Improved ionization chamber method for continuous measurement of DOPA decarboxylase activity

In the present study, we report a reliable method using an ionization chamber for continuous measurement of DOPA decarboxylase activities in vitro.     

Role of tyrosine, DOPA and decarboxylase enzymes in the synthesis of monoamines in the brain of the locust

The metabolic transformation of tyrosine (TYR) by the decarboxylase and hydroxylase enzymes was investigated in the central nervous system of the locust, Locusta migratoria. It has been demonstrated that the key amino acids, 3,4-dihydroxyphenylalanine (DOPA), 5-hydroxytryptophan (5HTP) and tyrosine are decarboxylated in all part of central nervous system. DOPA and 5HTP decarboxylase activities show parallel changes in the different ganglia, but the rank order of the activity of TYR decarboxylase is different. Enzyme purification has revealed that the molecular weights of TYR decarboxylase and DOPA/5HTP decarboxylase are 370,000 and 112,000, respectively. The decarboxylation of DOPA by DOPA/5HTP decarboxylase is stimulated, whereas the decarboxylation of DOPA by TYR decarboxylase is inhibited in the presence of the cofactor pyridoxal-5'-phosphate. TYR hydroxylase could not be detected and 3H-TYR is found to be metabolised to tyramine (TA), but not to DOPA. The haemolymph contains a significant concentration of DOPA (120 pmol/100 microl haemolymph), and the ganglia incorporates DOPA from the haemolymph by a high affinity uptake process (K(M)=12 microM and V(max)=24 pmol per ganglion/10 min). Our results suggest that no tyrosine hydroxylase is present in the locust CNS and the DOPA uptake into the ganglia by a high affinity uptake process as well as the DOPA decarboxylase enzyme may be responsible for the regulation of the ganglionic dopamine (DA) level. Two types of decarboxylases exist, one of them decarboxylating DOPA and 5HTP (DOPA/5HTP decarboxylase), other decarboxylating TYR (TYR decarboxylase). The DOPA/5HTP decarboxylase enzyme present in the insect brain may correspond to the 5HTP/DOPA decarboxylase in vertebrate brain, whereas TYR decarboxylase is characteristic only for the insect brain.     

Some aspects on L-dopa decarboxylase and p-tyrosine decarboxylase in the central nervous and peripheral tissues of the American cockroach Periplaneta americana

1. Aromatic amino acid decarboxylase activities toward L-DOPA (L-3,4-dihydroxyphenylalanine), 5-HTP (5-hydroxytryptophan) and p-tyrosine in different tissues of the sclerotized and newly ecdysed cockroach were analyzed. 2. The ratios of enzyme activity with regard to L-DOPA and p-tyrosine varied considerably in the tissues and between the two different growth stages. 3. A DOPA decarboxylase and a p-tyrosine decarboxylase were separated by gel filtration and ion exchange chromatography. 4. The optimal pH requirement for both enzymes was 7.5 with the exception of the one decarboxylating 5-HTP. 5. The molecular weights of the cockroach brain DOPA decarboxylase and tyrosine decarboxylase were estimated to be 120,000 and 100,000, respectively. 6. Unlike the mammalian aromatic amino acid decarboxylase, the cockroach DOPA decarboxylase cannot be activated by a small amount of benzene. 7. An increase of over 50-fold of DOPA decarboxylase activity and a 50% reduction of tyrosine decarboxylase activity in the epidermal tissue of the newly ecdysed animals was observed. 8. In the fully sclerotized cockroach, a reversible endogenous inhibitor(s) of DOPA decarboxylase in the integument was observed, suggesting that the DOPA decarboxylase is suppressed in the epidermal tissues when ecdysis does not occur.     

Ornithine decarboxylase activity and polyamines in relation to aging of human fibroblasts

A reduction of ornithine decarboxylase (ODC) activity is associated with the decreased growth rate in early senescence of human embryonic lung fibroblasts. Inhibition of ODC activity with α-methyl ornithine in younger cells reduces cell proliferation; inhibition of putrescine deamination with aminoguanidine increases intracellular putrescine concentration and stimulates cell proliferation. The data suggests that the decreased cell proliferation which occurs in senescence may, at least in part, be dependent upon the reduced ODC activity.     

Characterization of DOPA decarboxylase mRNA in rat pheochromocytoma

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.     

