Biochemical studies of the chromaffin granule. II. Properties of membrane-bound and water-soluble forms of chromogranin A and dopamine-β-hydroxylase activity

1. Chromogranin A has been isolated from the washed membrane fraction of highly purified chromaffin granules after solubilization in Triton X-100. A hydrated molecular weight of 2.9·10⁵ has been obtained for chromogranin A_M which in its electrophoretic mobility and molecular volume appears very similar to chromogranin A_I of the water-soluble fraction.     

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.     

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).     

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     

Membrane proteins of chromaffin granules. Dopamine β-hydroxylase, a major constituent

1. Soluble lysates and membranes were prepared from chromaffin granules isolated from bovine adrenal medulla. The detergent N-cetylpyridinium chloride was used for solubilizing the membrane proteins, including the membrane-bound dopamine (2,4-dihydroxyphenethylamine) beta-hydroxylase. The solubilized proteins were fractionated by Sephadex chromatography in the presence of N-cetylpyridinium chloride. The major component of the membrane proteins, i.e. chromomembrin A, was identified as the enzyme dopamine beta-hydroxylase. 2. The addition of N-cetylpyridinium chloride to the soluble lysate caused precipitation of up to 96% of the proteins, but only a small proportion of the dopamine beta-hydroxylase activity was precipitated. The only protein demonstrable in the supernatant by polyacrylamide-gel electrophoresis was the protein that has a lower mobility than chromogranin A in disc gel electrophoresis. This component has been identified previously as dopamine beta-hydroxylase. Thus, this method provides an extremely simple isolation procedure for dopamine beta-hydroxylase. 3. A comparison of the membrane-bound and soluble dopamine beta-hydroxylases revealed the identity of these two preparations. Both were activated by N-cetylpyridinium chloride, they migrated identically in polyacrylamide-gel electrophoresis, their amino acid composition was very similar and an immunological cross-reaction could be demonstrated.     

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.     

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.     

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.     

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     

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.     

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.     

Investigating the membrane orientation and transversal distribution of 17β-estradiol in lipid membranes by solid-state NMR

17β-Estradiol (E₂) is a potent estrogen, which modulates many important cellular functions by binding to specific estrogen receptors located in the cell nucleus and also on the plasma membrane. We have studied the membrane interaction of E₂ using a combination of solid-state NMR methods. ²H NMR results indicate that E₂ does not cause a condensation effect of the surrounding phospholipids, which is contrary to the effects of cholesterol, and only very modest E₂ induced alterations of the membrane structure were detected. ¹H magic-angle spinning NMR showed well resolved signals from E₂ as well as of POPC in the membrane-lipid layer. Two-dimensional NOESY spectra revealed intense cross-peaks between E₂ and the membrane lipids indicating that E₂ is stably inserted into the membrane. The determination of intermolecular cross-relaxation rates revealed that E₂ is broadly distributed in the membrane with a maximum of the E₂ distribution function in the upper chain region of the membrane. We conclude that E₂ is highly dynamic in lipid membranes and may undergo rotations as it exhibits two polar hydroxyl groups on either side of the molecule.     

Biochemical and biophysical studies on cytochrome c oxidase. XVII. An epr study of the photodissociation of cytochrome a32+ · CO

1. The photodissociation reaction of the cytochrome c oxidase-CO compound was studied by EPR at 15 °K. Illumination with white light at both room and liquid N₂ temperatures of the partially reduced cytochrome c oxidase (2 electrons per 4 metals) in the presence of CO, causes the appearance of a rhombic (g_x = 6.60, g_y = 5.37) high-spin heme signal.This signal disappears completely upon darkening of the sample and reappears upon illumination at room temperature; accordingly the photolytic process is reversible. Under these conditions, no great changes in the intensities are observed, neither of the copper signal at g = 2, nor of the low-spin heme signal at g = 3, 2.2 and 1.5.2. In the presence of ferricyanide (2 mM) and CO, both the low-spin heme signal (g = 3.0, 2.2 and 1.5) and the copper signal of the partially reduced enzyme have intensities about equal to those of the completely oxidized enzyme in the absence of CO. Upon illumination of the carboxy-cytochrome c oxidase in the presence of ferricyanide, it was found that the rhombic high-spin heme signal appears without affecting appreciably the copper of low-spin heme signals. Thus, in the presence of ferricyanide the EPR-detectable paramagnetism of the illuminated carboxy-cytochrome c oxidase is higher than in the untreated oxidized enzyme.3. The membrane-bound cytochrome c oxidase reduced with NADH in the presence of CO and subsequently oxidized with ferricyanide shows a similar rhombic high-spin heme signal (g_x = 6.62, g_y = 5.29) upon illumination at room temperature. This signal disappears completely upon darkening and reappears upon illumination at room temperature.     

