Highly Sensitive Assay for Dopamine-β-Hydroxylase Activity in Human Cerebrospinal Fluid by High Performance Liquid Chromatography-Electrochemical Detection: Properties of the Enzyme

Abstract: This paper describes a new, sensitive assay for dopamine-β-hydroxylase (DBH) activity in human cerebrospinal fluid (CSF), serum and brain tissues by high performance liquid chromatography (HPLC) with electrochemical detection (ED). Dopamine (DA) was used as a substrate and was incubated under optimal conditions. Norepinephrine (NE) formed enzymatically from DA was isolated by a double-column procedure, the first column of Dowex-50-H⁺ and the second column of aluminum oxide. NE was adsorbed on the second aluminum oxide column and then eluted with 0.5 M-hydrochloric acid and assayed by HPLC-ED. Epinephrine (EN) was added to each incubation mixture as an internal standard, and this assay was therefore highly reproducible. The peak height in HPLC was linear from 500 fmol to 100 pmol of NE and EN. The lower limit of detection for NE formed enzymatically was about 30 pmol, which indicated that the sensitivity of this procedure was comparable to that of radioassay procedures. We applied the method to measurement of the activity of and examination of some of the characteristics of DBH in human CSF. DBH activity in CSF of Parkinsonian patients was lower than that of control patients. The properties of DBH in human CSF were similar to those in serum and adrenal medulla.     

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.     

Genetic effect of mottled locus on reproduction and preweaning growth in laboratory mice

Summary An experiment was conducted to study the maternal and fetal effects of the sex-linked gene tortoise on litter size, birth weight, body weight from birth to 30-day of age, and mortality in normal (N) and mutant (M) mice (Mus musculus). The experiment involved two mating types: (1) N x N (dam x sire) which produced normal male and normal female offspring and (2) M X N which produced mutant males that died in utero, mutant females and normal male and female offspring. Comparison 1 consisted of all phenotypically normal male and female offspring from both N X N and M X N mating types born in 2 parities. The data supports the hypothesis that the tortoise gene, when present in the dam, did not significantly affect the body weight of normal progeny prior to 18 days old. There is also evidence for a negative maternal effect of the tortoise gene on body weight from 21 to 30 days of age postpartum. Mating type X parity interaction was not significant prior to 9 day postpartum. Sex of mice did not influence body weight of siblings prior to 18 day old, but males were heavier than females there-after. Normal and mutant females born in six parities from the M X N mating type constituted Comparison 2. The birth weight of the offspring in Comparison 2 was not significantly influenced by the presence of the tortoise gene. All other body weight measurements, however, were lower for mutant females when compared to normal females. Parity affected all body weight measurements in both comparisons. Mortality rate of the offspring was not influenced by parental mating type or parity, but sex differences were observed. Mutant females had higher mortality than normal sisters. This study provides evidence that the mottled locus in the tortoise dam and progeny influences growth and survival.Reference to a company and/or product named by the USDA is only for purposes of information and does not imply approval or recommendation of the product to the exclusion of others     

Restoration of Norepinephrine and Reversal of Phenotypes in Mice Lacking Dopamine β-Hydroxylase

Abstract: Mice with a targeted disruption of the dopamine β-hydroxylase (DBH) gene are unable to synthesize norepinephrine (NE) and epinephrine. These mice have elevated levels of dopamine in most tissues, although the levels are only a fraction of those normally found for NE. It is noteworthy that NE can be restored to normal levels in many tissues after a single injection of the synthetic amino acid precursor of NE, l -threo-3,4-dihydroxyphenylserine (DOPS). In other tissues, NE can be restored to normal levels after multiple injections of DOPS, whereas in the midbrain and cerebellum, restoration of NE is limited to 25–30% of normal. NE levels typically peak ∼5 h after DOPS administration and are undetectable by 48 h. Epinephrine levels are more difficult to restore. The elevated levels of dopamine fall modestly after injection of DOPS. S (−)-Carbidopa, which does not cross the blood-brain barrier, inhibits aromatic l -amino acid decarboxylase and effectively prevents restoration of NE by DOPS in the periphery, while allowing restoration in the CNS. Ptosis and reductions in male fertility, hind-limb extension, postdecapitation convulsions, and uncoupling protein expression in dopamine β-hydroxylase-deficient mice are all reversed by DOPS injection.     

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.     

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.     

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.     

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.     

