Biochemical studies of the chromaffin granule. III. Redistribution of lipid phosphate,dopamine-β-hydroxylase and chromogranin a after freezing and thawing of the isolated granule membranes

Freezing and thawing a dialysed suspension of lysed chromaffin granules and sedimented membrane preparations resulted in redistribution of lipid phosphate and protein. By this treatment the high ratios of lipid phosphate/protein in the membrane fragments, isolated on sucrose density gradient from the dialysed suspensions and the sedimented membrane preparation, were reduced from 1.56 to 1.03 μmoles/mg and from 1.97 to 0.83 μmoles/mg, respectively.Multilamellar, liposomal structures could be isolated from the frozen and thawed membrane preparations and were found to sediment in the 0.4 M sucrose layer by density gradient centrigugation. This fraction was without morphological resemblance to the intact chromaffin granules or their membranes and was found to account for 53% of the lipid phosphate, 35% of chromogranin A, 21% of dopamine-β-hydroxylase activity and 12% of the protein of the total preparation. The specific activities of chromogranin A and dopamine-β-hydroxylase in the artificially formed liposomal structures closely resembled that of the solubilized protein and was significantly higher than in the lipid phosphate-depleted membrane fragments recovered in the 1.1 M sucrose layers.It is concluded that freezing and thawing as a means of purifying the isolated granule membranes lead not only to the solubilization of chromogranin A, but also to removal of dopamine-β-hydroxylase activity and lipid phosphate from the labile membrane fragments.     

Immunofluorescent and biochemical studies on tyrosine hydroxylase and dopamine-β-hydroxylase of the bullfrog sciatic nerves

Summary Immunofluorescence specific for tyrosine hydroxylase (TH) or dopamine--hydroxylase (DBH) was accentuated in both proximal and distal segments of the sciatic nerve after ligation. Estimations of the enzyme activities confirmed the above results. Mean axoplasmic flow rates of TH and DBH in bullfrog sciatic nerve were found to be 8 and 123 mm/day, respectively. They were decreased by colchicine or by cold temperatures (4° C).     

Direct evidence for co-localization of adenylate cyclase,dopamine-β-hydroxylase and cytochrome b562 to bovine chromaffin granule membranes

Adenylate cyclase, dopamine-β-hydroxylase and cytochrome b₅₆₂ have been found to co-equilibrate on equilibrium sucrose gradients of lysed chromaffin granule membranes from bovine adrenal medulla. Peak activities for these enzymes, as well as maximum membrane protein concentration, were found to coincide at d = 1.11 gm cm^?3, and the ratios of adenylate cyclase to both dopamine-β-hydroxylase and cytochrome b₅₆₂ were constant across the entire peak. Adenylate cyclase activity has been reported previously to co-purify with chromaffin granule membranes, and we conclude, on the basis of these new data, that adenylate cyclase is also an intrinsic granule membrane enzyme.     

Consecutive demonstration of catecholamines and dopamine-β-hydroxylase within the same specimen

Summary A combined method was developed for characterization and differentiation of catecholamines in neuron populations containing more than one catecholamine, e.g. dopamine and norepinephrine. Its application to small intensely fluorescent (SIF-) cell clusters in sympathetic ganglia allows the successive demonstration of glyoxylic acid-induced catecholamine fluorescence and dopamine--hydroxylase by indirect immunofluorescence within the same tissue section. Applying this technique to an example, two types of SIF-cells were demonstrated in the guinea pig superior cervical ganglion.Supported by the Deutsche Forschungsgemeinschaft grant He 919/4     

A sensitive fluorometric assay for dopamine-β-hydroxylase activity by high-performance liquid chromatography

A sensitive and specific fluorometric assay for dopamine-β-hydroxylase (DBH) activity is described. The main natural substrate, dopamine (DA), was used and incubated under optimal conditions. Norepinephrine (NE) formed enzymatically from DA was isolated by an aluminum oxide column and was analyzed by high-performance liquid chromatography (HPLC) with trihydroxyindole fluorescence. Epinephrine (EN) was added to the incubation mixture as an internal standard after incubation, and this assay was therefore highly reproducible. HPLC conditions were settled to elute the product, NE, prior to the substrate, DA, and the internal standard, EN, between NE and DA. Only catecholamines produced significant peaks, and therefore, this assay is highly specific. We applied this method to measure the DBH activity in human serum and cerebrospinal fluid.     

Effect of tyramine and guanethidine on dopamine-β-hydroxylase activity and norepinephrine concentrations in vesicular fraction of the heart and plasma of rats

Repeated administration of high doses of tyramine to rats results in a striking increase in plasma levels of norepinephrine (NE) and a marked depletion in tissue content of NE. The drug also may produces a moderate increase in plasma levels of dopamine-β-hydroxylase (DBH) and a decrease in DBH in synaptic vesicles of sympathetic nerves in the heart. The latter effects are prevented by a ganglionic blocking agent, indicating that they may be mediated by neuronal activation secondary to the stress attending the drug administration. Chronic administration of guanethidine, which is reported to destroy most sympathetic nerves produces more marked decrease in plasma NE levels and plasma DBH activity. The possible sources of this activity are discussed.     

