Isolation and characterization of tetrahydropterin oxidation products generated in the tyrosine 3-monooxygenase (tyrosine hydroxylase) reaction

The catalytic mechanism of tyrosine 3-monooxygenase (tyrosine hydroxylase, EC 1.14.16.2), isolated from the cytosolic fraction of bovine adrenal medulla, was studied by new techniques of product isolation and characterization. Using either (6R)-tetrahydrobiopterin, (6RS)-tetrahydroneopterin, or 6-methyl-tetrahydropterin, as the cofactors, three enzymatic oxidation products could be isolated and identified from the reaction mixture by high-performance liquid chromatography and rapid-scanning spectroscopy: (a) the 4a-hydroxy derivatives, (b) the quinonoid dihydropterins, and (c) the stable 7,8-dihydropterins. Stopped-flow spectroscopy revealed that the formation of the 4a-hydroxy-tetrahydropterins preceded the formation of the quinonoid forms with both L-tyrosine and L-phenylalanine as the substrate. The formations of 4a-hydroxy-tetrahydropterins and hydroxylated amino acids were tightly coupled as recently shown in the phenylalanine 4-monooxygenase reaction [Haavik, J., D?skeland, A. P. & Flatmark, T. (1986) Eur. J. Biochem. 160, 1-8]. No detectable carbinolamine dehydratase activity was present in the enzyme preparation.     

Different acute effects of the tyrosine hydroxylase inhibitors alpha-methyl-p-tyrosine and 3-iodo-L-tyrosine on hypothalamic noradrenaline activity and adrenocorticotrophin release in the rat

Computerized gas chromatography-mass spectrometry techniques using selected ion monitoring and deuterated internal standards were used to assay simultaneously the medial basal hypothalamic concentrations of dopamine (DA) and noradrenaline (NA) and their major metabolites in individual rats 30 min after the administration of two different inhibitors of tyrosine hydroxylase, alpha-methyl-p-tyrosine (alpha-MT) and 3-iodo-L-tyrosine (MIT). Consistent with inhibition of DA synthesis, administration of both alpha-MT and MIT resulted in marked reductions (P less than 0.005) in the hypothalamic concentrations of DA and its metabolite homovanillic acid as well as in highly significant increases in prolactin secretion. alpha-MT administration, but not MIT, resulted in a highly significant decrease in NA concentration and a highly significant increase in the concentration of the NA metabolite 3,4-dihydroxyphenylethyleneglycol (DHPG). The hypothalamic ratio DHPG/NA was thus markedly increased (P less than 0.005) by alpha-MT indicating increased NA neuronal activity. alpha-MT administration also resulted in increased ACTH secretion (P less than 0.0005), an effect not observed following MIT. It is proposed that the effects on hypothalamic NA activity and ACTH secretion caused by alpha-MT are stress-mediated and unrelated to tyrosine hydroxylase inhibition. MIT is devoid of these effects but exhibits blockade activity, thus indicating it to be a preferable drug for the acute inhibition of tyrosine hydroxylase in neuroendocrine investigations.     

Histochemical studies on the adrenal glands of the marmosets (Callithrix jacchus and Callithrix penicillata).

The histrochemistry of the adrenal glands was studied in four adult male marmosets (two Callithrix jacchus and two Callithrix penicillata). It was impossible to demonstrate any reactivity to UDPG-GT, ADH, alanyl aminopeptidase, leucine aminopeptidase, xilitol (NAD-dependent) dehydrogenase, beta-glucuronidase and aryl-sulfatase in these glands. Total phosphorylase was found in scattered cells of the glomerulosa and adjacent outer fasciculata of one C. penicillata. The dehydrogenases (LDH, G-6-PDH,6-PGDH, NADPH2-TR,ICDH,SDH,NADH2-TR, alpha-GPDH, beta-OHBDH) as well as the hydrolases (except alkaline phosphatase, ATPase, and acetylcholinesterase) showed a stonger reactivity in the cortical part. Some hydrolases (naphthol acetate esterase, acid phosphatase) and cytochrome oxidase were less reactive in the zona glomerulosa, where the dehydrogenases were more abundant. The outer fasciculata and the reticularis also showed a strong dehydrogenase reactivity.     

