Oxidative deamination of S-(1-carboxyethyl)-L-cysteine and S-(1-carboxypropyl)-L-cysteine by L-aminoacid oxidase

S-(1-carboxyethyl)-L-cysteine (1-CEC) and S-(1-carboxypropyl)-L-cysteine (1-CPC) are oxidatively deaminated by L-aminoacid oxidase with consumption of half a mole of oxygen per mole of substrate in the presence of catalase. This reaction gives rise to the corresponding alpha-ketoacids, identified by some chemical and chromatographic tests and by comparison with synthetic compounds. It has been possible, therefore, to demonstrate that S-(1-carboxyethyl)-thiopvruvic acid (1-CETP) and S-(1-carboxypropyl)-thiopvruvic acid (1-CPTP) are the main products of oxidative deamination of 1-CEC and 1-CPC.     

Synthesis and chromatographic properties of S-(1-carboxyethyl)-L-cysteine and S-(1-carboxypropyl)-L-cysteine

Details are reported for the synthesis of S-(1-carboxyethyl)-L-cysteine (1-CEC) and S-(1-carboxypropyl)-L-cysteine (1-CPC) from cysteine and 2-bromopropionic acid or 2-bromobutyric acid, respectively. Some analytical data and the behaviour of these two compounds on paper and ion-exchange chromatography are also reported, which allow their identification.     

In vitro Dimroth rearrangement of 1-(2-carboxyethyl) adenine to N6-(2-carboxyethyl)adenine in single-stranded calf thymus DNA.

The new adduct N6-(2-carboxyethyl)adenine (N6-CEA) was prepared from 1-(2-carboxyethyl)adenine (1-CEA) by base catalyzed (Dimroth) rearrangement of 1-CEA. The structure of N6-CEA was assigned on the basis of UV spectra and electron impact and isobutane chemical ionization mass spectra. When the carcinogen beta-propiolactone was reacted in vitro with calf thymus DNA, 1-CEA but not N6-CEA was detected on paper chromatograms following acid hydrolysis of the DNA. When BPL-reacted single-stranded DNA was incubated at pH 11.7 (37 degrees C, 18 h) prior to acid hydrolysis, it was found that 1-CEA was completely converted to N6-CEA in DNA by Dimroth rearrangement, whereas no conversion occurred at pH 7.5. The extent of Dimroth rearrangement at various pHs and temperatures was determined for 1-CEA, 1-methyladenine (1-MeA), 1-(2-carboxyethyl)-deoxyadenosine-5'-monophosphoric acid (1-CEdAdo5'P) and the phosphodiester 5'-O-(2-carboxyethyl)phosphono-1-(2-carboxyethyl)deoxyadenosine (1-CE-Ado-5'-P-CE).     

Oxidative deamination of hydrolyzed fumonisin B(1) (AP(1)) by cultures of Exophiala spinifera.

Fumonisins are mycotoxins of world-wide distribution in maize infected by the fungus Fusarium verticillioides. They are highly toxic to certain livestock and are potential carcinogens. Exophiala spinifera, a black yeast fungus found on moldy maize kernels, was identified previously as capable of growing on fumonisin B1 as a sole carbon source and thus is a potential source for fumonisin detoxifying enzymes. Pure cultures of E. spinifera transform fumonisin B(1) to the amino polyol AP(1) plus free tricarballylic acid through the activity of a soluble extracellular esterase, and further transformation is evidenced by accumulation in culture supernatant of a less polar compound(s) lacking a fluorescamine-reactive amino group. A free amine is thought to be critical for biological activity of FB(1) or AP(1). As a first step towards characterizing this amine-modifying activity, we investigated the biotransformation of AP(1) by E. spinifera liquid cultures that had been previously grown in liquid medium containing AP(1) as a sole carbon source. Accumulation of AP(1)-derived metabolites was monitored by thin-layer chromatography of culture supernatants, and product metabolites were purified and evaluated by mass spectrometry and nuclear magnetic resonance. Two products of treatment of purified AP(1) with cultures of E. spinifera are shown to be N-acetyl AP(1) and a new compound, 2-oxo-12,16-dimethyl-3,5,10, 14,15-icosanepentol hemiketal (or 2-OP(1) hemiketal).     

Modified 5'-nucleotides resistant to 5'-nucleotidase: isolation of 3-(3-amino-3-carboxypropyl) uridine 5'-phosphate and N2, N2-dimethylguanosine 5'-phosphate from snake venom hydrolysates of transfer RNA.

