Anna Mura

Low doses of γ-hydroxybutyric acid stimulate the firing rate of dopaminergic neurons in unanesthetized rats

In unanesthetized rats the intravenous (i.v.) administration of gamma-hydroxybutyric acid (GHB) at the doses of 50-400 mg/kg produced a dose-related stimulation (10-56%) of the firing rate of dopaminergic (DA) neurons in the pars compacta of the substantia nigra. Doses of 1000 and 1500 mg/kg inhibited the firing rate almost completely. In unanesthetized rats the intraperitoneal injection of GHB at the dose of 750 mg/kg produced a brief initial stimulation (23%) followed by a modest reduction in the firing rate (29%). On the other hand, in chloral hydrate-anesthetized rats the i.v. administration of GHB at cumulative doses of up to 200 mg/kg failed to modify the firing rate of DA neurons, while a cumulative dose of 400 mg/kg suppressed neuronal firing. The results indicate that sub-anesthetic doses of GHB stimulate the firing rate of DA neurons in unanesthetized rats.

Haloperidol-induced vacuous chewing in rats : suppression by α-methyl-tyrosine

Abstract Chronic treatment of rats with haloperidol (1 mg/kg twice daily for 4 weeks) induced repetitive vacuous chewing movements (VC), that persisted for over 72 h after haloperidol withdrawal. Haloperidol-induced VC were inhibited by the s.c. administration of the specific dopamine D 1 receptor antagonist, SCH 23390 (0.025–0.100 mg/kg), in a dose-dependent manner, and were totally suppressed by an acute challenge with haloperidol (2 mg/kg i.p.) and by the dopamine synthesis inhibitor, α-methyl-tyrosine (AMT) (200 mg/kg i.p.). In AMT-treated rats, VC were reinstated by the administration of the selective D 1 agonist, SKF 38393. The results support the hypothesis that chronic haloperidol-induced VC are mediated by dopamine acting selectively upon D 1 receptors.

Tyramine oxidation by copper/TPQ amine oxidase and peroxidase from Euphorbia characias latex

Tyramine, an important plant intermediate, was found to be a substrate for two proteins, a copper amine oxidase and a peroxidase from Euphorbia characias latex. The oxidation of tyramine took place by two different mechanisms: oxidative deamination to p-hydroxyphenylacetaldehyde by the amine oxidase and formation of di-tyramine by the peroxidase. The di-tyramine was further oxidized at the two amino groups by the amino oxidase, whereas p-hydroxyphenylacetaldehyde was transformed to di-p-hydroxyphenylacetaldehyde by the peroxidase. Data obtained in this study indicate a new interesting scenario in the metabolism of tyramine.

Catalase and antiquitin from Euphorbia characias: Two proteins involved in plant defense?

Here we report the cDNA nucleotide sequences of a calmodulin-binding catalase and an antiquitin from the latex of the Mediterranean shrub Euphorbia characias. Present findings suggest that catalase and antiquitin might represent additional nodes in the Euphorbia defense systems, and a multi-enzymatic interaction contributing to plant’s protection against biotic and abiotic stresses is proposed to occur in E. characias laticifers.

Catalytic pathways of Euphorbia characias peroxidase reacting with hydrogen peroxide.

Abstract The reaction of Euphorbia characias latex peroxidase (ELP) with hydrogen peroxide as the sole substrate was studied by conventional and stopped-flow spectrophotometry. The reaction mechanism occurs via three distinct pathways. In the first (pathway I), ELP shows catalase-like activity: H2O2 oxidizes the native enzyme to compound I and subsequently acts as a reducing substrate, again converting compound I to the resting ferric enzyme. In the presence of an excess of hydrogen peroxide, compound I is still formed and further reacts in two other pathways. In pathway II, compound I initiates a series of cyclic reactions leading to the formation of compound II and compound III, and then returns to the native resting state. In pathway III, the enzyme is inactivated and compound I is converted into a bleached inactive species; this reaction proceeds faster in samples illuminated with bright white light, demonstrating that at least one of the intermediates is photosensitive. Calcium ions decrease the rate of pathway I and accelerate the rate of pathways II and III. Moreover, in the presence of calcium the inactive stable verdohemochrome P670 species accumulates. Thus, Ca2+ ions seem to be the key for all catalytic pathways of Euphorbia peroxidase.

Properties of copper‐free pig kidney amine oxidase: Role of topa quinone

Copper removal from pig kidney amine oxidase containing Cu/topaquinone (TPQ) has been obtained using CN− in the presence of the poor substrate p‐(dimethylamino)benzylamine. Upon removal of copper, the enzyme loses its activity while the TPQ cofactor remains in its oxidized form. The addition of copper to the apo‐form fully restores the active enzyme. The CN− treatment in the presence of sodium dithionite or good substrates (cadaverine or benzylamine) also removes copper but the TPQ cofactor is irreversibly reduced and the addition of copper does not regenerate the active enzyme. NiII and ZnII do not bind the apo‐protein in contrast to CoII which is incorporated to the same extent as CuII. However, Co‐reconstituted enzyme only shows a very low activity. These results demonstrate that copper is essential for the catalytic mechanism because it maintains the correct active site geometry.

