RESUMO
The coenzyme-linked fluorescence of aromatic-L-amino-acid decarboxylase decays non-exponentially. The decay of both native and NaBH4 reduced samples can only be fitted by two exponentials each roughly accounting for about half of the total fluorescence. Denaturation of the reduced protein with 8 M urea makes the fluorescence decay mono-exponential, like that observed for the reference compound pyridoxamine-5-phosphate. An extra pyridoxyl moiety can be bound to the enzyme after incubation with excess pyridoxal phosphate and reduction with NaBH4. This sample is almost twice as fluorescent and shows also two lifetimes. After denaturation only one fluorescence lifetime is observed. The presence of two non-equivalent pyridoxal sites in the native enzyme can be postulated. The heterogeneous decay behaviour of the pyridoxyl moiety in the enzyme together with the variability of lifetime shown, makes this fluorophore an even more interesting fluorescent probe for proteins.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/análise , Fosfato de Piridoxal/análise , Animais , Sítios de Ligação , Fluorescência , Rim/enzimologia , Desnaturação Proteica , SuínosRESUMO
DOPA decarboxylase from pig kidney, an alpha 2 dimeric enzyme of Mr = 107,000, has been crystallized by the vapour diffusion method with ammonium sulphate as precipitant. The crystals belong to the space group P6(2) (or its enantiomer P6(4)) and have unit cell dimensions of a = b = 155.9 A, c = 87.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. They diffract to 2.6 A resolution. There is one dimeric molecule per asymmetric unit. Rotation function studies have revealed the orientation of the non-crystallographic 2-fold axis of the dimer in the asymmetric unit.
Assuntos
Dopa Descarboxilase/ultraestrutura , Animais , Cristalografia , Rim/enzimologia , Suínos , Difração de Raios XRESUMO
This study reports for the first time that binding of aromatic methyl ester analogs to Dopa decarboxylase in the native and inactive nicked forms causes the appearance of a dead-end quinonoidal species absorbing at 500 nm, in addition to an external aldimine absorbing at 398 nm. The equilibrium mixture of these species varies depending on both the analog structure and the enzyme form. The above mentioned intermediates are also characterized with respect to their CD properties and the equilibria for their formation are determined as a function of pH. The results have provided evidence that the establishment of proper contacts between the active site and hydroxyl groups of the ligand are indispensable in order to limit unwanted side reactions.
Assuntos
Aminoácidos/metabolismo , Dopa Descarboxilase/metabolismo , Aminoácidos/química , Ésteres/metabolismo , Concentração de Íons de Hidrogênio , Metilação , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/metabolismo , Fosfato de Piridoxal/metabolismo , Espectrofotometria , Tirosina/análogos & derivados , Tirosina/química , Tirosina/metabolismoRESUMO
The topographic distribution of aromatic L-amino acid decarboxylase (AADC)-immunoreactive (IR) neurons was investigated in the cat hypothalamus, limbic areas, and thalamus by using specific antiserum raised against porcine kidney AADC. The perikarya and main axons were mapped on an atlas in ten cross-sectional drawings from A8 to A16 of the Horsley Clarke stereotaxic plane. AADC-IR neurons were widely distributed in the anterior brain. They were identified in the posterior hypothalamic area, rostral arcuate nucleus of the hypothalamus, dorsal hypothalamic area, and periventricular complex of the hypothalamus, which contain tyrosine hydroxylase (TH)-IR cells and are known as A11 to A14 dopaminergic cell groups. AADC-IR perikarya were also found in the other hypothalamic areas where few or no TH-IR cells have been reported: the supramamillary nucleus, tuberomamillary nucleus, pre- and anterior mamillary nuclei, caudal arcuate nucleus, dorsal hypothalamic area immediately ventral to the mamillothalamic tract, anterior hypothalamic area, area of the tuber cinereum, retrochiasmatic area, preoptic area, suprachiasmatic and dorsal chiasmatic nuclei. We also identified them in the anterior commissure nucleus, bed nucleus of the stria terminalis, stria terminalis, medial and central amygdaloid nuclei, lateral septal nucleus, and nucleus of the diagonal band of Broca. AADC-IR neurons were localized in the ventromedial part of the thalamus, lateral posterior complex, paracentral nucleus and lateral dorsal nucleus of the thalamus, medial habenula, parafascicular nucleus, subparafascicular nucleus, and periaqueductal gray. Conversely, we detected only a few AADC-IR cells in the supraoptic nucleus whose rostral portion contains TH-IR perikarya. Comments are made on the relative localizations of the AADC-IR and TH-IR neurons, on species differences between the cat and rat, as well as on the possible physiological functions of the enzyme AADC.