A procedure for the quantitative measurement of the O2'-methylnucleoside constitutents of RNA has recently been developed in this laboratory (Gray, M.W. Can. J. Biochem. 53, 735-746 (1975)). This assay method is based on the resistance of O2'-methylnucleoside 5'-phosphates (pNm) (generated by phosphodiesterase hydrolysis of RNA) to subsequent dephosphorylation by venom 5'-nucleotidase (EC 3.1.3.5). In the present investigation, two base-modified 5'-nucleotides, each displaying an unusual resistance to 5'-nucleotidase, have been identified. These compounds have been characterized by a variety of techniques as N2, N2-dimethylguanosine 5'-phosphate (pm2/2G) and 3-(3-amino-3-carboxypropyl)uridine 5'-phosphate (p4abu3U). Because of their resistance to 5'-nucleotidase, pm2/2G and p4abu3U are isolated along with the pNm in the mononucleotide fraction of venom hydrolysates of transfer RNA. Under hydrolysis conditions, the stability of p4abu3U is comparable to that of a pNm, allowing quantitative assay of the nucleotide. The proportion (mean +/- SD) of p4abu3U in venom hydrolysates of wheat embryo and Escherichia coli tRNA has been determined to be 0.35 +/- 0.03 (n=5) and 0.14 +/- 0.02 (n=4) mol%, respectively. The absence of p4abu3U in venom hydrolysates of yeast tRNA implies the absence of the corresponding nucleoside in yeast tRNA, in agreement with existing data. The variable recovery of pm2/2G from venom hydrolysates of wheat embryo and yeast tRNA indicates that under hydrolysis conditions, this base-modified nucleotide is only partially resistent to 5'-nucleotidase. The complete absence of pm2/2G in venom hydrolysates of E. coli tRNA is consistent with the known absence of N2, N2-dimethylguanosine in this RNA. These observations demonstrate that resistance to 5'-nucleotidase is a necessary but not sufficient criterion for concluding that a 5'-nucleotide is O2'-methylated. When applied to wheat embryo ribosomal RNA, the analytical methods described in this report failed to reveal any compound having the distinctive charge properties of p4abu3U. It therefore appears that 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, recently characterized as a constituent of the 18 S rRNA of Chinese hamster cells (Saponara, A.G. & Enger, M.D. Biochim. Biophys. Acta 349, 61-77 (1974)), may not be present in wheat embryo ribosomal RNA.     

Isolation, structural, and functional characterization of an apoptosis-inducing L-amino acid oxidase from leaf-nosed viper (Eristocophis macmahoni) snake venom

The enzyme L-amino acid oxidase (LAO) from the leaf-nosed viper (Eristocophis macmahoni) snake venom was purified to homogeneity in a single step using high performance liquid chromatography on a Nucleosil 7C18 reverse phase column. The molecular mass of the purified enzyme was 58734.0 Da, as determined by matrix-assisted laser desorption/ionization mass spectrometry. The N-terminal amino acid sequence (ADDKNPLEEAFREADYEVFLEIAKNGL) and the chemical composition of the purified LNV-LAO shows close structural homology with other L-amino acid oxidases isolated from different snake venoms. The secondary structural contents analysis of LAO, established by means of circular dichroism, revealed ca. 49% alpha-helix, 19% beta-sheet, 10% beta-turn, and 22% random coil structure. The purified LNV-LAO not only retained its specific enzymatic activity (73.46 U/mg), determined against L-leucine as a substrate, but also exhibited potent haemolytic (1-10 microg/ml), edema- (MED 4.8 microg/ml) and human platelet aggregation-inducing (ED50 33 microg/ml) properties. Unlike other haemorrhagic snake venom L-amino acid oxidases, the LNV-LAO does not produce haemorrhage. In addition to these local effects, the purified LNV-LAO showed apoptosis-inducing activity in the MM6 cell culture assay. After 18 h treatment with 25-100 microg/ml of LAO, the typical DNA fragmentation pattern of apoptotic cells was observed by means of fluorescent microscopy and agarose gel electrophoresis.     

