Congenital Copper Deficiency: Copper Therapy and Dopamine-β-Hydroxylase Activity in the Mottled (Brindled) Mouse

Abstract: The mottled (Mo) mouse is an animal model of the human congenital copper (Cu) deficiency disorder, Menkes' kinky hair syndrome. Intraperitoneal Cu chloride injections have been shown to produce clinical and morphological improvements in this mutant mouse. Cu injections (10 μg/g) on postnatal days 7 and 10 are shown to increase endogenous activity of the Cu-dependent enzyme dopamine-β-hydroxylase in the brains of Mo mice.     

Amphiphilic and Nonamphiphilic Forms of Bovine and Human Dopamine β-Hydroxylase

We show that human and bovine dopamine beta-hydroxylases (DBH) exist under three main molecular forms: a soluble nonamphiphilic form and two amphiphilic forms. Sedimentation in sucrose gradients and electrophoresis under nondenaturing conditions, by comparison with acetylcholinesterase (AChE), suggest that the three forms are tetramers of the DBH catalytic subunit and bind either no detergent, one detergent micelle, or two detergent micelles. By analogy with the Gna4 and Ga4 AChE forms, we propose to call the nonamphiphilic tetramer Dna4 and the amphiphilic tetramers Da4I and Da4II. In addition to the major tetrameric forms, DBH dimers occur as very minor species, both amphiphilic and nonamphiphilic. Reduction under nondenaturing conditions leads to a partial dissociation of tetramers into dimers, retaining their amphiphilic character. This suggests that the hydrophobic domain is not linked to the subunits through disulfide bonds. The two amphiphilic tetramers are insensitive to phosphatidylinositol phospholipase C, but may be converted into soluble DBH by proteolysis in a stepwise manner; Da4II----Da4I----Dna4. Incubation of soluble DBH with various phospholipids did not produce any amphiphilic form. Several bands corresponding to the catalytic subunits of bovine DBH were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but this multiplicity was not simply correlated with the amphiphilic character of the enzyme. In the case of human DBH, we observed two bands of 78 and 84 kDa. As previously reported by others, the presence of the heavy subunit characterizes the amphiphilic forms of the enzyme. We discuss the nature of the hydrophobic domain, which could be an uncleaved signal peptide, and the organization of the different amphiphilic and nonamphiphilic DBH forms. We present two models in which dimers may possess either one hydrophobic domain or two domains belonging to each subunit; in both cases, a single detergent micelle would be bound per dimer.     

Orthograde, Retrograde, and Turnaround Axonal Transport of Dopamine-β-Hydroxylase: Response to Axonal Injury

Reversal of the direction (turnaround) of orthograde axonal transport of dopamine-beta-hydroxylase (DBH) activity was studied at a ligature placed on rat sciatic nerve. DBH was allowed to accumulate at a ligature in vivo for selected intervals, at which time a second ligature was placed proximal to the first and turnaround transport measured just distal to the second tie after incubation in vivo or in vitro. Orthograde accumulation of DBH activity proximal to a ligature peaked at 2 days, and then rapidly decreased as a result of turnaround transport and injury-induced reduction of orthograde transport. Destruction of postganglionic sympathetic axon terminals in vivo with 6 hydroxydopamine resulted in a decrease in orthograde transport similar to that seen after axotomy and turnaround at or proximal to the site of chemical injury. Turnaround transport of DBH in vitro was blocked by incubation in the cold and in the presence of NaCN and vinblastine. Orthograde transport of DBH appeared to reverse direction within a few millimeters of a ligature.     

