the matrix and membrane associated forms of the enzyme, respectively.     

Concomitant elevation of tyrosine hydroxylase and dopamine beta-hydroxylase by cyclic AMP in cultured mouse neuroblastoma cells.

The effects of cyclic AMP analogues and of phosphodiesterase inhibitors were investigated in neuroblastoma cells (NBD-2) cloned from the C-1300 tumor. 8Br-cAMP and phosphodiesterase inhibitors that elevated cAMP induced large (greater than 15 fold) and specific increases in tyrosine hydroxylase and dopamine beta-hydroxylase activity. In contrast, catechol O-methyltransferase, monoamine oxidase and aromatic-l -amino-acid decarboxylase were unaffected by the cAMP altering drugs. Similarly, AChE was unaffected and only a small increase in choline acetyltransferase (3 fold) was observed. The increases in tyrosine hydroxylase and dopamine beta-hydroxylase were similar with respect to dose response relationships and with respect to time course of onset. Only those phosphodiesterase inhibitors that elevated cAMP (papaverine and Ro20-1724 as opposed to theophylline) were effective in elevating tyrosine hydroxylase and dopamine beta-hydroxylase. Further, the doses optimal for elevating cAMP coincided with the optimal doses for elevating the two enzymes. Theophylline had no influence either upon NBD-2 cell cAMP levels or upon tyrosine hydroxylase and dopamine beta-hydroxylase activity. The changes in protein synthesis rates produced by the cAMP altering drugs were temporally distinct from the changes in either tyrosine hydroxylase or dopamine beta-hydroxylase. These results suggest that the intracellular messenger compound cAMP is involved in the specific regulation of both tyrosine hydroxylase and dopamine beta-hydroxylase in adrenergic cells.     

Distribution of tyrosine hydroxylase in human and animal brain

The activity of tyrosine hydroxylase (EC 1.10.3.1) when assayed under ideal conditions in young human brains, was comparable to that in brains of other species in level of activity and distribution. The highest levels of activity were in the putamen, caudate nucleus and substantia nigra, in keeping with data on other species. The caudate activity in human brain appeared to decrease substantially with increasing age. In both humans and baboons, the enzyme in the neostriatum was particle-bound and inhibited by the 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine cofactor system. In the substantia nigra it was soluble and stimulated by the 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine cofactor system. The data suggest that tyrosine hydroxylase may be produced in a soluble form in the cell bodies of the substantia nigra but become bound as it moves toward the nerve endings in the putamen and caudate nucleus. The bound form of the enzyme was unstable but the soluble form exhibited considerable stability.     

Regulation of Tyrosine Hydroxylase and Dopamine β-Hydroxylase mRNA Levels in Rat Adrenals by a Single and Repeated Immobilization Stress

Adrenal catecholamines are known to mediate many of the physiological consequences of the "fight or flight" response to stress. However, the mechanisms by which the long-term responses to repeated stress are mediated are less well understood and possibly involve alterations in gene expression. In this study the effects of a single and repeated immobilization stress on mRNA levels of the adrenal catecholamine biosynthetic enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase, were examined. A repeated 2-hr daily immobilization for 7 consecutive days markedly elevated both tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels (about six- and fourfold, respectively). In contrast, tyrosine hydroxylase but not dopamine beta-hydroxylase mRNA levels were elevated immediately following a single immobilization. The elevation in tyrosine hydroxylase mRNA with a single immobilization was as high as with seven daily repeated immobilizations. This elevation was not sustained and returned toward control values 24 hr later. Both tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels were elevated immediately following two daily immobilizations to levels similar to those observed after seven immobilizations and were maintained 24 hr later. The results indicate that both tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels are elevated by stress; however, the mechanism and/or timing of their regulation are not identical.     

An acetylenic mechanism-based inhibitor of dopamine beta-hydroxylase

The catalytic action of dopamine beta-hydroxylase on 1-phenyl-1-propyne results in concomitant loss of enzyme activity. At pH 5.5 and 25 degrees C, 1-phenyl-1-propyne inactivates dopamine beta-hydroxylase in a mechanism-based fashion. The inactivation rate is first-order, follows saturation kinetics, and is strictly dependent on catalysis (oxygen and ascorbate are essential). The inactivation rate of saturating 1-phenyl-1-propyne (kinact) increases from 0.08 to 0.22 min-1 when the oxygen saturation increases from 21 to 100%, respectively. Inactivation also requires a copper-containing catalytically competent enzyme. Tyramine and norepinephrine (respectively, substrate and product of the normal catalytic reaction) protect against inactivation, and no regain of enzyme activity occurs after prolonged dialysis. Experiments with ether-extracted incubation solutions (+/- enzyme) showed no difference in their gas chromatography-mass spectral patterns implying that inactivation of dopamine beta-hydroxylase by 1-phenyl-1-propyne occurs through a kinetic process with a partition ratio (kcat/kinact) equal to or near 1. Thus, this acetylenic substrate analog appears to be a very efficient mechanism-based inhibitor of dopamine beta-hydroxylase. We propose that inactivation of this enzyme by 1-phenyl-1-propyne proceeds by formation of a reactive intermediate that occurs prior to product formation and that alkylates an amino acid residue at the active site of the enzyme.     

