Bovine Dopamine Beta-hydroxylase Research Articles

Complete assignment of disulfide bonds in bovine dopamine beta-hydroxylase.

Peptide mapping, chemical sequencing, microbore HPLC/electrospray ionization mass spectrometry (LC/ESI/MS), and matrix-assisted laser desorption mass spectrometry (MALDI/MS) were used to identify the sites of intra- and intermolecular disulfide linkages in bovine dopamine beta-hydroxylase. The enzyme contains 14 cysteines and seven disulfides per monomer. Edman sequencing of tryptic and peptic peptides determined linkages at positions Cys140-Cys582, Cys218-Cys269, Cys255-Cys281, Cys452-Cys474, Cys514-Cys514, and Cys516-Cys516, where cysteines at positions 514 and 516 on one monomer disulfide pair with their homologs on a second monomer. These linkages were confirmed by LC/ESI/MS and MALDI/MS. Further analysis by LC/ESI/MS and MALDI/MS identified linkages at positions Cys376-Cys489 and Cys380-Cys551. Cysteines 140 and 582 form a disulfide linkage that folds the C-terminus back in proximity to the N-terminus. The remaining intramolecular disulfides occur along two separate internal regions of the protein. The density of histidine residues in these two regions suggests binding sites for two Cu2+ atoms per monomer. In addition, previously identified amino acids that react with mechanism-based inactivators occur in these two regions. We propose that these five internal disulfide bonds define two Cu2+ binding domains that make up the active site of a dopamine beta-hydroxylase monomer. Considering previous data on the location of glycosylation sites, mechanism-based inactivation sites, and the disulfide linkages presented here, the data suggest an overall topology were the N- and C-termini are in close proximity and are solvent exposed and where the Cu2+ binding sites are buried in two interior domains stabilized by five disulfide bonds.

Effects of cAMP, glucocorticoids, and calcium on dopamine beta-hydroxylase gene expression in bovine chromaffin cells.

To better understand the molecular mechanism underlying regulation of bovine dopamine beta-hydroxylase (DBH), the effects of elevated intracellular cAMP, glucocorticoids, and calcium were studied in primary cultured chromaffin cells. Elevation of intracellular cAMP by forskolin and treatment with its analog 8-bromo-cAMP caused an increase in the bovine DBH mRNA level by 3.5 +/- 0.5- and 7.8 +/- 0.9-fold, respectively, which was maximal at 6 h after the treatments. On the other hand, dexamethasone elicited no apparent change in DBH gene expression at various concentrations and time. The combined treatment with forskolin and dexamethasone resulted in the same degree of increase as that with forskolin alone. Increased intracellular calcium by the ionophore A23187 ranging from 50 to 500 nM caused DBH mRNA to decrease, which began to be observed after 6 h and was undetectable by 48 h. The results demonstrate the existence of coordinate and differential regulations among the enzymes involved in catecholamine biosynthesis in bovine adrenomedullary cells.

Expression of dopamine beta-hydroxylase in Drosophila Schneider 2 cells. Evidence for a mechanism of membrane binding other than uncleaved signal peptide

To characterize the mechanism of membrane attachment of dopamine beta-hydroxylase, an expression system producing the processed form of this enzyme has been developed. We have replaced the endogenous signal peptide of bovine dopamine beta-hydroxylase with a heterologous signal peptide which is efficiently recognized and cleaved in Drosophila Schneider 2 cells. A cDNA encoding this chimeric recombinant bovine enzyme has been stably transfected into Schneider 2 cells. The inducible expression of active dopamine beta-hydroxylase in these cells has been verified by Western blotting and enzyme activity assays. N-terminal sequence analysis of purified recombinant enzyme demonstrates complete removal of the signal peptide. Subcellular analysis shows that the recombinant enzyme exists as both a soluble and a membrane-bound form in these cells. These data demonstrate that the endogenous signal peptide is not required for the formation of the membranous dopamine beta-hydroxylase and further that the enzyme can be bound to membranes via a mechanism other than uncleaved signal sequence.

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.