Dopamine-β-Hydroxylase: Structural Comparisons of Membrane-Bound Versus Soluble Forms from Adrenal Medulla and Pheochromocytoma

Dopamine-beta-hydroxylase (DBH) in membrane-bound (mDBH) and water-soluble (sDBH) forms was isolated from chromaffin granules of bovine adrenal medullae and a human pheochromocytoma tumor. sDBH was purified by concanavalin A-agarose column chromatography followed by DEAE-Sepharose column chromatography. The final bovine preparation had a specific activity of 16.27 IU/mg; the human preparation had a specific activity of 9.16 IU/mg. mDBH was isolated in enzymatically inactive form by preparative polyacrylamide gel electrophoresis. The proteins were subjected to amino acid analysis, as well as digestion with trypsin, followed by separation of the resulting peptides by two-dimensional TLC/electrophoresis. No intraspecies differences between sDBH and mDBH were found from comparisons of amino acid composition or peptide maps. Thus the basis of the difference between sDBH and mDBH cannot easily be explained by differences in primary structure, within the resolution of these techniques.     

Dopamine-β-Hydroxylase Activity of the Cat Carotid Body Under Different Arterial O2 and CO2 Conditions

To determine the importance of dopamine and noradrenaline as neurotransmitters during chemoreception in the cat carotid body we investigated the contents of both compounds as well as the activity of dopamine-beta-hydroxylase (DBH) under different arterial PO2 and PCO2 conditions. The superior cervical ganglion was used as a control organ. In the carotid body and the ganglion an inverse relationship exists between the catecholamine content and the DBH activity. The carotid body has a high catecholamine content with a low DBH activity whereas the superior cervical ganglion has a low catecholamine content and high DBH activity. Hypercapnia did not produce any significant change in the catecholamine content or in the DBH content of the carotid body. However, in comparison with hyperoxia, hypoxia produced a significant change (p less than 0.05) in the noradrenaline content without changing the DBH activity. The dopamine content under these conditions did not change significantly. The results may indicate that the high catecholamine content of the carotid body is the result of a high retention and/or low rate of degradation rather than of a high rate of synthesis.     

A Sensitive and Reliable Assay for Dopamine (β-Hydroxylase in Tissue

A new assay procedure for dopamine β-hydroxylase (DBH) in tissue extracts is described. Solubilized DBH was adsorbed from crude extracts on Concanavalin A-Sepharose (Con A-Sepharose), resulting in enrichment of the enzyme as well as removal of endogenous catecholamines and inhibitory substances. The enzymatic assay was carried out with DBH still adsorbed to Con A-Sepharose. The adsorption of the DBH to Con A-Sepharose offers three advantages over previous assay procedures. (1) Because of removal of the endogenous inhibitory substances, a single Cu²⁺ concentration can be used for the determination of DBH activity, regardless of the tissue dilution or inhibitor content of the analysed sample. Using this procedure, the optimal Cu²⁺ concentration for DBH of bovine adrenal gland extracts was 3 μM and for rat brain 10 μM. (2) Because of removal of endogenous catecholamines, dopamine, the main physiological substrate of DBH in noradrenergic neurons, can be used for the assay. The enzymatic reaction product, noradrenaline, was determined by high performance liquid chromatography and electrochemical detection (hplc-ec). This procedure resulted in an approx. 10-fold increase in sensitivity of the assay compared with other procedures, e.g., the radioenzymatic assay. (3) Direct determination of the immediate product of the enzymatic reaction (noradrenaline) permits kinetic analysis. It was found that the Michaelis constants for the substrate (dopamine) and co-factor (ascorbic acid) (2 mM and 0.65 mM, respectively) determined in bovine adrenal tissue extracts by the described procedure were identical with the values for the purified DBH preparation.     

Carbohydrate Structures of β-Trace Protein from Human Cerebrospinal Fluid: Evidence for "Brain-Type"N-Glycosylation