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/metabolismo , Gatos/metabolismo , Diencéfalo/enzimologia , Telencéfalo/enzimologia , Animais , Feminino , Hipotálamo/enzimologia , Imuno-Histoquímica , Sistema Límbico/enzimologia , Masculino , Área Pré-Óptica/enzimologia , Tálamo/enzimologia , Distribuição TecidualRESUMO
By indirect immunohistochemistry, the present study examined the distribution of neuronal structures in the cat medulla oblongata, pons, and midbrain, showing immunoreactivity to aromatic L-amino acid decarboxylase (AADC), which catalyzes the conversion of L-3, 4-dihydroxyphenylalanine (L-DOPA) to dopamine, and 5-hydroxytryptophan to serotonin (5HT). With simultaneous and serial double immunostaining techniques, immunoreactivity to this enzyme was demonstrated in most of the catecholaminergic and serotonergic neurons. We could also demonstrate AADC-IR cell bodies that do not contain tyrosine hydroxylase (TH-) or 5HT-immunoreactivity (called "D-type cells") outside such monoaminergic cell systems. At the medullo-spinal junction, very small D-type cells were found within and beneath the ependymal layer of the 10th area of Rexed surrounding the central canal. D-type cells were localized in the caudal reticular formation, nucleus of the solitary tract, a dorsal aspect of the lateral parabrachial nucleus, and pretectal areas as have been reported in the rat. Furthermore, the present study describes, in the cat brainstem, new additional D-type cell groups that have not been reported in the rat. Dense or loose clusters of D-type cells were localized in the external edge of the laminar trigeminal nucleus, dorsal motor nucleus of the vagus, external cuneate nucleus, nucleus praepositus hypoglossi, central, pontine, and periaqueductal gray, superficial layer of the superior colliculus, and area medial to the retroflexus. D-type cells were loosely clustered in the lateral part of the central tegmental field dorsal to the substantia nigra, extending dorsally in the medial division of the posterior complex of the thalamus and medial side of the brachium of the inferior colliculus. They extended farther rostrodorsally along the medial side of the nucleus limitans and joined with the pretectal cell group. Almost all these cells were very small and ovoid to round with 1-2 short processes with the exception of dorsal motor vagal cells. AADC-IR axons were clearly identified in the vagal efferent nerves, longitudinal medullary pathway, dorsal tegmental bundle rostral to the locus coeruleus. Serotonergic axons were identified not only in the central tegmentum field and lateral side of the central superior nucleus, but also in the ventral surface of the medulla oblongata. We describe principal densely stained fiber plexuses in the cat brainstem. The findings of the present study provide a morphological basis for neurons that decarboxylate endogenous and exogenous L-DOPA, 5HTP, and other aromatic L-amino acids.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/análise , Gatos/anatomia & histologia , Bulbo/enzimologia , Mesencéfalo/enzimologia , Proteínas do Tecido Nervoso/análise , Ponte/enzimologia , Animais , Dopamina/metabolismo , Feminino , Técnicas Imunoenzimáticas , Masculino , Bulbo/anatomia & histologia , Mesencéfalo/anatomia & histologia , Ponte/anatomia & histologia , Ratos/anatomia & histologia , Serotonina/metabolismo , Especificidade da EspécieRESUMO