N6-(1-carboxyethyl)lysine formation by Streptococcus lactis. Purification, synthesis, and stereochemical structure

During growth in an arginine-deficient (chemically defined) medium, cells of Streptococcus lactis K1 formed significant amounts of a previously undetected ninhydrin-positive compound. This intracellular compound did not cochromatograph with any of a wide range of amino acids or amino acid analogs tested. However, by two-dimensional thin layer chromatography, the unknown compound migrated close to the recently discovered N5-(1-carboxyethyl)ornithine (Thompson, J., Curtis, M. A., and Miller, S. P. F. (1986) J. Bacteriol. 167, 522-529; Miller, S. P. F., and Thompson, J. (1987) J. Biol. Chem. 262, 16109-16115). The purified compound behaved as a neutral amino acid and eluted between valine and methionine in the amino acid analyzer. The results of 1H NMR spectroscopy suggested the presence of a lysine backbone and a coupled methyl-methine unit in the molecule, and 13C NMR showed that there were nine carbon atoms, of which two (C-1 and C-7) were carboxyl carbons. The simplest structure compatible with the physicochemical data was that of an alkylated derivative of lysine. The identity of this new amino acid, N6-(1-carboxyethyl)lysine, was confirmed by chemical synthesis. In vivo labeling experiments conducted using L[U-14C]lysine and [epsilon-15N]lysine showed that exogenous lysine served as the precursor of intracellular N6-(1-carboxyethyl)lysine and that the epsilon-amino N atom was conserved during biosynthesis of the lysine derivative. Of the two possible diastereomers (2S,8S or 2S,8R) of N6-(1-carboxyethyl)lysine, comparative 13C NMR spectroscopy established that the amino acid produced by S. lactis K1 was exclusively of the 2S,8S configuration.     

Therapeutic application of natural inhibitors against snake venom phospholipase A(2)

Natural inhibitors occupy an important place in the potential to neutralize the toxic effects caused by snake venom proteins and enzymes. It has been well recognized for several years that animal sera, some of the plant and marine extracts are the most potent in neutralizing snake venom phospholipase A(2) (svPLA(2)). The implication of this review to update the latest research work which has been accomplished with svPLA(2) inhibitors from various natural sources like animal, marine organisms presents a compilation of research in this field over the past decade and revisiting the previous research report including those found in plants. In addition to that the bioactive compounds/inhibitor molecules from diverse sources like aristolochic alkaloid, flavonoids and neoflavonoids from plants, hydrocarbones -2, 4 dimethyl hexane, 2 methylnonane, and 2, 6 dimethyl heptane obtained from traditional medicinal plants Tragia involucrata (Euphorbiaceae) member of natural products involved for the inhibitory potential of phospholipase A(2) (PLA(2)) enzymes in vitro and also decrease both oedema induced by snake venom as well as human synovial fluid PLA(2). Besides marine natural products that inhibit PLA(2) are manoalide and its derivatives such as scalaradial and related compounds, pseudopterosins and vidalols, tetracylne from synthetic chemicals etc. There is an overview of the role of PLA(2) in inflammation that provides a rationale for seeking inhibitors of PLA(2) as anti-inflammatory agents. However, more studies should be considered to evaluate antivenom efficiency of sera and other agents against a variety of snake venoms found in various parts of the world. The implications of these new groups of svPLA(2) toxin inhibitors in the context of our current understanding of snake biology as well as in the development of new novel antivenoms therapeutics agents in the efficient treatment of snake envenomations are discussed.     

N2-(1-carboxyethyl)methionine. A ''pseudo-opine'' in octopine-type crown-gall tumours.

A novel methionine-containing plasmid-determined compound, N2-(1-carboxyethyl)methionine (NCEM) has been identified in crown-gall tumours induced by octopine-type strains of Agrobacterium tumefaciens. NCEM is probably synthesized by octopine synthase. Cell-free preparations from octopine-type strains of A. tumefaciens can degrade NCEM; however, the bacterium cannot transport the compound into the cell, although these strains can take up and degrade the octopine family of opines.     

Enzymatic activity of L-amino acid oxidase from snake venom Crotalus adamanteus in supercritical CO2

L-amino acid oxidase (L-AAO) from snake venom Crotalus adamanteus was successfully tested as a catalyst in supercritical CO₂ (SC-CO₂). The enzyme activity was measured before and after exposure to supercritical conditions (40°C, 110 bar). It was found that L-AAO activity slightly increased after SC-CO₂ exposure by up to 15%. L-AAO was more stable in supercritical CO₂ than in phosphate buffer under atmospheric pressure, as well as in the enzyme membrane reactor (EMR) experiment. 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) oxidation was performed in a batch reactor made of stainless steel that could withstand the pressures of SC-CO₂, in which L-amino acid oxidase from C. adamanteus was able to catalyze the reaction of oxidative deamination of L-DOPA in SC-CO₂. For the comparison L-DOPA oxidation was performed in the EMR at 40°C and pressure of 2.5 bar. Productivity expressed as mmol-s of converted L-DOPA after 3?h per change of enzyme activity after 3?h was the highest in SC-CO₂ (1.474?mmol?U^?1), where catalase was present, and the lowest in the EMR (0.457?mmol?U^?1).