Trilobite cuticle thickness in relation to palaeoenvironment

100 thickness measurements from thin sections of cephala or pygidia of early Ordovician trilobites occurring across an onshore to offshore environmental gradient show that progressively greater maximum cuticle thickness was characteristic of increasingly inshore sites. There is a 40-fold difference between the thinnest and thickest cuticles, and exclusively thin cuticles are confined to the offshore Olenid Biofacies. Variability in cuticle thickness increases offshore to onshore. Environmental control is shown to be more influential on cuticle thickness than is the overall length of the trilobite: some comparatively large trilobites having thin cuticles and small trilobites thick cuticles. The environmental factors which might be responsible for the pattern are briefly discussed. The thin cuticles dominating the offshore Olenid Biofacies were probably appropriate for dysaerobic conditions. Thick cuticles in the most inshore biofacies may have offered protection against predators and turbulence, but the additional presence there of trilobites with thinner cuticles is considered to reflect the greater heterogeneity of the epeiric habitat.     

Purification and characterisation of tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase

Microbial tyrosine decarboxylase (EC 4.1.1.25) and mammalian aromatic-L-amino-acid decarboxylase (EC 4.1.1.28) catalyse the formation of tyramine from L-tyrosine. These enzymes were characterised after isolation to purity by methods including fast polymer liquid chromatography (FPLC). Tyrosine decarboxylase was isolated from Streptococcus faecalis by FPLC anion exchange chromatography (11-times purification; 72% recovery; 23.2 U/mg protein). FPLC on Phenyl-Superose resulted in purification to 115 U/mg protein. Aromatic-L-amino-acid decarboxylase was isolated from pig kidney by ammonium sulfate fractionation, DEAE chromatography, and FPLC anion exchange chromatography (21-times purification; 22% recovery; 0.71 U/mg protein). By FPLC chromatofocusing, tyrosine decarboxylase eluted at pH 4.3 and aromatic-L-amino-acid decarboxylase at pH 5.0. Isoelectric focusing of tyrosine decarboxylase gave two bands (pI 4.4 and 4.5). With pyridoxal 5'-phosphate removed by ultrafiltration, only one band (pI 4.4) appeared, and SDS polyacrylamide electrophoresis confirmed the purity. FPLC gel filtration resulted in molecular weights 143,000 and 86,000, respectively, for tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase. In SDS electrophoresis, tyrosine decarboxylase had the monomer molecular weight 75,000, showing a dimer structure for the enzyme.     

Ornithine decarboxylase activity in rat organs and tissues under artificial hypobiosis

The influence of hypothermia-hypoxia-hypercapnia on ornithine decarboxylase (ODC, EC 4.1.1.17) activities in rat organs and tissues and also on the thymocyte distribution throughout the cell cycle stages was studied. The state of artificial hypobiosis in rats on decrease in the body temperature to 14.4–18.0°C during 3.0–3.5 h was accompanied by drops in the ODC activities in the neocortex and liver by 50–60% and in rapidly proliferating tissues (thymus, spleen, and small intestine mucosa) by 80% of the control value. In kidneys the ODC activity raised to 200% of the control level. Twenty-four hours after termination of the cooling and replacing the rats under the standard conditions, the ODC activities in the neocortex, liver, kidneys, spleen, and intestinal mucosa returned to the control values, but remained decreased in the thymus. Forty-eight hours later the ODC activities in the thymus and spleen exceeded the normal level. The distribution of thymocytes throughout the cell cycle stages did not change in rats in the state of hypothermia (hypobiosis); 24 and 48 h after termination of the cooling the fraction of thymocytes in the S stage was decreased and the fraction of the cells in the G₀+G₁ stage was increased. The normal distribution of thymocytes throughout the cell cycle stages recovered in 72 h. Thus, in the thymus the diminution of the ODC activity preceded the suppression of the cell proliferation rate. The tissue-specific changes in the ODC activity are suggested to reflect adaptive changes in the functional and proliferative activities of organs and tissues during the development of hypobiosis under conditions of hypothermia-hypoxia-hypercapnia.     

Fasting-induced changes of tyrosine transaminase activity in rat tissues

Tyrosine transaminase activity in liver, kidney, intestine, stomach, skin, adipose tissue, striated muscle and brain in fed and 24-hour fasted rats, has been studied. Maximal activity has been found in liver, with only fractional activity in the other tissues. 24 hour fasting induced significant decrease in liver and adipose tissue activity, while no changes have been detected in the other tissues. The possible implications of these facts are discussed.     