Abstract: The carbohydrate structures of β-trace protein from human cerebrospinal fluid have been elucidated. This protein carries exclusively N-linked oligosaccharides at two sites (Asn²⁹ and Asn⁵⁶). Enzymatically released N -glycans were studied by compositional and methylation analyses, high-pH anion-exchange chromatography, and liquid secondary ion mass spectrometry. All glycans were found to be of the complex type, and most (90%) of them were biantennary with no (40%), one (40%), or two (20%) N -acetylneuraminic acid residues. The rest were triantennary chains or biantennary chains with intact or truncated lactosamine repeats. The innermost N -acetylglucosamine residues of nearly all structures were found to be α1,6-fucosylated. Peripheral fucose (about 20%α1,3-linked to N -acetylglucosamine) was also detected. Seventy percent of the oligosaccharides contained a bisecting N -acetylglucosamine. Especially in the neutral, but also in the monosialylated oligosaccharide fractions, many incomplete antennae consisting of N -acetylglucosamine only were present. At least 20 different N -glycans were identified. Analysis of the site-specific glycosylation patterns at Asn²⁹ and Asn⁵⁶ revealed only minor differences. According to the structural features (a high degree of fucosylation, high amounts of bisecting N -acetylglucosamine, as well as terminal N -acetylglucosamine and galactose residues, and significant amounts of N -acetylneuraminic acid in α2,3 linkage), this protein can be classified as "brain-type" glycosylated.     

Fluorometrical Analysis of Guanidino Compounds in Human Cerebrospinal Fluid

Abstract: Guanidino compounds in CSF of 57 human subjects were determined fluorometrically after reaction with phenanthrenequinone in alkali solution, using HPLC. Creatinine (65.2 ± 13.4 nmol/ml), arginine (24.7 ± 6.4 nmol/ml), and homoarginine (0.7 ± 0.3 nmol/ml) were found in all subjects. Trace amounts of guanidinosuccinic acid and guanidinoacetic acid were detected in some of the subjects. Brain guanidino compounds, taurocyamine, N -acetylarginine, and methylguanidine were not detected in CSF.     

Mutagenesis of Rat Dopamine β-Hydroxylase: Examination in Cell-Free System

Abstract: Dopamine β-hydroxylase (DBH; EC 1.14.17.1) exists as membrane-bound and soluble forms in neurosecretory vesicles. The features of DBH required for glycosylation and incorporation into membranes were studied in a cell-free system. Translation of full-length DBH with microsomal membranes generated two glycosylated products (GH and GL) depending on the magnesium concentration. Carboxyl-terminal, in contrast to amino-terminal, truncations gave translation products that were glycosylated by microsomal membranes. Site-directed mutants were generated with the second AUG codon and the region of a putative signal sequence cleavage site modified. Translation without membranes indicated that the second AUG is not used to initiate translation. The mutant with Glu⁴¹→ Leu⁴¹ and Ser⁴³→ Thr⁴³ yielded only the GH form with membranes, whereas mutation of Ser⁴³→ Ala⁴³ generated both GH and GL forms. Both glycosylated forms comigrated with the microsomal membranes on sucrose gradients. Endoglycosidase H digestion indicated that the differences between the GH and GL forms are not due to the sugar moiety. The results suggest a role for cleavage of a signal sequence in the formation of different forms of DBH. The possibility that these mutations change the secondary structure near the signal cleavage site, affecting processing, is discussed.     

Purification and Chemical Characterization of β-Trace Protein from Human Cerebrospinal Fluid: Its Identification as Prostaglandin D Synthase

Abstract: β-Trace protein from pooled human CSF was purified to homogeneity. An apparent molecular mass of 23–29 kDa was determined for the polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequencing of the polypeptide yielded the unique amino acid sequence APEAQVSVQPNFQQDKFLGRWFSA²⁴. Alignment of amino acid sequences obtained from tryptic peptides with the sequence previously deduced from a cDNA clone isolated by other investigators allowed the identification of β-trace protein as prostaglandin D synthase [prostaglandin-H₂ D-isomerase; (5 Z , 13 E )-(15 S )-9α, 11 a-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase; EC 5.3.99.2]. A conservative amino acid exchange (The instead of Ser) was detected at amino acid position 154 of the β-trace polypeptide chain in the corresponding tryptic peptide. The two N -glycosylation sites of the polypeptide were shown to be almost quantitatively occupied by carbohydrate. Carbohydrate compositional as well as methylation analysis indicated that Asn²⁹and Asn⁵⁶ bear exclusively complex-type oligosaccharide structures (partially sialylated with α2–3- and/or α2–6-linked N -acetylneuraminic acid) that are almost quantitatively α1-6 fucosylated at the proximal N -acetylglucosamine; ∼70% of these molecules contain a bisecting N -acetylglucosamine. Agalacto structures as well as those with a peripheral fucose are also present.     