Multiple Molecular Forms of Dopamine β-Hydroxylase in the C1300 Mouse Neuroblastoma Tumor and in the Serum of Tumor-Bearing Mice

Abstract: The distribution of the enzymatic activity of dopamine β-hydroxylase (DBH) in linear sucrose gradients was studied for a soluble fraction of the C₁₃₀₀ mouse neuroblastoma tumor, for the serum of tumor-bearing A/J mice, and for adrenal tissue and serum of control mice. In controls (adrenal gland and serum of A/J mice), about 75% of the DBH activity was associated with a high-molecular-weight form, denoted as DBHA_A, with an apparent sedimentation coefficient of 11.3 S. About 25% of the DBH activity was attributable to a slower-sedimenting species (7.1 S), denoted as DBHB_B. In tumor supernatants and in the serum of tumor-bearing mice, about 55% of the DBH activity was present as the 7.1 S species (DBHR_B), while only 35% was recovered as the high-molecular-weight form (DBHA_A). Approximately 5% of the activity could be attributed to a separate form, with a sedimentation coefficient of about 4.5 S. This form is designated DBH_C. The ratio DBHR/DBHA is significantly higher in tumor tissue and in serum of tumor-bearing mice than in controls. The three enzymically active forms of DBH in the C₁₃₀₀ tumor are considered to represent the tetrameric (DBHA_C), dimeric (DBH_B), and monomeric (DBH_C) forms of the enzyme.     

Effects of Systemic Administration of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine to Mice on Tyrosine Hydroxylase, l-3,4-Dihydroxyphenylalanine Decarboxylase, Dopamine β-Hydroxylase, and Monoamine Oxidase Activities in the Striatum and Hypothalamus

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg/kg subcutaneously per day for 8 days) to C57BL/6N mice were studied on tyrosine hydroxylase (TH), L-3,4-dihydroxyphenylalanine decarboxylase (DDC), and monoamine oxidase (MAO) activities in the striatum, and TH, DDC, dopamine-beta-hydroxylase (DBH), and MAO activities in the hypothalamus. Treatment with MPTP led to a large decrease in TH activity and a parallel decrease in DDC activity in the striatum, as compared with the saline controls. In contrast, MPTP administration did not cause a decrease of the activities of TH, DDC, and DBH in the hypothalamus. There was also no reduction in MAO activities of striatum and hypothalamus. These data indicate that MPTP administration to mice results in specific degeneration of the dopaminergic nigrostriatal pathway and that DDC in the mouse striatum may mainly be localized in the dopaminergic neurons with TH.     

β(1,3)-Glucanases from Geotrichum lactis: activity on its own nascent and preformed β(1,3)-glucan

Abstract Actively growing mycelium of Geotrichum lactis contains at least three β(1,3)-glucanase activities. Two of the activities have been characterized as exo- and the third as endo-hydrolytic. The action of the activities on β(1,3)-glucan synthesized in vitro by the β(1,3)-glucan synthase system from G. lactis has been studied. One of the exo-β-glucanases and the endo-β-glucanase were active on this β(1,3)-glucan and the degradation rates were higher on nascent than on preformed β(1,3)-glucan.     

Variation in plasma glucose and pancreatic β cells in the turtle, Lissemys punctata (order: Chelonia; family: Trionychidae)

The present study relates to the determination of the plasma glucose level and volumetric analysis of β cells in pancreatic islets of the soft‐shelled turtle Lissemys punctata during different phases of its reproductive cycle. Reproductive events play a vital role in influencing the plasma glucose level and β‐cell behaviour in the pancreatic islets. The colour of the pancreas is either yellowish or pinkish, depending on endocrine activity. Islets are present throughout the gland and range from individual cells to small or large clumps, depending on the seasonal cycle. Splenic islets are dense with more blood capillaries and nerve innervations irrespective of sex and season. The endocrine cell mass forms irregular patches without connective tissue capsule. β cells occupy the inner region of the islets, being surrounded by other cell types. Lissemys punctata exhibits higher β‐cell activity during hibernation. Most insulin‐secreting cells acquire a larger size during the regressive period. An analysis indicates that β cells outnumber the non‐β endocrine cell mass in both number and per cent volume. There is negative correlation between islet mass and animal weight. Between the periods of reproductive cycles, a difference exists with respect to fasting plasma glucose and β‐cell volume.     

Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-β (1–42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators

Aggregation of amyloid-β (Aβ) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Aβ aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Aβ₄₂ fibrillization and initiate formation of non-fibrillar Aβ₄₂ aggregates, and that the inhibitory effect of Zn(II) (IC₅₀ = 1.8 μmol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Aβ₄₂ aggregation. Moreover, their addition to preformed aggregates initiated fast Aβ₄₂ fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Aβ₄₂. H13A and H14A mutations in Aβ₄₂ reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-β core structure within region 10–23 of the amyloid fibril. Cu(II)-Aβ₄₂ aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Aβ₄₂ aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Aβ aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.     