Diphenols in hemolymph and cuticle during development and cuticle tanning of Periplaneta americana (L.) and other cockroach species

Diphenolic compounds in cockroach hemolymph and cuticle were extracted with 1.2 N HCI, partially purified by alumina adsorption, and analyzed by liquid chromatography. Dopamine (DA) is the major catecholamine in hemolymph of Periplaneta americana, Blatta orientalis, Blattella germanica, Gromphadorhina portentosa, and Blaberus craniifer at adult ecdysis, while N-acetyldopamine (NADA) predominates in hemolymph of Leucophaea maderae. In P. americana, NADA is the second most abundant catecholamine, while N-β-alanyldopamine (NBAD), norepinephrine (NE), 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylethanol, 3,4-dihydroxyphenylacetic acid, and 3,4-dihydroxybenzoic acid occur in lesser quantities. Catecholamines occur mainly as acid labile conjugates in hemolymph. Dopamine, conjugated primarily as the 3-sulfate ester, increases in hemolymph from 0.1 to 0.8 mM during the last instar. Concentrations decrease by 75% in pharate adults, partially because of an increase in hemolymph volume. A second smaller peak of DA sulfate occurs after ecdysis followed by a rapid disappearance as the cuticle tans. A conjugate of catechol (o-dihydroxybenzene) is also present in relatively high concentrations at all ages examined. In cuticle, N-β-alanylnorepinephrine accumulates during the early period of adult tanning, while NBAD, NADA, N-acetylnorepinephrine, and DA increase more slowly. The N-β-alanyl and N-acetyl derivatives of DA and NE occure in relatively high concentrations in tanned cutical of P. americana and probably play an important role in the stablization process.     

DOPA compared with dihydroxyfumarate as co-factor in peroxidase-mediated oxidation of tyrosine to melanin

Summary Dihydroxyfumarate was used as a co-factor in the histochemical demonstration of peroxidase-mediated oxidation of tyrosine to melanin in eosinophils, mast cells, melanoma cells and neurons. The use of dihydroxyfumarate as co-factor provides a direct method for demonstrating peroxidase-mediated oxidation of tyrosine to melanin in tissues, and allows for the ultrastructural localization of this pigment.Supported by USPHS Grant T1 AM 5220 and by the St. Vincent Hospital Research Foundation.     

ATP's impact on the conformation and holoenzyme formation in relation to the regulation of brain glutamate decarboxylase

To investigate ATP as a potential factor in the regulation of brain glutamate decarboxylase (GAD), the impact of ATP on the enzyme conformation and holoenzyme formation was investigated. ATP at 100 microM quenches fluorescence emission intensity of the holoenzyme of GAD (holoGAD) by 18% after a correction for the inner filter effect and enhances fluorescence steady-state polarization from 0.158 to 0. 183 when excited at 280 or 295 nm. These findings suggest that ATP moderately changes the microenvironment of one or more tryptophan or tyrosine residues in holoGAD and alters these residues from a more mobile state to a less mobile one. A moderate ATP-induced conformational change in holoGAD is also supported by the observations that ATP increases the thermal denaturation temperature of holoGAD by 2 degrees C, as derived from temperature-dependent fluorescence spectra, and decreases the alpha-helical content of holoGAD by 8-10%, as determined by circular dichroism. Moreover, ATP does not affect the keto-enol tautomerization of holoGAD and has little or no direct effect on its activity, implying that the ATP interacting domain in holoGAD is not at the active site. Kinetics studies, as demonstrated by stopped-flow fluorescence and UV/visible spectroscopy, demonstrate that formation of holoGAD involves two steps: a fast reaction forming an apoGAD-cofactor intermediate complex, followed by a slow reaction involving the conformational change in the intermediate complex. ATP reduces the rate constant of the fast step to one-third and decreases the rate of the slow step and the intermediate complex formation constant to 60% of their original values. The present data suggest that ATP may regulate the interconversion between apoGAD and holoGAD by interacting with apoGAD rather than holoGAD. By slowing down the rate of intermediate complex formation, ATP reduces the amount of holoGAD formed.     

Ornithine decarboxylase activity in relation to DNA synthesis in mouse interfollicular epidermis and hair follicles.

The relationship between ornithine decarboxylase (L-ornithine carboxylyase, EC 4.1.1.17) activity and DNA synthetic activity was studied in mouse epidermis. Interfollicular epidermis and hair follicles were investigated separately. It was found that, in hair follicles, the variations of DNA replicative activity, which are reflected in the cyclic growth of hair, are paralleled by corresponding changes in ornithine decarboxylase activity. In both interfollicular epidermis and hair follicles, stimulation of DNA synthetic activity by plucking of hair induced a rapid and marked increase in ornithine decarboxylase activity. The relationship of steady-state and induced ornithine decarboxylase activity to DNA synthetic activity was compared in hair follicles and interfollicular epidermis. A correlation between the activity of this enzyme and DNA replication was found thereby in each of these tissues.