Laura Pecci

Novel findings on the copper catalysed oxidation of cysteine

SummaryThe oxidation of cysteine (RSH) has been studied by using O2, ferricytochrome c (Cyt c) and nitro blue tetrazolium (NBT) as electron acceptors. The addition of 200μM CuII to a solution of 2mM cysteine, pH 7.4, produces an absorbance with a peak at 260 nm and a shoulder at 300 nm. Generation of a cuprous bis-cysteine complex (RS-CuI-SR) is responsible for this absorbance. In the absence of O2 the absorbance is stable for long time while in the presence of air it vanishes slowly only when the cysteine excess is consumed. The neocuproine assay and the EPR analysis show that the metal remains reduced in the course of the oxidation of cysteine returning to the oxidised form at the end of reaction when all RSH has been oxidised to RSSR. Addition of CuII enhances the reduction rate of Cyt c and of NBT by cysteine also under anaerobiosis indicating the occurrence of a direct reduction of the acceptor by the complex. It is concluded that the cuprous bis-cysteine complex (RS-CuI-SR) is the catalytic species involved in the oxidation of cysteine. The novel finding of the stability of the complex together with the metal remaining in the reduced form during the oxidation suggest sulfur as the electron donor in the place of the metal ion.

Prevention of peroxynitrite-dependent damage by carnosine and related sulphonamido pseudodipeptides.

Abstract. The naturally occurring dipeptides carnosine and anserine have been proposed to act as antioxidants in vivo. We investigated whether these compounds can act as protective agents able to counteract peroxynitrite-dependent reactions. The results showed that the dipeptides efficiently protect tyrosine against nitration, α1-antiproteinase against inactivation and human low-density lipoprotein against modification by peroxynitrite. Carnosine exerts its protective effect at concentrations similar to those found in human tissues. In addition, some synthetic pseudodipeptides, stucturally related to carnosine but stable to hydrolytic enzymes, possess protective properties against peroxynitrite-dependent damage similar to the natural dipeptides. These pseudodipeptides may represent stable mimics of the biologically active carnosine suitable for pharmacological applications.

Hydrogen peroxide involvement in the rhodanese inactivation by dithiothreitol.

Abstract The conditions required to obtain rhodanese inactivation in the presence of dithiothreitol indicate the involvement of hydrogen peroxide produced by metal-ion catalyzed oxidation of dithiothreitol. Inhibition of dithiothreitol oxidation by a chelating agent, or by removal of hydrogen peroxide by catalase prevents the enzyme inactivation. The inactivated enzyme contains a disulfide bond resulting from the oxidation of the catalytic sulfhydryl group and another sulfhydryl group close to it. This disulfide might be formed via a sulfenic intermediate.

Bovine brain ketimine reductase

We report the purification from bovine brain of an NAD(P)H-dependent reductase which actively reduces a new class of cyclic unsaturated compounds, named ketimines. Ketimines arise from the transamination of some sulphur-containing amino acids, such as L-cystathionine, S-aminoethyl-L-cysteine and L-lanthionine. The enzyme also reduces delta 1-piperidine 2-carboxylate, the carbon analog of aminoethylcysteine ketimine. Some kinetic and molecular properties of this enzyme have been determined. Subcellular localization and regional brain distribution have also been studied. The ketimine reductase activity was found to be associated with the soluble fraction, and was located prevalently in the cerebellum and cerebral cortices. Cyclothionine and 1,4-thiomorpholine-3,5-dicarboxylic acid, the enzymatic reduction products of cystathionine ketimine and lanthionine ketimine, respectively, have been detected in bovine brain, thus suggesting a role of this enzyme in their biosynthesis.

Antioxidant properties of sulfinates: protective effect of hypotaurine on peroxynitrite-dependent damage.

Fig. 3. Prevention of ONOO-mediated a1AP inactivation by hypotaurine and cysteine sulfinic acid (CSA). Sulfinates, at the indicated concentrations, were incubated with a1AP in 0.5 M K-phosphate buffer, pH 7.4 before ONOO ) addition (0.5 mM). The a1AP inactivation was measured by its ability to inhibit elastase activity as described in experimental procedure. Results are the mean ± SEM of three experiments.