Biochemical and biological properties of cortexillin III,a component of Dictyostelium DGAP1–cortexillin complexes

Cortexillins I–III are members of the α-actinin/spectrin subfamily of Dictyostelium calponin homology proteins. Unlike recombinant cortexillins I and II, which form homodimers as well as heterodimers in vitro, we find that recombinant cortexillin III is an unstable monomer but forms more stable heterodimers when coexpressed in Escherichia coli with cortexillin I or II. Expressed cortexillin III also forms heterodimers with both cortexillin I and II in vivo, and the heterodimers complex in vivo with DGAP1, a Dictyostelium GAP protein. Binding of cortexillin III to DGAP1 requires the presence of either cortexillin I or II; that is, cortexillin III binds to DGAP1 only as a heterodimer, and the heterodimers form in vivo in the absence of DGAP1. Expressed cortexillin III colocalizes with cortexillins I and II in the cortex of vegetative amoebae, the leading edge of motile cells, and the cleavage furrow of dividing cells. Colocalization of cortexillin III and F-actin may require the heterodimer/DGAP1 complex. Functionally, cortexillin III may be a negative regulator of cell growth, cytokinesis, pinocytosis, and phagocytosis, as all are enhanced in cortexillin III–null cells.     

Biochemical,Mössbauer,and EPR studies of the diiron cluster of phenol hydroxylase from Pseudomonas sp. strain CF 600

Phenol hydroxylase of Pseudomonas sp. strain CF600 comprises three components: DmpP is an FAD- and [2Fe-2S]-containing reductase; DmpM is a cofactorless activator protein; and DmpLNO is the oxygenase. Single turnover experiments established that DmpLNO contains the active site, but requires DmpM for efficient turnover: the steady-state turnover rate reaches a maximum at 1.5 DmpM:1 DmpLNO. Chemical cross-linking experiments showed that DmpM interacts with the large subunit of the DmpLNO oxygenase complex. M?ssbauer studies revealed that the active site of the oxygenase can accommodate two types of diiron clusters, each of these cluster types having two equivalent sites. Cluster form I, representing typically around 85% of total Fe, has DeltaE(Q) = 1.73 mm/s and delta = 0.54 mm/s, while cluster II exhibits DeltaE(Q) = 0.79 mm/s and delta = 0.48 mm/s. Studies in strong applied magnetic fields suggest that the two iron sites of cluster I are bridged by an oxo group while sites in cluster II appear to be hydroxo-bridged. Reduction of the samples with dithionite yields the diferrous forms of the clusters. Air oxidation of the reduced samples leads to an increase of the cluster II fraction, accompanied by a corresponding decrease in catalytic activity. The reduced oxygenase samples exhibit at X-band an integer spin EPR signal centered, in parallel mode, at g = 16.6. Quantitative analysis showed that 19% of the clusters contribute to the EPR signal, suggesting that cluster II is the EPR-active species. Incubation with dithiothreitol (DTT) inactivated the oxygenase by a mechanism apparently involving H(2)O(2) generation. In addition, M?ssbauer studies of DTT-inactivated enzyme showed that all ferric iron belonged to one diamagnetic diferric cluster with parameters that indicate that DTT coordinates to the cluster.     

Biochemical detection of a metallo-β-lactamase in carbapenem resistant strain ofStreptomyces sp. CN229 isolated from soil

Streptomyces sp. CN229 was isolated from Tunisia soil. This strain displayed antimicrobial activity against Gram positive and Gram negative bacteria. In addition it is resistant to most β-lactam antibiotics including imipenem and meropenem (MIC imipenem >70 μg/ml). Metallo-β-lactamase (MβL) production was confirmed by either imipenem MIC decrease in the presence of ethylene diamine tetraactic acid (EDTA) or the inhibition zone enhancement around EDTA-impregnated imipenem, or meropenem discs. Isolectric focusing analysis demonstrated the production of β-lactamase with pI of 5.8 that is inhibited by EDTA.Streptomyces sp. CN229 was screened for the imipenem resistance genes,bla _VIM andbla _IMP previously identified inPseudomonas aeruginosa. The presence of these genes was not confirmed by specific PCR analysis. We concluded that carbapenem resistance inStreptomyces sp. CN229 strain is mainly due to production of a novel carbapenemase. Our data show for the first time that MβL is produced byStreptomyces sp. MβL-mediated imipenem and meropenem resistance inStreptomyces is a cause for concern in the study of resistance evolution and antibiotic cluster biosynthetic genes.     