An important lysine residue in copper/quinone‐containing amine oxidases

The interaction of xenon with copper/6‐hydroxydopa (2,4,5‐trihydroxyphenethylamine) quinone (TPQ) amine oxidases from the plant pulses lentil (Lens esculenta) and pea (Pisum sativum) (seedlings), the perennial Mediterranean shrub Euphorbia characias (latex), and the mammals cattle (serum) and pigs (kidney), were investigated by NMR and optical spectroscopy of the aqueous solutions of the enzymes. 129Xe chemical shift provided evidence of xenon binding to one or more cavities of all these enzymes, and optical spectroscopy showed that under 10 atm of xenon gas, and in the absence of a substrate, the plant enzyme cofactor (TPQ), is converted into its reduced semiquinolamine radical. The kinetic parameters of the analyzed plant amine oxidases showed that the kc value of the xenon‐treated enzymes was reduced by 40%. Moreover, whereas the measured Km value for oxygen and for the aromatic monoamine benzylamine was shown to be unchanged, the Km value for the diamine putrescine increased remarkably after the addition of xenon. Under the same experimental conditions, the TPQ of bovine serum amine oxidase maintained its oxidized form, whereas in pig kidney, the reduced aminoquinol species was formed without the radical species. Moreover the kc value of the xenon‐treated pig enzyme in the presence of both benzylamine and cadaverine was shown to be dramatically reduced. It is proposed that the lysine residue at the active site of amine oxidase could be involved both in the formation of the reduced TPQ and in controlling catalytic activity.

Structural Changes and Aggregation Process of Cu/Containing Amine Oxidase in the Presence of 2,2,2-Trifluoroethanol

Conformational and structural changes of lentil seedlings amine oxidase (LSAO) were studied in the presence of trifluoroethanol (TFE) by spectroscopic and analytical techniques. At TFE concentrations up to 5%, the induction of a structural transition from beta-sheet to alpha-helix and up to 10% TFE a structural transition from alpha-helix to beta-sheet as well as inactivation of the enzyme are observed. At TFE concentrations between 10-35%, LSAO proves to be prone to aggregation and beyond 35% TFE leads to a non-native protein structure with a high alpha-helix content. The obtained results revealed that the aggregation of LSAO is strongly linked to the nature of secondary structures.

Allosteric modulation of Euphorbia peroxidase by nickel ions

A class III peroxidase, isolated and characterized from the latex of the perennial Mediterranean shrub Euphorbia characias, contains one ferric iron–protoporphyrin IX pentacoordinated with a histidine ‘proximal’ ligand as heme prosthetic group. In addition, the purified peroxidase contained 1 mole of endogenous Ca2+ per mole of enzyme, and in the presence of excess Ca2+, the catalytic efficiency was enhanced by three orders of magnitude. The incubation of the native enzyme with Ni2+ causes reversible inhibition, whereas, in the presence of excess Ca2+, Ni2+ leads to an increase of the catalytic activity of Euphorbia peroxidase. UV/visible absorption spectra show that the heme iron remains in a quantum mechanically mixed‐spin state as in the native enzyme after addition of Ni2+, and only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ni2+. In the presence of H2O2 and in the absence of a reducing agent, Ni2+ decreases the catalase‐like activity of Euphorbia peroxidase and accelerates another pathway in which the inactive stable species accumulates with a shoulder at 619 nm. Analysis of the kinetic measurements suggests that Ni2+ affects the H2O2‐binding site and inhibits the formation of compound I. In the presence of excess Ca2+, Ni2+ accelerates the reduction of compound I to the native enzyme. The reported results are compatible with the hypothesis that ELP has two Ni2+‐binding sites with opposite functional effects.

Electroactive Centers in Euphorbia Latex and Lentil Seedling Amine Oxidases

The electrochemical behavior of redox centers in the active site of amine oxidases from lentil seedlings and Euphorbia characias latex was investigated using a mercury film electrode. Tyrosine-derived 6-hydroxydopa quinone (TPQ) and copper ions in the active site are redox centers of these amine oxidases. The enzymes undergo two reduction processes at negative potentials related to the reduction of the TPQ cofactor to the corresponding hydroquinones and the reduction of copper ions, (Cu(II)→Cu(I)). Copper depleted enzymes, prepared by reduction with dithionite followed by dialysis against cyanide, undergo only one reduction process. Nyquist diagrams, recorded at potentials corresponding to the reduction of cofactors as dc-offset, represent charge transfer impedance followed by a Warburg-type line at low frequencies, indicating the occurrence of a diffusion controlled process in the rate-limiting step of the reduction process.