Total poly (A+) RNA has been extracted from rat pheochromocytoma and translated in vitro by means of a reticulocyte lysate system. We show that two antisera, prepared against pig kidney DOPA decarboxylase (DDC) or rat pheochromocytoma DDC, immunoprecipitate an in vitro synthetized 50 kDa polypeptide identified as DDC by competition experiments with pure DDC. The proportion of specific mRNA has been calculated and represents 0.05% of total poly A+ mRNA. Its size has been established by electrophoresis in methylmercuric hydroxide containing agarose gel, corresponding to a 2.2 kb length mRNA.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/genética , Dopa Descarboxilase/genética , Feocromocitoma/enzimologia , RNA Mensageiro/análise , Animais , Dopa Descarboxilase/biossíntese , Técnicas In Vitro , Rim/enzimologia , Poli A/análise , Testes de Precipitina , Ratos , SuínosRESUMO
Using a technique by which binding sites for two antibodies can be visualized in single tissue sections, we have studied the distribution of neurones containing DOPA decarboxylase-like and dopamine beta-hydroxylase-like immunoreactivity in ganglia of dog sympathetic chain. Three types of neurones could be distinguished: those that contained both enzymes, and were presumably noradrenergic; those that contained neither enzyme, and were presumably not catecholaminergic; and a group that contained DOPA decarboxylase but lacked dopamine beta-hydroxylase. The numbers of cells of each type were counted in serially-sectioned ganglia from regions of the sympathetic chain thought to contain dopaminergic neurones (T12-L1 and L7-S2). The percentages of total cell numbers contributed by the DOPA decarboxylase-positive, dopamine beta-hydroxylase-negative cells in these regions were similar to the estimates of dopaminergic neurone numbers that can be made from previously obtained biochemical data. Our results are consistent with the presence of dopaminergic neurons in regions of the paravertebral chain supplying the kidney and the distal hindlimb.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/análise , Dopa Descarboxilase/análise , Dopamina beta-Hidroxilase/análise , Gânglios Simpáticos/enzimologia , Neurônios/enzimologia , Animais , Cães , Feminino , Gânglios Simpáticos/citologia , Histocitoquímica , Técnicas Imunológicas , Masculino , Neurônios/classificação , Distribuição TecidualRESUMO
Tyrosine hydroxylase-like immunoreactive (TH-IR) neurons with morphological features of interneurons were found throughout the human cerebral cortex. Quantitative estimates in 14 different cytoarchitectonic areas revealed a specific regional distribution pattern, neurons being less dense in primary cortical areas and denser in higher order associative areas and some limbic related areas. A partial relationship was noted between the density of labeled neurons and that of the known dopaminergic innervation. The role of the cortical TH-IR neurons in catecholaminergic function, however, remains unclear since the presence of other catecholaminergic synthesizing enzymes, dopamine-beta-hydroxylase and DOPA decarboxylase, could not be demonstrated at their level. Similar neurons have been observed transiently in the rodent cortex during development; their persistence and topographical extension in the human brain warrants further study on their possible functional role.
Assuntos
Catecolaminas/fisiologia , Córtex Cerebral/análise , Tirosina 3-Mono-Oxigenase/análise , Adulto , Idoso , Catecolaminas/metabolismo , Córtex Cerebral/citologia , Humanos , Imuno-Histoquímica , Pessoa de Meia-IdadeRESUMO
To determine whether dihydroxyphenylalanine (DOPA) decarboxylase (DDC) activity in the terminal regions of noradrenergic axons varies with axonal length, we compared the pattern of immunohistochemical staining for DDC in the 'short' terminal nerves of dog vas deferens with that in the 'long' nerves of spleen and atrium. The terminal nerves supplying the muscular coats of the vas deferens were, like those in spleen and heart, devoid of DDC immunoreactivity. The presence of this enzyme is therefore not characteristic of either short or long noradrenergic axons, in support of previous evidence that it is a specific marker for dopaminergic terminal nerves. Many axons supplying the mucosal epithelial cells in the vas deferens were DDC-positive, suggesting the existence of a dopaminergic innervation.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/metabolismo , Dopa Descarboxilase/metabolismo , Sistema Nervoso Simpático/enzimologia , Ducto Deferente/inervação , Animais , Cães , Dopa Descarboxilase/imunologia , Imuno-Histoquímica , Masculino , Sistema Nervoso Simpático/citologia , Ducto Deferente/citologiaRESUMO