EPR and 1H-NMR spectroscopic studies on the paramagnetic iron at the active site of phenylalanine hydroxylase and its interaction with substrates and inhibitors.

The paramagnetic iron at the active site of highly purified, catalytically active phenylalanine hydroxylase was studied by EPR at 3.6 K and one-dimensional 1H-NMR spectroscopy at 293 K. The EPR-detectable iron of the bovine enzyme was found to be present as a high-spin form (S = 5/2) in different ligand field symmetries depending on medium conditions (buffer ions) and the presence of ligands known to bind at the active site. At 3.6 K and in phosphate buffer, the paramagnetic iron is coordinated in an environment of rhombic symmetry (g = 4.3), whereas Tris buffer favours an environment of axial ligand field symmetry (g = 6.7, 5.3 and 2.0). The latter axial type of signals resembles those observed at g = 7.0, 5.2 and 1.9 for the enzyme in phosphate buffer when L-noradrenaline is added as an active-site ligand (inhibitor). The same proportion of iron that coordinates to L-noradrenaline seems to be reduced by the pterin cofactor and participate in catalysis. Experimental evidence is presented that Tris inhibits the enzyme by interacting with the enzyme-bound ferric iron and decreases its rate of reduction by the tetrahydropterin cofactor. Preincubation with dithiothreitol also inhibits the enzyme activity and prevents the reduction of its catalytically active ferric iron by pterin cofactors as well as binding of catecholamines to the enzyme. 1H-NMR spectroscopy revealed that the substrate (L-phenylalanine) and L-noradrenaline bind close to the paramagnetic iron, and that the catecholamine displaces the substrate from its binding at the active site. The results support our recently proposed model for the cooperative binding of inhibitor and substrate at the active site [Martínez, A. et al. (1990) Eur. J. Biochem. 193, 211-219].     

Evidence from studies on segregated nucleoli that nucleolar silver staining proteins C23 and B23 are in the fibrillar component

Several procedures for the silver staining of nucleoli have been evaluated at the electron microscopic level to determine optimal conditions for ultrastructural preservation and staining specificity. The present study shows that a brief fixation with 1% buffered formaldehyde followed by methanol: acetic acid (3 : 1) fixation yielded optimal preservation and silver staining of nucleoli. Using this procedure for electron microscopic studies of interphase nucleoli, it was found that the punctate silver grains observed by light microscopy were composed of fine silver granules, of approx. 100 Å diameter, organized in discrete clusters. In similar studies on adriamycin-induced segregated nucleoli, it was observed that the silver staining reaction was mainly limited to the fibrillar portion of the nucleolus. Accordingly, nucleolar proteins C23 and B23, found earlier to be the major silver binding proteins of the nucleolus, are mainly concentrated in the fibrillar nucleolar component.     

Mössbauer studies on protoporphyrin IX iron(II) frozen solutions containing ligands that cause the iron to be in a five coordinate high spin iron(II) environment

Mössbauer parameters are presented for a number of protoporphyrin IX iron(II) complexes containing ligands that allow the iron to be in a five coordinate high spin iron(II) electronic environment. Such environments are characterised by large quadrupole splittings in the range 4.0 to 4.4 mm s^?1. These compounds have characteristic electronic spectra.The implications of catechol type ligands binding protoporphyrin IX iron II/III moieties are discussed.     

Nitrate (NO3) and nitrite (NO2) are endocrine disruptors to downregulate expression of tyrosine hydroxylase and motor behavior through conversion to nitric oxide in early development of zebrafish

With a view to consider the increasing concern over nitrogen pollution in the aquatic environment, we investigated effects of nitrate (NO₃⁻) and nitrite (NO₂⁻) on the activity of dopaminergic neuron in zebrafish embryos and larvae. Both nitrate and nitrite exposure decreased the expression of tyrosine hydroxylase (TH) in dopaminergic neurons at 48 hpf. Only nitrite decreased the response to tactile stimulation at 72 hpf, whereas both nitrate and nitrite decreased the swimming activity at 6 dpf. When the embryos were exposed to nitrate or nitrite together with an estrogen receptor blocker (ICI 182,780), the decreases in TH expression and motor behavior caused by nitrate or nitrite alone were reversed suggesting the effects of nitrate and nitrite were mediated through estrogen receptor (ER). The result of co-incubation with an oxidoreductase inhibitor, diphenyleneiodonium, indicated the conversion to nitric oxide (NO) is likely to be responsible for the effects of nitrate and nitrite, which was further supported by the increased staining for NO after exposure. The present study demonstrates that nitrate and nitrite are neurotoxicants acting as an endocrine disruptor possibly through conversion to NO to downregulate the activity of dopaminergic neuron in early development of zebrafish.     