Production and immunocytochemical application of a highly sensitive and specific monoclonal antibody against rat dopamine-?-hydroxylase

A highly sensitive and specific monoclonal antibody against the enzyme dopamine beta-hydroxylase (DBH) from rat was produced and coded DBH 41. The generated hybridoma secreted immunoglobulins of mouse IgG1 subtype, as determined by radial immunodiffusion. This antibody, characterized by immunoblotting against a crude rat DBH preparation, was found to specifically recognize two bands of molecular weight 70 and 75 kDa corresponding to the soluble and membrane bound forms of the enzyme, respectively. With regard to species specificity, the anti-DBH antibody recognizes only the rat DBH molecule as it exhibits no cross-reactivity with either mouse, human, rabbit, guinea pig, cat or bovine DBH. Comparative immunocytochemical localization of DBH and TOH immunoreactivity was performed in different brain regions and we found that the DBH 41 antibody specifically stained DBH-containing neurons and fibers in the rat central nervous system (CNS). The high sensitivity of the DBH 41 antibody permitted us to detect immunologically the presence of the enzyme even in areas where only scattered DBH-containing fibers were present.

Bovine dopamine .beta.-hydroxylase, primary structure determined by cDNA cloning and amino acid sequencing

A cDNA clone encoding bovine dopamine beta-hydroxylase (DBH) has been isolated from bovine adrenal glands. The clone hybridizes to two oligonucleotide probes, one based on a previously reported active site peptide [DeWolf, W. E., Jr., et al. (1988) Biochemistry 27, 9093-9101] and the other based on the human DBH sequence [Lamouroux, A., et al. (1987) EMBO J. 6, 3931-3937]. The clone contains a 1.9-kb open reading frame that codes for the soluble form of bovine DBH, with the exception of the first six amino acids. Direct confirmation of 93% of the cDNA-derived sequence was obtained from cleavage peptides by protein sequencing and mass spectrometry. Differences were found between these two sequences at only two positions. Of the four potential N-linked carbohydrate attachment sites, two, Asn-170 and Asn-552, were shown to be partially and fully glycosylated, respectively. Within the 69% of the protein sequence confirmed by mass spectrometry, no other covalent modifications were detected.

Primary amino acid sequence of bovine dopamine beta-hydroxylase.

Fifty-eight tryptic and Staphylococcus aureus V8 protease generated peptides from bovine dopamine beta-hydroxylase were isolated by reverse-phase high pressure liquid chromatography and sequenced. These peptide sequences were compared with the deduced amino acid sequences of bovine and human dopamine beta-hydroxylase obtained from the cloned cDNAs. Bovine peptide sequences had five differences with the sequence derived from the bovine cDNA, and four of the changes could be accounted for by a single base change in the DNA. N-terminal sequence analysis of the bovine enzyme indicated that it contained two N termini, one of which is 3 amino acids longer than the other and begins with the sequence Ser-Ala-Pro. The amino acid sequences deduced from the bovine and human cDNAs are 19 and 25 amino acids longer, respectively, and these additional amino acids represent leader peptide sequences. Two bovine peptide sequences contained glycosylation sites and gave positive tests for carbohydrate residues, and two others contained the consensus sequence for a glycosylation site but were negative in the carbohydrate test. The bovine enzyme contains 6 Trp, as compared with 7 in the bovine cDNA and 8 in the human cDNA. The protein and bovine cDNA contain 24 Tyr each, as compared with 26 in the human cDNA. These numbers indicate that the true epsilon 1% 280 = 8.95, and, therefore, that it is 28% lower than the previously determined value. The data also identify 5 His-containing regions that may be involved in Cu2+ coordination at the active site.

Bovine dopamine beta-hydroxylase cDNA. Complete coding sequence and expression in mammalian cells with vaccinia virus vector.

We have isolated cDNA clones for bovine dopamine beta-hydroxylase from an adrenal medulla cDNA library and have determined the complete coding sequence. The largest cDNA clone isolated from the library is 2.4 kilobase pairs (kb) and contains an open reading frame of 1788 bases, coding for a protein of 597 amino acids and Mr = 66,803. The predicted amino acid sequence of the bovine cDNA contains 85% identity with human dopamine beta-hydroxylase (Lamouroux, A., Vingny, A., Faucon Biquet, N., Darmon, M. C., Franck, R., Henry, J.P., and Mallet, J. (1987) EMBO J. 6, 3931-3937; Kobayashi, K., Kurosawa, Y., Fujita, K., and Nagatsu, T. (1989) Nucleic Acids Res. 17, 1089-1102). Northern blot analysis reveals that the cDNA hybridizes to an mRNA of 2.4 kb present in bovine adrenal medulla, but not in kidney, heart, or liver. In addition, the cDNA hybridizes to a second RNA species of 5.5 kb, which is 4-fold less abundant than the 2.4-kb RNA. In vitro translation of a synthetic RNA transcribed from the 2.4-kb cDNA produces a 68-kDa protein, which is specifically immunoprecipitated by antiserum to bovine dopamine beta-hydroxylase. The 2.4-kb cDNA was cloned into a vaccinia virus vector, and the recombinant virus was used to infect the rat pheochromocytoma PC12 and monkey BSC-40 fibroblast cell lines. In both cell lines, infection with recombinant virus produces a protein of Mr = 75,000, which reacts with antiserum to bovine dopamine beta-hydroxylase. These results indicate that the 2.4-kb cDNA contains the genetic information necessary to code for the bovine dopamine beta-hydroxylase subunit.