1,4-Thiomorpholine-3,5-dicarboxylic acid, a novel cyclic imino acid detected in bovine brain

Gas‐liquid chromatography of enriched bovine brain extract revealed the occurrence of several sulfur‐containing compounds. By co‐chromatography with authentic product and by mass‐spectrometric analysis, one of these compounds has been identified as 1,4‐thiomorpholine‐3,5‐dicarboxylic acid (TMDA). The possible derivation of TMDA from lanthionine is discussed. This represents the second S‐containing cyclic imino acid so far discovered in a mammalian brain whose physiological significance has not yet been explored.

Detection of 2H-1,4-thiazine-5,6-dihydro-3-car☐ylic acid (aminoethylcysteine ketimine) in the bovine brain

2H-1,4-Thiazine-5,6-dihydro-3-carboxylic acid (trivial name: aminoethylcysteine ketimine) is a cyclic sulfur-containing imino acid detected in bovine brain extracts by means of three different procedures. Gas liquid chromatography of protein-free extracts of five bovine brains revealed the presence of this compound at concentrations ranging from 2 to 3 nmol/g wet weight of tissue. The enzymatic method based on the inhibition of D-amino acid oxidase activity by aminoethylcysteine ketimine together with an high-performance liquid chromatography procedure confirm the identification and quantitations obtained with gas liquid chromatography. The discovery of this compound structurally similar to pipecolic acid opens the question of its physiological role in the central nervous system.

Biochemical properties of new synthetic carnosine analogues containing the residue of 2,3-diaminopropionic acid: the effect of N-acetylation

Summary.Three novel carnosine analogues 7–9 containing the residue of L(+)2,3-diaminopropionic acid with different degree of N-acetylation instead of β-alanine have been synthesized and characterized. Comparative analysis of hydrolysis by carnosinase revealed that the mono- and bis-acetylated compounds 8 and 9 are resistant to enzymatic hydrolysis and act as competitive inhibitors of this enzyme. The hydroxyl radical scavenging potential of the three analogues was evaluated by their ability to inhibit iron/H2O2-induced degradation of deoxyribose. The second-order rate constants of the reaction of compounds 7–9 with hydroxyl radical were almost identical to that of carnosine. These compounds were also found to act as protective agents against peroxynitrite-dependent damage as assessed by their ability to prevent nitration of free tyrosine induced by this species.

Antioxidant properties of the decarboxylated dimer of aminoethylcysteine ketimine: Assessment of its ability to scavenge peroxynitrite

The natural sulfur compound aminoethylcysteine ketimine decarboxylated dimer (AECK dimer) has been investigated for its ability to act as peroxynitrite scavenger. It has been found that the product efficiently protects against the nitration of tyrosine and the inactivation of alpha1-antiproteinase by peroxynitrite. The tyrosine nitration can be completely prevented by 100 microM AECK dimer which appears as effective as the antioxidants glutathione and N-acetylcysteine. The AECK dimer was also found to limit surface charge alteration of low density lipoprotein induced by peroxynitrite. These findings indicate that the AECK dimer is a strong protective agent against peroxynitrite damage and that it could play an important role in the defence against oxidative stress in human diseases.

Detection of Cystathionine Ketimine in Bovine Cerebellum

Abstract: A new sulfur‐containing cyclic imino acid, cystathionine ketimine, has been detected in bovine cerebellum by gas chromatography, gas chromatography‐mass spectrometry, and high pressure liquid chromatography procedures. Gas chromatography and gas‐mass analyses are based on derivatization of endogenous cystathionine ketimine with diazomethane after a simple enrichment procedure. The high pressure liquid chromatography procedure takes advantage of the selective absorbance at 380 nm of the phenyl isothio‐cyanate‐ketimine interaction product. The concentration of this new sulfur imino acid found in a pool of four bovine cerebella is ∼0.5 nmol/g.