The effect of low temperature on the pressor response to noradrenaline in a hibernating (hedgehog) and a nonhibernating mammal (rat).

Hypothermia was found to affect the α-adrenoceptor in the resistance vessels differently in the hibernating and in the nonhibernating mammalian species. Low temperature increased the affinity for NA in the rat, while the efficacy of NA was reduced. In the hedgehog no changes were observed either in affinity or efficacy, suggesting that the regulation of the vascular smooth muscle by the sympathetic nervous system may be independent of temperature in the hibernators.     

Model studies on a synthetically facile series of N-substituted phenyl-N'-pyridin-3-yl ureas leading to 1-(3-pyridylcarbamoyl) indolines that are potent and selective 5-HT(2C/2B) receptor antagonists

A model series of 5-HT_2C antagonists have been prepared by rapid parallel synthesis. These N-substituted phenyl-N′-pyridin-3-yl ureas were found to have a range of 5-HT_2C receptor affinities and selectivities over the closely related 5-HT_2A receptor. Extrapolation of simple SAR, derived from this set of compounds, to the more active but synthetically more complex 1-(3-pyridyl-carbamoyl)indoline series allowed us to target optimal substitution patterns and identify potent and selective 5-HT_2C/2B antagonists.     

Definition of mouse chromosome 1 and 3 gene linkage groups that are conserved on human chromosome 1: Evidence that a conserved linkage group spans the centromere of human chromosome 1

Comparative mapping between the human and the mouse genomes allows characterization of linkage groups that have been conserved over evolution. In this study, genes previously localized to adjacent regions of human chromosome 1 were mapped to discrete regions on distal mouse chromosomes 1 and 3 using an interspecific cross. Linkage analysis in mouse defined two groups in which the gene order appears to be the same as that in humans: 15 genes localized between human chromosome 1q21 and 1q32 were found to span 29.5 cM on distal mouse chromosome 1; 6 genes localized between human chromosome 1q21 and 1p22 spanned 15.6 cM on distal mouse chromosome 3. These data suggest that gene order within large chromosome segments may remain stable over long periods of evolution and that the position of the centromere may reflect a late event in the evolution of higher eukaryotic organisms. These studies provide a model for examination of specific evolutionary events.     

Evidence that the requirements for ATP and wheat germ initiation factors 4A and 4F are affected by a region of satellite tobacco necrosis virus RNA that is 3' to the ribosomal binding site

A cDNA containing the complete genome of satellite tobacco necrosis virus (STNV) RNA was constructed and cloned into a plasmid vector containing the T7 polymerase promotor. A second clone containing the first 54 nucleotides from the 5' end, which includes the ribosome binding site, was also constructed. RNAs were transcribed from these plasmids (pSTNV1239 and pSTNV54) and tested for their ability to bind to wheat germ 40 S ribosomal subunits in the presence of wheat germ initiation factors eIF-4A, eIF-4F, eIF-4G, eIF-3, eIF-2, Met-tRNA, ATP, and guanosine 5'-(beta, gamma-imino)triphosphate (GMP-PNP). Maximal binding of the STNV RNA transcribed from pSTNV1239 is obtained only in the presence of all the initiation factors and ATP. In contrast, close to maximal binding of STNV RNA transcribed from pSTNV54 is obtained in the absence of eIF-4A, eIF-4F, eIF-4G, and ATP. A series of deletion clones from the 3' end of the STNV cDNA was prepared, and the requirements for binding to 40 S ribosomal subunits were determined. STNV RNAs containing more than 134 nucleotides from the 5' end require eIF-4A, eIF-4F, eIF-4G, and ATP for maximal binding to 40 S ribosomal subunits, whereas STNV RNAs containing 86 nucleotides or less no longer require ATP and these factors. These findings indicate that a region 3' to the initiation codon affects the requirements for eIF-4A, eIF-4F, eIF-4G, and ATP.     