Biochemical and genetic studies on rabbit hemoglobin. II. Electrophoretic polymorphism of theα-chain

A genetic polymorphism of rabbit (Oryctolagus cuniculus) hemoglobin chain is demonstrated by means of acid starch gel electrophoresis. The polymorphism is not detected by isoelectric focusing and may be based upon neutral for neutral amino acid substitutions in accordance with previous findings by means of amino acid sequencing. Segregation analysis was performed on 15 matings with 49 offspring and confirmed the initial genetic hypothesis of three common codominant alleles at an autosomal locus. The calculated gene frequencies in a random sample of 86 unrelated individuals areHBA*1=0.73,HBA*2=0.22, andHBA*3=0.05.     

Amino acid and carbohydrate compositions of human serum dopamine-β-hydroxylase

Dopamine--hydroxylase has been purified from human serum. The amino acid composition has been determined and found to be similar to that of the enzyme purified from human pheochromocytoma. Human serum dopamine--hydroxylase is a glycoprotein, containing 13.11 g carbohydrates/100 g protein. Individual sugar determinations showed the presence of fucose, galactose, glucose, mannose,N-acetylglucosamine,N-acetylgalactosamine andN-acetylneuraminic acid.     

Phenylethanolamine-N-methyltransferase and dopamine-β-hydroxylase immunoreactivity and the occurrence of noradrenaline and adrenaline in the nervous system of the snail Helix aspersa

Summary The presence of dopamine--hydroxylase (DBH) and phenylethanol-amine-N-methyltransferase (PNMT) immunoreactivity in specific neurones of the snail Helix aspersa has been demonstrated. In addition, high performance liquid chromatography and electrochemical detection have revealed the presence of noradrenaline and adrenaline in the snail central nervous system, although the major catecholamine is dopamine. These results suggest that adrenaline, and perhaps noradrenaline, have transmitter or modulatory functions in the snail nervous system.     

Biochemical and structural characterization of recombinant hyoscyamine 6β-hydroxylase from Datura metel L.

Hyoscyamine 6β-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6β-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. metel (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. metel (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6β-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K_m values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50 μM each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of α-helicity in the secondary structure. From the fluorescence studies, Stern–Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14 M^?1 and 0.56 M^?1, respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes.     

Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine-β-hydroxylase

Presynaptic muscarinic receptors labeled with [³H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [³H]quinuclidinyl benzilate or [³H]dexetimide binding revealed a distribution profile similar to that of dopamine-β-hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine-β-hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine-β-hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle.     

Hypothalamic dopamine-β-hydroxylase activity: Fluctuations with time of day and their modifications by intracranial surgery,adrenalectomy, and pinealectomy

Hypothalamic and plasma dopamine--hydroxylase (DBH, EC 1.14.2.1) activities were measured by a coupled radioenzymatic method. Animals representing five experimental groups (intact controls, adrenalectomized, pinealectomized, adrenalectomized + pinealectomized, doubly sham-operated) were killed and sampled at 8 times through the 24-hr daily cycle, 15 days postoperation, and at 50–52 days of age. Hypothalamic DBH in intact control animals had statistically significant fluctuations in relation to time of day. These changes were lost or dampened in groups that had had intracranial surgery and were characteristically shifted by adrenalectomy, either alone or with pinealectomy. Plasma DBH fluctuations in the same animals resembled those in hypothalamus in some features (e.g., peak near mid-dark; shift in daily maxima and minima after adrenalectomy) and differed in others (e.g., no effect of intracranial surgery or of sham operation; adrenalectomized + pinealectomized animals resembled the solely pinealectomized). Although temporal patterns in hypothalamic DBH activity thus differed in the experimental animal groups, the daily means of hypothalamic DBH activity were similar.     

Electron microscopic analysis of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat

Summary The catecholaminergic innervation of the hypothalamic paraventricular nucleus (PVN) of the rat was studred by preembedding immunocytochemical methods utilizing specific antibodies which were generated against catecholamine synthesizing enzymes. Phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive terminals contained 80–120 nm dense core granules and 30–50 nm clear synaptic vesicles. The labeled boutons terminated on cell bodies and dendrites of both parvo- and magnocellular neurons of PVN via asymmetric synapses. The parvocellular subnuclei received a more intense adrenergic innervation than did the magnocellular regions of the nucleus. Dopamine--hydroxylase (DBH)-immunopositive axons were most numerous in the periventricular zone and the medial paryocellular subnucleus of PVN. Labeled terminal boutens contained 70–100 nm dense granules and clusters of spherical, electron lucent vesicles. Dendrites, perikarya and spinous structures of paraventricular neurons were observed to be the postsynaptic targets of DBH axon terminals. These asymmetric synapses frequently exhibited subsynaptic dense bodies. Paraventricular neurons did not demonstrate either PNMT or DBH immunoreactivity. The fibers present within the nucleus which contained these enzymes are considered to represent extrinsic afferent connections to neurons of the PVN.Tyrosine hydroxylase (TH)-immunoreactivity was found both in neurons and neuronal processes within the PVN In TH-cells, the immunolabel was associated with rough endoplasmic reticulum, free ribosomes and 70–120 nm dense granules. Occasionally, nematosome-like bodies and cilia were observed in the TH-perikarya. Unlabeled axons established en passant and bouton terminaux type synapses with these TH-immunopositive cells. TH-immunoreactive axons terminated on cell bodies as well as somatic and dendritic spines of paraventricular parvocellular neurons. TH-containing axons were observed to deeply invaginate into both dendrites and perikarya of magnocellular neurons.These observations provide ultrastructural evidence for the participation of central catecholaminergic neuronal systems in the regulation of the different neuronal and neuroendocrine functions which have been related to hypothalamic paraventricular neurons.Supported by NIH Grant NS 19266 to W.K. Paull     

Immunocytochemical localization of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase in the adrenal glands of the frog and rat by a peroxidase-antiperoxidase method

Summary The subcellular locilazations of tryrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in the adrenal glands of the frog and rat have been examined by a peroxidase-antiperoxidase (PAP) method. TH was localized in the ground substance of the adrenaline-containing cells and noradrenaline-containing cells, but not in the nucleus or in the mitochondria. TH was also located on the outside of the membrane of the chromaffin granules. DBH was observed only inside the granules. PNMT was found not only in the ground substance but also on the membrane of some adrenaline-containing granules. Cortical lipid cells of the frog adrenals did not show TH-, DBH-, and PNMT-reactions. The negative reactions to TH-, DBH-, and PNMT-antiserum exhibited by the summer cells of the frog adrenals prove that they belong to the cortical cells.     

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

Summary A highly sensitive and specific monoclonal antibody against the enzyme dopamine -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.     

A. A. Ukhtomskiĭ's ideas in studies on the cognitive activity

Experimental data on spatial and temporal organisation of the human EEG from birth to maturity, at resting and during cognitive activity, is presented. The data obtained is interpreted in light of a confirmation of the Ukhtomsky's ideas on the "functional working organ" appearing in the brain for realising cognitive processes. Role of the alpha-rhythm in formation of plastic intracortical neuronal network is elucidated. A hypothesis is advanced stating that the gradually developing brain system exerting a local regulated activating effects upon the cortical level, is a significant factor of the functional intercentre integration.     

Zwittermicin A: Synthesis of analogs and structure–activity studies

Analogs and diastereomers of the natural product zwittermicin A were prepared. SAR studies of these compounds reveal the antifungal activity to be dependent singularly upon the natural constitution and configuration.     

Synthetic studies on selective adenosine A2A receptor antagonists. Part II: Synthesis and structure–activity relationships of novel benzofuran derivatives

Based on the previously reported lead compound, a series of benzofuran derivatives were prepared to study their antagonistic activities to A_2A receptor. The replacement of the phenyl group at the 4-position with a heterocyclic ring improved the PK profile and aqueous solubility. From these studies, we discovered a potent new A_2A antagonist, 12a, which has both a good oral bioavailability and in vivo efficacy on motor disability in MPTP-treated common marmosets.     

Screening of mycelial fungi for 7α- and 7β-hydroxylase activity towards dehydroepiandrosterone

In total, 481 fungal strains were screened for the ability to carry out 7(α/β)-hydroxylation of dehydroepiandrosterone (DHEA, 3β-hydroxy-5-androsten-17-one). Representatives of 31 genera of 15 families and nine orders of ascomycetes, 17 genera of nine families and two orders of zygomycetes, two genera of two families and two orders of basidiomycetes, and 14 genera of mitosporic fungi expressed 7(α/β)-hydroxylase activity. The majority of strains were able to introduce a hydroxyl group to position 7α. Active strains selectively producing 3β,7α-dihydroxy-5-androsten-17-one were found among Actinomucor, Backusella, Benjaminiella, Epicoccum, Fusarium, Phycomyces and Trichothecium, with the highest yield of 1.25 and 1.9 g L^?1 from 2 and 5 g L^?1 DHEA, respectively, reached with F. oxysporum. Representatives of Acremonium, Bipolaris, Conidiobolus and Curvularia formed 3β,7β-dihydroxy-5-androsten-17-one as a major product from DHEA. The structures of the major steroid products were confirmed by TLC, gas chromatography (GC), mass spectra (MS), and ¹H-NMR analyses.