Spectral Studies of Bovine Dopamine β-Hydroxylase: Absence of covalently bound pyrroloquinoline quinone

Abstract Bovine dopamine beta-hydroxylase was examined spectroscopically for the presence of covalently bound pyrroloquinoline quinone (PQQ). Pure dopamine beta-hydroxylase had a featureless UV-visible spectrum above 300 nm. An equimolar solution of dopamine beta-hydroxylase and exogenously added PQQ (1 PQQ/active site) had a strong absorption maximum at 333 nm. Dialysis removed the added PQQ, indicating that dopamine beta-hydroxylase does not bind PQQ irreversibly. Reaction of dopamine beta-hydroxylase with 6 mM phenylhydrazine in the presence of 15 mM ascorbate caused 96% inactivation within 20 min and did not produce any spectrally detectable amounts of the phenylhydrazone adduct of PQQ, as reported by van der Meer et al. (van der Meer, R.A., Jongejan, J.A., and Duine, J.A. (1988) FEBS Lett. 231, 303-307). The peptide profile of phenylhydrazine inactivated dopamine beta-hydroxylase was monitored at 316 nm and did not reveal any peptides that might contain a PQQ-phenylhydrazone adduct. Thus, the absence of any spectrally detectable PQQ-phenylhydrazone adducts under these conditions demonstrates that the mechanism of phenylhydrazine inactivation does not involve covalent modification of PQQ at the active site of dopamine beta-hydroxylase and provides strong evidence that the native enzyme does not contain PQQ.

The copper sites of dopamine beta-hydroxylase: an X-ray absorption spectroscopic study.

X-ray absorption edge and extended X-ray absorption fine structure (EXAFS) spectra are reported for the Cu(I) and Cu(II) forms of bovine dopamine beta-hydroxylase (DBH; EC 1.14.17.1) and for the Cu(I) form of DBH bound either to tyramine substrate or to a multisubstrate inhibitor [Kruse, L. I., DeWolf, W. E., Jr., Chambers, P. A., & Goodhart, P. J. (1986) Biochemistry 25, 7271-7278]. A significant change in the structure of the copper sites occurs upon ascorbate-mediated reduction of Cu(II) DBH to the Cu(I) form. While the average Cu(II) site most likely consists of a square-planar array of four (N,O)-containing ligands at 1.98 A, the average Cu(I) site shows a reduction in (N,O) coordination number (from approximately 4 to approximately 2) and the addition of a S-containing ligand at 2.30 A. No change in the average Cu(I) ligand environment accompanies binding of tyramine substrate, whereas binding of a multisubstrate inhibitor, 1-(3,5-difluoro-4-hydroxybenzyl)-1H-imidazole-2(3H)-thione, causes an increase in the Cu-S coordination, consistent with inhibitor binding to the Cu(I) site through the S atom. Although excellent signal-to-noise ratio in the EXAFS spectra of ascorbate-reduced DBH facilitated analysis of outer-shell scattering for a Cu..Cu interaction, the presence of a binuclear site could not be proven or disproven due to interference from Cu...C scattering involving the carbons of imidazole ligands.

The primary structure of human dopamine-beta-hydroxylase: insights into the relationship between the soluble and the membrane-bound forms of the enzyme.

A full length dopamine-beta-hydroxylase (DBH) cDNA clone was isolated from a human pheochromocytoma lambda gt11 library. Both structural and functional evidence confirms the authenticity of the clone: (i) antibodies selected with fusion proteins generated by positive clones precipitate DBH activity, (ii) the sequence of three internal DBH tryptic peptides are included in the deduced DBH sequence, (iii) the previously reported N-terminal 15 amino acids of bovine DBH exhibits a nearly complete identity with that predicted for human DBH. The polypeptide chain of DBH comprises 578 amino acids corresponding to an unmodified protein of 64 862 daltons and is preceded by a cleaved signal peptide of 25 residues. DBH exists in both membrane-bound and soluble forms. The hydropathy plot reveals no obvious hydrophobic segment, except the signal peptide. S1 mapping analysis indicates no diversity in the 5' and 3' extremities of the DBH mRNA. Taken together with available biochemical data, these observations suggest that the membrane attachment of DBH probably results from a post-translational modification, glypiation being the most likely candidate. Comparative amino acid sequence analysis establishes that DBH shares no homology with the other catecholamine synthesizing enzymes, tyrosine hydroxylase and phenylethanolamine-N-methyl transferase.