Biochemical studies in a eunicid (polychaete) of the littoral of São Paulo,Brazil

• 1.1. The sodium, potassium, chloride, calcium, magnesium, phosphorus, sulphur, copper, iron, total carbon dioxide, uric acid, creatinine, urea, glucose, erythrocruoin, nitrogen, total iodine, protein-bound iodine, total lipids, triglycerides, alkaline phosphatase activity, acid phosphatase activity and copper oxidase activity contents of the blood of the giant Polychaete, Eunice sp., were determined.
• 2.2. The osmolarity of the blood was 997 mOsm/1 and the pH was 6·49, a very low value. The bicarbonate concentration estimated by the Henderson-Hasselbach equation was 4·70 mM/1.
• 3.3. The values of the sea water, sediments, water contained in the tube, tube, cuticle, muscle and faeces are also given.
• 4.4. The chemical composition of the mucus was determined.

     

Immunohistochemical demonstration of peptides,serotonin and dopamine-β-hydroxylase-like material in the nervous system of the leechHirudo medicinalis

Summary The central ganglia of the leech,Hirudo medicinalis, were processed for the immunohistochemical localisation of bombesin-, substance P-, cholecystokinin-, vasoactive intestinal polypeptide-, enkephalin-, serotonin- and dopamine--hydroxylase-related substances. To varying extents all of the substances were localised in neuropile processes, and all, with the exception of substance P, were associated with specific perikarya. The most prominent neuropeptides, in terms of the number of immunoreactive neurones, were cholecystokinin and vasoactive intestinal peptide. The dopamine--hydroxylase positive neurones are thought to be octopaminergic, and the serotonin monoclonal antibody revealed positive staining in the Retzius cells. We were unable to demonstrate the coexistence of pairs of substances in any neurones in the leech ganglia.     

Effect of the ω-acylceramides on the lipid organization of stratum corneum model membranes evaluated by X-ray diffraction and FTIR studies (Part I)

The lipid organization in the outermost layer of the skin, the stratum corneum, is important for the skin barrier function. The stratum corneum lipids are composed of ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL). In the present study Fourier transform infrared (FTIR) and small-angle X-ray scattering (SAXS) techniques were utilized to evaluate the effect of three C18 fatty acid esterified ω-acylceramides (CER EOS) on the lipid organization of stratum corneum model membranes. FTIR spectra (scissoring and rocking bands) showed as a function of temperature significant line-shape changes for both components assigned to the orthorhombic phase. Second-derivative analyzes revealed a significant decrease in the interchain coupling strength (Δν values) for the samples formed by CER EOS with the linoleate (CER EOS-L) and oleate (CER EOS-O) moiety around 28.5 °C. However, only a gradual decrease in the Δν values was noticed for the mixture formed with CER EOS with the stearate moiety (CER EOS-S) over the whole temperature range. In the absence of CER EOS the decrease started already at 25.5 °C, demonstrating that CER EOS stabilized the orthorhombic lattice. This stabilization was most pronounced for the CER EOS-S. Spectral fittings allowed to evaluate the orientation changes of the skeletal plane within the orthorhombic unit cell (θ values) for a given temperature range. From the best-fit parameters (peak area values), a decrease in the orthorhombic phase contribution to the scissoring band was also monitored as a function of the temperature. SAXS studies showed the coexistence of two lamellar phases with a periodicity of ∼5.5 nm (short periodicity phase, SPP) and ∼12 nm (LPP) in the presence of the CER EOS-L and CER EOS-O. However, no diffraction peaks associated to the LPP were detected for CER EOS-S. While CER EOS-S most efficiently stabilized the orthorhombic phase, CER EOS-L and CER EOS-O promoted the presence of the LPP. Therefore, the presence of all three CER EOS as observed in human stratum corneum may contribute to a proper skin barrier function.