The molecular shape of dopamine beta-hydroxylase from chromaffin granules of bovine adrenal medulla

The structural features of the soluble dopamine beta-hydroxylase from chromaffin granules of bovine adrenal medulla were studied using negative staining and platinum shadowing electron microscopic methods. The enzyme was shown to be highly asymmetric as suggested in earlier hydrodynamic studies. The tetramer of the enzyme appeared as four subunits arranged in the shape of a planar rose with an estimated width of 15 nm. A minimum thickness of 3.0 nm for the enzyme monomer was calculated from the shadow length of unidirectionally shadowed molecules. A model composed of four oblate ellipsoid monomers in a tetrameric rose arrangement is proposed for the shape of the dopamine beta-hydroxylase molecule. Two monomers associate edge to edge to form an in-plane dimer and two dimers associate side-by-side with their respective long axes at a slight angle to form a tetramer. Theoretical calculations based on the model are consistent with previous hydrodynamic studies.     

Kinetic and spectroscopic studies of the interaction of copper with dopamine beta-hydroxylase

The question of the stoichiometry of copper bound to dopamine beta-hydroxylase and the number of copper atoms required for maximal activity was addressed in this study. Incubation of tetrameric enzyme from bovine adrenal medulla with 64Cu2+ followed by rapid gel filtration yielded an enzyme containing 8.3-8.9 mol of Cu/mol of tetramer. An identical stoichiometry was obtained by analysis of bound copper by atomic absorption methods. NMR and EPR were used to monitor titrations of the enzyme with Cu2+ and showed that the longitudinal relaxation rate of solvent water protons and the amplitude of the signal at g approximately 2 increased linearly up to a copper to protein ratio of approximately 8. Additional titrations also indicate that an enzyme-Cu2+-tyramine-CN- inhibitory complex was formed when 8 mol of Cu2+ are bound per mol of enzyme. The rate of inactivation of dopamine beta-hydroxylase by the mechanism-based inhibitor 2-Br-3-(p-hydroxyphenyl)-1-propene was measured and used as a method to follow enzymatic catalysis. An increase in rate was observed with increasing Cu2+ up to a protein to Cu2+ ratio of 8 Cu/tetramer. The rate becomes constant after this ratio is achieved. These data indicate that dopamine beta-hydroxylase specifically binds 8 mol of Cu/tetramer and that this stoichiometry is required for maximal activity.     

Gamma-glutamyltransferase release by the post ischemic kidney: multiple forms and cellular pH

The release of gamma-glutamyltransferase from renal tubule cells was studied in situ following 30 minutes of ischemia. The ischemic kidney enzyme level fell 33 percent after 15 minutes of reflow of which only 1.2 percent was recovered in the urine; none was released into the renal vein. At this time the overwhelming majority of the enzyme appears bound to membranes in both the kidney and the urine. In the subsequent 15 minutes renal levels continue to decline while urinary excretion accounts for 5 percent of that disappearing from the kidney. Interestingly the form of the enzyme present in kidney and urine shifts to a soluble form coinciding with cellular alkalosis, urinary alkalinization and a rise in ATP levels. Alkalinization of renal homogenates result in a 2-fold increase in the soluble enzyme form. The results are consonant with the immediate loss of brush border enzyme via uptake into the cell or release into the urine with the former pathway predominating; subsequent appearance of the soluble enzyme appears to reflect intracellular alkaline proteinase activity and exocytosis. The form in which the enzyme is excreted may provide a useful clinical index: membranous reflecting cellular necrosis and soluble reflecting cellular recovery.     

Mechanism of biosynthesis of soluble and membrane-bound forms of dopamine beta-hydroxylase in PC12 pheochromocytoma cells

Dopamine beta-hydroxylase was present as 2 subunit forms (apparent Mr = 77,000 and 73,000) in the PC12 pheochromocytoma cell line as detected by immunoprecipitation from [35S]methionine-labeled cultures, and analyzed by sodium dodecyl sulfate gel electrophoresis and fluorography. The Mr = 77,000 form was present in a crude membrane fraction, while the Mr = 73,000 form was soluble. Both forms appeared to be present in approximately equal amounts, and both were glycosylated. Treatment of PC12 cells with tunicamycin, a potent inhibitor of core glycosylation in the endoplasmic reticulum, completely inhibited the appearance of the Mr = 77,000 and Mr = 73,000 forms, and 2 new immunoreactive polypeptides were obtained (apparent Mr = 67,000 and 63,000). Pulse-chase experiments suggested that the Mr = 77,000 form is initially synthesized (by 5 min) and a portion is converted in 15-90 min to the Mr = 73,000 form. Thereafter, the ratio between forms remains relatively constant, at least for several hours. Translation of mRNA from bovine and rat adrenals, and immunoprecipitation, indicated that dopamine beta-hydroxylase is initially synthesized as a single polypeptide (apparent Mr = 67,000). The subcellular site of biosynthesis of dopamine beta-hydroxylase was determined by isolation of mRNA from free and membrane-bound polysomes from bovine adrenal medulla. Translation in a cell free system and immunoprecipitation localized the synthesis of dopamine beta-hydroxylase on membrane-bound polysomes. These experiments suggest that both soluble and membrane-bound forms of dopamine beta-hydroxylase are synthesized and core glycosylated in the endoplasmic reticulum, and that there probably is a precursor-product relationship between the Mr = 77,000 and the Mr = 73,000 subunit forms of dopamine beta-hydroxylase.