We have compared the pattern of neural catecholamine fluorescence with that of immunoreactivity for the catecholamine-synthesizing enzymes tyrosine hydroxylase (TH) and DOPA decarboxylase (DDC) in dog atrium, which is innervated by noradrenergic nerves, and in dog kidney, which is thought to be supplied by dopaminergic nerves as well. In both tissues the distribution of nerves containing catecholamine fluorescence was similar to that of nerves exhibiting TH-like immunoreactivity. By contrast, DDC-like immunoreactivity was present in some (but not all) of the nerves associated with the intrarenal blood vessels, but was not detectable in any atrial nerves. High DDC activity provides further confirmation of the existence of sympathetic dopaminergic neurons supplying the kidney.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/metabolismo , Dopa Descarboxilase/metabolismo , Dopamina/fisiologia , Norepinefrina/fisiologia , Sistema Nervoso Simpático/enzimologia , Animais , Cães , Feminino , Átrios do Coração/inervação , Histocitoquímica , Imunoquímica , Rim/inervação , Masculino , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The present study has explained the general reaction mechanism of the bacterial tyrosine decarboxylase. The rate equation for this mechanism has been presented. The steady state kinetics of tyrosine decarboxylase, as for tyrosine transaminase, have shown that the apoenzyme can bind not only the coenzyme, but also the non-enzymatically formed Schiff base between the coenzyme and the substrate. Our data then have confirmed the importance of the non-enzymatically formed Schiff base in the B6-dependent enzymes, possibly in all of them which have a low affinity constant for the coenzyme, such that the coenzyme must be present in excess in respect to the protein to saturate the active center. The interaction between apotyrosine decarboxylase with pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate has been studied.
Assuntos
Enterococcus faecalis/enzimologia , Tirosina Descarboxilase/metabolismo , Cinética , Matemática , Compostos Organofosforados/metabolismo , Fosfato de Piridoxal/metabolismo , Piridoxamina/análogos & derivados , Piridoxamina/metabolismo , Tirosina/metabolismoAssuntos
Haptoglobinas , Acrilatos , Alquilação , Animais , Antígenos , Fenômenos Químicos , Química , Cromatografia em Gel , Eletroforese , Géis , Haptoglobinas/análise , Haptoglobinas/isolamento & purificação , Hemoglobinas , Humanos , Soros Imunes , Imunodifusão , Imunoeletroforese , Mercaptoetanol , Oxirredução , Peptídeos , Peroxidases/análise , Desnaturação Proteica , Coelhos , Amido , Compostos de Sulfidrila/análiseAssuntos
Aspartato Aminotransferases , Piridoxamina/análogos & derivados , Animais , Apoenzimas/metabolismo , Aspartato Aminotransferases/metabolismo , Citosol/enzimologia , Dinitrofluorbenzeno/análogos & derivados , Dinitrofluorbenzeno/farmacologia , Mitocôndrias/enzimologia , Ligação Proteica , Piridoxamina/farmacologia , EspectrofotometriaAssuntos
Descarboxilases de Aminoácido-L-Aromático/metabolismo , Dopa Descarboxilase/metabolismo , Rim/enzimologia , Sequência de Aminoácidos , Animais , Drosophila/enzimologia , Dados de Sequência Molecular , Cofator PQQ , Fosfato de Piridoxal/metabolismo , Quinolonas/metabolismo , Homologia de Sequência do Ácido Nucleico , Espectrofotometria , SuínosRESUMO
Phosphopyridoxyl derivatives, which are stable analogues of a substrate-coenzyme complex, are bound at the active site with great affinity. From a comparison of the interaction of a number of such compounds with the apoenzyme the delta G0 values for the binding of the substrate carboxy and phenyl groups and of the coenzyme aldehydic group were determined to be equal to (or more negative than) -3.8. -8.4 and -12.5kJ/mol (-0.9, -1.9 and -3kcal/mol) respectively; the delta G0 for the binding of the coenzyme phosphate group was shown to be more negative than -20.5kJ/mol (-4.9kcal/mol). Two features of the binding process of the coenzyme-substrate analogues to tyrosine decarboxylase have already been found in the case of tyrosine aminotransferase [Borri-Voltattorni, Orlacchio, Giartosio, Conti & Turano (1975) Eur. J. Biochem. 53, 151-160]: (1) in the binding of the substrate to the enzyme a significant fraction of the instrinsic delta G0 appears to be used for some associated endoergonic process; (2) the delta H0 and delta S0 of binding appear to be very sensitive indicators of the correct alignment of the substrate-coenzyme and analogues at the active site.