Evidence for a third allele at the β-lactoglobulin (β-Lg) locus of sheep milk and its occurrence in different breeds

Summary. Milk samples from 189 Merinoland Sheep, 145 Black Faced Mutton Sheep, 89 East Friesian Milk Sheep, 36 Rhön, 36 Pleven, 23 Tsigaja, 25 Black Razka and 86 Hungarian Merino x Pleven (F1) sheep were analysed by polyacrylamide gel electrophoresis under acid conditions and isoelectric focusing in ultrathin layer polyacrylamide gels with carrier ampholytes. Six different β-lactoglobulin (β-Lg) phenotypes (A, AB, B, AC, BC and C) were observed by both methods. The occurrence of three codominant alleles (β-Lg^A, β-Lg^B, β-Lg^c) at an autosomal locus (β-Lg) was supported by family and population data on genetic equilibrium. Differences in gene frequencies between the breeds were observed.     

α-N-Acetyl β-Endorphins in the Pituitary: Immunohistochemical Localization Using Antibodies Raised Against Dynorphin(1-13)

Abstract: Intense immunohistochemical staining of the intermediate lobe of the pituitary was observed by using an antiserum raised against synthetic dynorphin(1-13) treated with a water-soluble carbodiimide (CDI). Subsequent studies showed that the immunostaining was blocked by preincubation of the antiserum with acetylated derivatives of both β-endorphin and dynorphin(1-13) as well as by CDI-treated dynorphin(1-13), but only weakly by authentic dynorphin(1-13). Neither nonacetylated β-endorphin nor any other fragments of the ACTH/endorphin precursor blocked the immunostaining of the intermediate lobe. Analysis of the CDI-treated dynorphin(1-13) used as an antigen showed that most of the peptide was acetylated at primary amino groups. CDI treatment of dynorphin(1-13) results in the formation of an acetyl derivative because the commercially available peptide is supplied as the acetate salt. The antibodies responsible for the intermediate lobe staining were isolated by affinity chromatography, using a column containing partially purified intermediate lobe extract linked to an affinity resin and a radioimmunoassay (RIA) was developed with CDI-treated dynorphin(1-13) used as a trace and as a standard. Competition studies showed 0.5-1% cross-reactivity with α-N-acetyl β-endorphin(1-31), α-N-acetyl β-endorphin(1-27), and totally acetylated β-endorphin(1-31). Nonacetylated β-endorphins did not cross-react. Posterior-intermediate lobe extracts from rat and beef were fractionated by gel filtration. Rat posterior-intermediate lobe extracts were also fractionated by cation-exchange chromatography. Fractionated extracts were analyzed by RIAs for β-endorphin, CDI-treated dynorphin(1-13), and authentic dynorphin(1-13). The results suggested that the peptides responsible for the intermediate lobe staining were mainly four different derivatives of β-endorphin bearing an acetyl group at the amino terminus. No immunostaining was seen in the posterior and anterior lobes of the pituitary. This suggests that the intermediate lobe is the main source of acetylated β-endorphins in the pituitary.     

Lipopolysaccharide and Interleukin-1β Augmented Histidine Decarboxylase Activity in Cultured Cells of the Rat Embryonic Brain

Abstract: We investigated the effect of lipopolysaccharide (LPS) and various inflammatory cytokines on the histidine decarboxylase (HDC) activity in cultured cells of the rat embryonic brain. Histaminergic neuronal cell bodies were supposed to exist in cultured cells of the diencephalon but not in those of the cortex. The HDC activity was elevated by adding LPS and interleukin-1 β (IL-1β) but not by tumor necrosis factor-α (TNF-α) and IL-6 to the mixed primary cultures of diencephalon. In the adherent cell fraction of the cultured diencephalon cells, HDC activity was also enhanced by LPS and IL-1β. In a similar manner, LPS augmented HDC activity in the mixed primary culture of cerebral cortical cells and in its adherent cell fraction. The effects of IL-1β but not LPS in the mixed primary culture of diencephalon were canceled by a prior exposure to cytosine-β- d -arabinofuranoside. The changes in HDC activity after exposure to LPS for 12 h were not accompanied by increased mRNA levels. In these cell cultures, mast cells were not detected by Alcian Blue staining. These results indicated the presence of the third type of HDC-bearing cell besides neurons and mast cells in the brain. The increase of HDC activity by IL-1β might be due to cell proliferation.     

Bovine haemoglobin βA Zebu, βA43(CD3)Ser Thr: an intermediate globin type between the βA and βD Zambia is present in Indian zebu cattle

Two bovine haemoglobin beta chains, electrophoretically identical with the beta A chain of Herefords, were obtained from Ongole and Banteng, Bos javanicus, cattle. The amino acid residue differences of the two beta chains were compared by electrophoresis, cation-exchange and reverse-phase chromatography, amino acid analyses, and Edman degradation in comparison with beta A chain. The results showed that two beta chains differed from the beta A chain of the Hereford breed by the substitution of serine with threonine at the beta 43 position. No other difference was found between the two chains and beta A. This new beta chain type was termed beta A Zebu, which forms a possible evolutionarily transitional type between the beta A and the rare variant beta D Zambia found previously in African zebu cattle. The beta A Zebu differentiates from the previous beta B by at least four amino acid substitutions involving five codon-base changes.