Further studies on the covalent crosslinking of thyrotropin to its receptor: evidence that both the alpha and beta subunits of thyrotropin are crosslinked to the receptor

Highly purified alpha- and beta-subunits of thyrotropin were individually radioiodinated and, subsequently, recombined with their unlabeled complementary subunits. This procedure resulted in the formation of [125I]thyrotropin(TSH) hybrid molecules which were labeled on only one hormone subunit. Characterization of the binding properties of these two hybrid molecules demonstrated that both yielded nonlinear Scatchard plots with Kd and Bmax values similar to those obtained with radioiodinated native TSH and that both were capable of interaction with the high- and low-affinity binding components of the TSH receptor. The recombined [125I]TSH molecules were then crosslinked to the TSH receptor using disuccinimidyl suberate. Following electrophoresis and autoradiography, two labeled TSH-receptor complexes with Mr of 68,000 and 80,000 were observed. These two complexes exhibited hormone specificity and electrophoretic mobility identical to those previously observed using native [125I]TSH. Crosslinking with increasing concentrations of disuccinimidyl suberate suggested that the formation of the 68,000 and 80,000 complexes was sequential with the 68,000 appearing before the 80,000. Furthermore, the two bands were labeled regardless of which TSH subunit of the hybrid TSH was radioiodinated. These data strongly suggest that the 68,000 and 80,000 TSH-receptor complexes are the result of crosslinking to the TSH alpha-beta dimer and not to one subunit in the case of the 68,000 complex and to the TSH alpha-beta dimer in the case of the 80,000 complex, as had been hypothesized previously.     

Thermodynamics of triple helix formation: spectrophotometric studies on the d(A)10.2d(T)10 and d(C+3T4C+3).d(G3A4G3).d(C3T4C3) triple helices.

We have stabilized the d(A)10.2d(T)10 and d(C+LT4C+3).d(G3A4G3).d(C3T4C3) triple helices with either NaCl or MgCl2 at pH 5.5. UV mixing curves demonstrate a 1:2 stoichiometry of purine to pyrimidine strands under the appropriate conditions of pH and ionic strength. Circular dichroic titrations suggest a possible sequence-independent spectral signature for triplex formation. Thermal denaturation profiles indicate the initial loss of the third strand followed by dissociation of the underlying duplex with increasing temperature. Depending on the base sequence and ionic conditions, the binding affinity of the third strand for the duplex at 25 degrees C is two to five orders of magnitude lower than that of the two strands forming the duplex. Thermodynamic parameters for triplex formation were determined for both sequences in the presence of 50 mM MgCl2 and/or 2.0 M NaCl. Hoogsteen base pairs are 0.22-0.64 kcal/mole less stable than Watson-Crick base pairs, depending on ionic conditions and base composition. C+.G and T.A Hoogsteen base pairs appear to have similar stability in the presence of Mg2+ ions at low pH.     

Time-resolved resonance Raman and time-resolved step-scan FTIR studies of nitric oxide reductase from Paracoccus denitrificans: comparison of the heme b3-FeB site to that of the heme-CuB in oxidases

Time-resolved resonance Raman (TR(3)) and time-resolved step-scan (TRS(2)) FTIR spectroscopies have been used to probe the structural dynamics at the heme b(3) proximal and distal sites after carbon monoxide photolysis from fully reduced CO-bound nitric oxide reductase. The Raman spectra of the transient species exhibit structural differences relative to the equilibrium geometry of heme b(3). The most significant of these is a shift of 8 cm(-1) to higher frequency of the 207 cm(-1) mode, and a shift of 7 cm(-1) to lower frequency of the nu(4) mode. Our results indicate that the 207 cm(-1) mode observed in the equilibrium-reduced heme b(3) originates from nu(Fe-His). Its behavior in the photolytic transients indicates that the relaxed Fe-His state is not significantly populated. We suggest that relaxation along the tilt angle (theta) of the proximal histidine with respect to the heme plane and the out-of-plane displacement of the Fe (q) are coupled, and ligand binding and dissociation are accompanied by significant changes in the angular orientation of the His ligand. The results are compared to those obtained for the aa(3)-cytochrome c oxidase from Paracoccus denitrificans. The results are compared to those obtained for the aa(3)-cytochrome c oxidase from P. denitrificans. The TR(3) and TRS(2) FTIR data demonstrate significant alterations in the nature of the heme-protein dynamics between nitric oxide reductase and heme-copper oxidases resulting from specific structural differences in their respective hemepockets.     