Micropurification of Two Human Cerebrospinal Fluid Proteins by High Performance Electrophoresis Chromatography

Abstract: Using C8 reversed-phase HPLC in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we have fractionated proteins contained in human CSFs obtained from patients with schizophrenic disorders. When these proteins were electrophoretically blotted onto polyvinylidene difluoride membrane for direct N-terminal amino acid sequencing, several CSF proteins were identified; these included albumin, transferrin, apolipoprotein A-l, β₂-microglobulin, and prealbumin. We have also identified two structurally related human CSF proteins designated cerebrin 28 (M_r 28,000) and cerebrin 30 (M_r 30,000) that have an N-terminal amino acid sequence of NH₂-APPAQVSVQPNF and NH₂-APEAQVSVQPLFXQ, respectively. Comparison of these sequences with existing database at Protein Identification Resource (R 32.0), GenBank (R 72.0), SWISS-PROT (R 22.0), and EMBL (R 31.0) indicated that they are unique proteins. These proteins were subsequently purified by high performance electrophoresis Chromatography (HPEC) using an Applied Biosystems 230A HPEC system. A specific polyclonal antibody was prepared and an ELISA was established for cerebrin 30. It was noted that HPEC is a powerful tool to purify microgram quantities of proteins from human, rabbit, and rat CSFs. Using such a system, we have been able to micropurify as many as 10 proteins simultaneously in a single experiment because the elution of proteins occurred strictly according to their molecular weights. More importantly, we routinely obtained a recovery of >90%. The potential use of this technology for micropurification of proteins was discussed.     

Dopamine β-Hydroxylase and Other Glycoproteins from the Soluble Content and the Membranes of Adrenal Chromaffin Granules: Isolation and Carbohydrate Analysis

Abstract: Chromogranin A and two other proteins (A₁ and A₂) of the soluble proteins of bovine chromaffin granules were isolated by extraction from polyacrylamide gels after electrophoresis. The carbohydrate content of these proteins was 5%, with galactose, N -acetylgalactosamine, and sialic acid as the main sugars. Membranes of chromaffin granules were solubilized with sodium dodecyl sulphate (SDS) and three glycoproteins were isolated by sequential affinity chromatography on Concanavalin A (Con A) and wheat germ lectin (WGL) Sepharose columns. Two glycoproteins, designated GP II and III, were found to have a high carbohydrate content of about 30%. Mannose, galactose, N -acetylgalactosamine, and sialic acid were the main sugars. In addition membrane-bound dopamine β-hydroxylase was isolated by this procedure. No significant differences between the carbohydrate composition of the membrane-bound and the soluble enzyme were revealed. It was shown that all four subunits of dopamine β-hydroxylase possess carbohydrate chains with an affinity for Con A. The isolation methods established in this study will be useful for immunological studies on these glycoproteins.     

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.     

Purifications of Rat Adrenal Dopamine-β-Hydroxylase: Immunological Analysis of Its Soluble and Membrane-Bound Forms with the Use of an Antibody Raised Against the Soluble Form