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.

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.

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.

Dopamine β-hydroxylase: Determination of half-life and appearance in lymph fluid after IV injection of 125I-DBH into rats

A 4 day half-life of dopamine beta-hydroxylase (DBH) was determined for rats injected IV with 125I-rat DBH from the slow exponential component of radioactivity appearing in plasma, urine, feces and combined urine and feces. Half-life estimates for 125I-rat DBH injected IV into WKY and SHR animals did not differ from Sprague Dawley (Zivic Miller) rats. Radioactivity declined in parallel in plasma, urine and feces following IV 125I-rat DBH administration and each radioactivity falloff curve could be resolved into two components. The slow phase of the decline of radioactivity excreted into urine and feces from which DBH half-life was calculated occurred between 5 and 25 days after 125I-rat DBH injection. The early fast phase which is associated with distribution of the exogenous protein in body fluids and tissues continued for approximately the first 140 hr after DBH injection. The distribution characteristics of IV administered active bovine DBH and 125I-rat DBH into the lymphatic system were examined. After active bovine DBH or 125I-rat DBH was injected IV into rats, active DBH or radioactivity, respectively, appeared in lymph fluid (thoracic duct) within 20 min; reached peak concentrations within 90 min, and thereafter, declined in parallel with the plasma concentration. The concentration of radioactivity in plasma and lymph fluid were found to be unequal at 9 hr but were equivalent 68–75 hrs after IV injection of 125I-rat DBH. Based on the amount of active DBH or radioactivity which accumulates in lymph fluid it is clear that'a substantial amount (> 50%) of the DBH in blood circulates through the lymphatic channels. Analysis of parallel experiments with labelled serum albumin indicate that use of these methods to study plasma proteins do provide sensitive measures of biological half-life and lymphatic distribution characteristics. Specifically for DBH, the results of our study suggest that DBH normally circulates in plasma and lymph fluid with a biological half-life of 4 days.

The Inverse Relationship between Serum Dopamine-β-Hydroxylase Activity and Thyroid Function*

Serum dopamine-beta-hydroxylase (DBH) activity is inversely related to thyroid status in both humans and rats. The low level of serum DBH activity in hyperthyroid rats is accompanied by a rapid rate of disappearance of enzyme activity after the injection of exogenous bovine DBH. Conversely, the high level of serum DBH activity in hypothyroid rats is accompanied by a slow rate of exogenous DBH disappearance. These results suggest that the extraneuronal disposal pathway for DBH is an important factor in the regulation of the level of DBH activity in altered thyroid function in both humans and rats.

Rat adrenal dopamine-beta-hydroxylase: purification and immunologic characteristics.

Dopamine-beta-hydroxylase (DBH) was purified from rat adrenal medulla by a series of steps including sedimentation of membranes, extraction with n-butanol, ammonium sulfate fractionation, gel chromatography and ion-exchange chromatography. Disk gel electrophoresis revealed two protein bands, both of which were active. Antiserum was prepared against homogeneously purified bovine adrenal and rat adrenal DBH; Ouchterlony immunodiffusion, enzyme neutralization and complement fixation tests demonstrated that the respective homologous antisera were monospecific and of high titer. Antiserum to bovine DBH was only 2- to 3-fold more potent than pre-immune serum in inhibition of rat DBH activity. Complement fixation tests demonstrate that antiserum to bovine DBH has a 25,000-fold lower immunoreactivity with rat DBH than with bovine DBH.

Ascorbate 2-Sulfate Inhibits Dopamine β-Hydroxylase Reaction, but not Ascorbate Oxidase Reaction

Bovine adrenal dopamine beta-hydroxylase [EC 1.14.17.1] was considerably inhibited by ascorbate 2-sulfate. The inhibition was competitive with regard to ascorbate. The Ki value was 3.44 mM. The possibility that ascorbate 2-sulfate may play a regulatory role in the biosynthesis of norepinephrine is suggested. Another copper-containing oxidase, squash ascorbate oxidase [EC 1.10.3.3], was not inhibited by the same compound at a concentration of 150 mM.