Fc epsilon RI-induced protein tyrosine phosphorylation of pp72 in rat basophilic leukemia cells (RBL-2H3). Evidence for a novel signal transduction pathway unrelated to G protein activation and phosphatidylinositol hydrolysis.

Recently, we demonstrated that aggregation of the high affinity IgE receptor in rat basophilic leukemia (RBL-2H3) cells results in rapid tyrosine phosphorylation of a 72-kDa protein (pp72). Here we investigated the relationship of pp72 phosphorylation to guanine nucleotide-binding protein (G protein) activation and phosphatidylinositol hydrolysis. The activation of G proteins by NaF in intact cells or by guanosine 5'-O-(3-thiotriphosphate) in streptolysin O-permeabilized cells induced both phosphatidylinositol hydrolysis and histamine release without tyrosine phosphorylation of pp72. Similarly, in RBL-2H3 cells expressing the G protein-coupled muscarinic acetylcholine receptor, carbachol activated phospholipase C and induced secretion without concomitant pp72 phosphorylation. Therefore, pp72 phosphorylation was not induced by G protein activation or as a consequence of phosphatidylinositol hydrolysis. To investigate whether pp72 tyrosine phosphorylation precedes the activation of phospholipase C, we studied the effect of the tyrosine kinase inhibitor genistein. Preincubation of cells with genistein decreased, in parallel, antigen-induced tyrosine phosphorylation of pp72 (IC50 = 34 micrograms/ml) and histamine release (IC50 = 31 micrograms/ml). However, genistein at concentrations of up to 60 micrograms/ml did not inhibit phosphatidylinositol hydrolysis nor did it change the amount of the secondary messenger inositol (1,4,5)-triphosphate. Previous observations showed that there was no pp72 tyrosine phosphorylation after activation of protein kinase C or after an increase in intracellular calcium. Taken together, these results suggest that pp72 tyrosine phosphorylation represents a distinct, independent signaling pathway induced specifically by aggregation of the Fc epsilon RI.     

Further studies on the biochemical characterization of the MC-29 virus derived transplantable hepatoma (VTH). I. Binding of 3H-cortisol to cytoplasmic receptor and DNA

The molecular mechanisms underlying the failure of steroids to stimulate glucose-6-Pase and arylhydrocarbonhydroxylase activities in the MC-29-virus-derived transplantable hepatoma (VTH) were investigated. Following cellular uptake of 3H-Cortisol, its subcellular distribution, binding to a specific cytoplasmic receptor and the interaction between steroid-bound receptor and DNA were compared in VTH and in normal chicken liver. No appreciable difference was observed either in 3H-Cortisol uptake or in binding to cytoplasmic receptors. However, compared with normal liver, only half as much hepatoma steroid receptor was able to interact with DNA at the protein/DNA ratio of 60. This reduced DNA binding of VTH 3H-Cortisol-receptor was irrespective of the source of DNA (VTH or liver). It is concluded that one of the causes for abnormal gene regulation in VTH may lie at the level of DNA-protein interaction.     

Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDE3). Two N-terminal domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum

Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of approximately 50 aa). To investigate the role of regions 1 and 2 in membrane association, we examined the subcellular localization of a series of catalytically active, Flag-tagged N-terminal-truncated human (H) PDE3A and MPDE3B recombinants, as well as a series of fragments from regions 1 and 2 of MPDE3B synthesized as enhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 cells, the localization of a mutant HPDE3A, lacking the first 189 amino acids (aa) and therefore four of the six predicted transmembrane helices (H3A-Delta189), was virtually identical to that of the wild type. M3B-Delta302 (lacking region 1) and H3A-Delta397 (lacking region 1 as well as part of region 2) retained, to different degrees, the ability to associate with membranes, albeit less efficiently than H3A-Delta189. Proteins that lacked both regions 1 and 2, H3A-Delta510 and M3B-Delta604, did not associate with membranes. Consistent with these findings, region 1 EGFP-MPDE3B fusion proteins colocalized with the ER, whereas region 2 EGFP fusion proteins were diffusely distributed. Thus, some portion of the N-terminal hydrophobic domain in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDE3.