Soluble and membrane-bound dopamine-beta-hydroxylases (sDBH and mDBH, respectively) from rat adrenal glands have been purified through concanavalin A-Sepharose chromatography, gel filtration, and ion-exchange high-performance chromatographies. Both sDBH and mDBH were composed by four subunits of apparent molecular weight of 75,000 and showed a native molecular weight of 300,000. This procedure has not allowed us to obtain a sufficient amount of enzyme to immunize a rabbit. A second, more rapid procedure was designed to isolate sDBH, including concanavalin A-Sepharose chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A rabbit antiserum was raised against this purified protein. The specificity of the antiserum was demonstrated by neutralization of rat adrenal gland DBH activity, labeling of rat adrenal medulla on histological sections, and, after Western blot, labeling of the 75,000-molecular-weight band in the different fractions associated with DBH activity during purification. The antiserum had a higher affinity for the sDBH denatured by sodium dodecyl sulfate than for the native protein. It had a higher affinity for sDBH than for mDBH. These results strongly suggest the presence of specific hydrophilic epitopes on the sDBH, revealing structural differences between the two hydroxylase forms. Two protein bands were stained on Western blots of crude rat adrenal gland extract. One band had an apparent molecular weight of 75,000, and the other of 82,000. Our results showed that the two proteins contained similar epitopes, an observation suggesting a close structural relationship. The higher-molecular-weight protein could be the 75,000 protein before covalent modifications and cleavage.     

Selective Aggregation of Endogenous β-Amyloid Peptide and Soluble Amyloid Precursor Protein in Cerebrospinal Fluid by Zinc

Abstract: Zinc added to buffered solutions of synthetic β-amyloid peptide (Aβ) has been reported to induce accelerated formation of insoluble aggregates. This observation suggests that zinc may play a role in the formation of senile plaques, which contain Aβ, in Alzheimer's disease. To test this hypothesis under conditions more representative of the brain, we investigated the ability of zinc to induce aggregation of Aβ in freshly drawn canine CSF, which contains the same sequence as human Aβ. Aggregates were separated from CSF by ultracentrifugation before and after incubation with zinc and assayed by quantitative western blotting and ELISA. We found that zinc induced the rapid aggregation of endogenous Aβ in CSF, with an EC₅₀ of 120–140 µ M . The reaction was specific, because most (≥95%) CSF protein remained soluble under conditions where most Aβ was insoluble, as assayed by scanning densitometry of Coomassie-stained gels. Staining of the precipitated material resulted in the visualization of punctate regions that were thioflavin positive or birefringent when stained with Congo red, suggesting the formation of amyloid-related structures. These results suggest that zinc could play a role in amyloid deposition, because there is overlap between the regions of the brain where zinc concentrations are highest and regions with the highest amyloid content. It is surprising that zinc induced the aggregation of endogenous soluble APP at lower concentrations than required for Aβ (EC₅₀ 80 µ M ). The possibility that zinc-induced aggregation of APP may precede the deposition of Aβ into plaques is discussed. Investigation of aggregation of Aβ in CSF will aid in assessing the biological relevance of other agents that have been reported to accelerate amyloid formation.     

Relationship Between the Regulation of Enkephalin-Containing Peptide and Dopamine β-Hydroxylase Levels in Cultured Adrenal Chromaffin Cells

Adrenal medullary chromaffin cells were maintained under conditions known to increase their cellular levels of enkephalin-containing peptides and the effects of these treatments on another chromaffin vesicle component, dopamine beta-hydroxylase, were examined. Catecholamine-depleting drugs, such as tetrabenazine, and cyclic nucleotide-elevating drugs, including forskolin, 8-bromo-cyclic AMP, and theophylline, increase chromaffin cell enkephalin-containing peptide levels but fail to increase dopamine beta-hydroxylase. In contrast, insulin treatment increases the levels of both enkephalin-containing peptides and dopamine beta-hydroxylase. The increased amounts of enkephalin-containing peptides produced by tetrabenazine and by insulin are stored in subcellular particles with properties identical to chromaffin vesicles on density-gradient centrifugation. These results suggest that following insulin treatment chromaffin cells synthesize new chromaffin vesicles with a full complement of enkephalin-containing peptides, but that after treatment with catecholamine-depleting or cyclic nucleotide-related agents enkephalin-containing peptides can be inserted into preexisting vesicles or that new vesicles, made as a part of the normal turnover of cellular components, contain elevated peptide levels.