Search

L-Amino acid oxidases (L-ААО, EC 1.4.3.2) comprise a group of flavoproteins, catalyzing oxidative deamination of L-alpha amino acids to the corresponding alpha-keto acids, NH3 and Н2О2. In most cases these enzymes present homodimeric molecules with a molecular mass of 100-150 kDa, which were shown to possess antiviral, antifungal and antitumor activity. L-lysine alpha-oxidase (LO) holds an outstanding place among this group of enzymes and its biological role may differ significantly from the other L-AAO, because it cleaves an essential amino acid - L-lysine without significant action on the other amino acids. Although much research has examined LO effects in the organism, the molecular basis of these effects is yet to be identified. To fill this gap, the present work addressed one of hypothetical mechanisms of LO biological action using the enzyme from Trichoderma cf. aureoviride Rifai ВКМF-4268D and rat pheochromocytoma PC-12 as a model cell line. Using flow cytometry a dose-dependent cytotoxicity of LO was shown. The significant growth of intracellular reactive oxygen species levels, detected by 2,7-dichlorodihydrofluorescein assay, implies generation of peroxide as one of the molecular mechanisms of LO cytotoxic action, although this does not rule out other probable ways of LO action in the organizm.

Synergism effects of cisplatin and L-lysine-alpha-oxidase (LO), while sequential (no interval) administration of drugs depends on the tumor model and duration of treatment. Synergism is identified at intraperitoneal daily (during 3 days) administration of cisplatin to experimental animals in single doses of 1.5 or 3.0 mg/kg and intravenously 5-fold after 48 h administration of LO and also administered intravenously in cumulative doses of 300-600 E / kg discretely, the first dose--doubled. Synergism of cisplatin and LO is showed by significant (p < 0.05) therapeutic gain against cisplatin at such indicators as increased survival of mice with P388 tumor and increased inhibition of primary tumor melanoma B16.

During previous decade L-amino acid oxidases (LAAO) attracted the steady interest of researchers due to their poly functional effects on different biological systems. The review summarizes information concerning the sources, structure, phisico-chemical and catalytical properties of LAAO which exhibit antibacterial, antifungal, antiprotozoal, antiviral effects as well as the ambiguous action on platelet aggregation. Special attention is devoted to the elucidation of molecular mechanisms of LAAO action. It is proposed that the unique properties of LAAO) are based on their catalytic reaction, which causes the decrease of L-amino acid levels, including the essential amino acids and formation of hydrogen peroxide. The action of liberated H2O2 on cells involves the synthesis of oxygen reactive species and the development of necrotic and apoptotic pathways of cell death. The presence of carbohydrate moieties in LAAO molecules promotes their attachment to cell's surface and creation of high H2O2 local concentrations. The wide range of LAAO biological effects is undoubtedly connected with their important functional roles in the organism. In particular, it was shown that in the mice brain the LAAO-catalyzed reaction is the single pathway of L-lysine degradation, while in the mice milk LAAO carry out the antibacterial effect and in human leucocytes LAAO take part in fulfilling their defending role. Protector action may be also attributed to the oxidases from the other numerous sources: microscopic fungi, snake venoms and sea inhabitants.

Full-thickness skin wounds (460 mm(2)) in rats were associated with increased blood chemiluminescence and neutrophil infiltration of the wound tissue and surrounding skin (recorded by myeloperoxidase activity). Activities of glutathione peroxidase and glutathione S-transferase in the skin and wound tissue increased on days 4 and 8. A correlation was revealed between activities of these enzymes and myeloperoxidase activity. Activities of myeloperoxidase and catalase increased in patient's skin excised during plastic surgeries of more than 2.5 h duration.

The significant difference between biological properties of L-lysine-alpha-oxidase from Trichoderma harzianum Rifai (LO) and L-asparaginase from E. coli has been observed in vitro and in vivo. High antitumor activity was shown against 8 types of murine and rat transplanted tumors with a wide range of LO therapeutic doses: 35-350 U/mg. The LO conjugates with monoclonal antibodies CD5 specific to the surface of cell line Yurkat were obtained without significant loss of either enzymatic and cytotoxic activity or immunological specificity. The further perspective investigation for the clinical application of the native or conjugated enzymes is discussed.

The conjugates of L-lysine alpha-oxidase and monoclonal antibodies ICO-80 towards CD-5 receptor were produced using glutaraldehyde. The cytotoxic effect of conjugates on Yurkat cells line appeared to be lower in comparison with the native enzyme. Negligible decrease of conjugate biological activity may be explained by the large molecular weight of conjugate, which is several times higher than the molecular weight of the native enzyme. Such conjugates can not penetrate into the cells. So they catalyze the hydrogen peroxide formation, the main damaging agent, probably only outside the cells. We suppose also that the free native enzyme penetrates into the cell and activates there the oxidative deamination of L-lysine and correspondingly the hydrogen peroxide formation. This may be the proper explanation for the higher cytotoxic effect of L-lysine alpha-oxidase on Yurkat cell line.

The conjugation of the drugs with vector molecules enables to obtain therapeutic preparation, which may be transported to the selected target organ. In the present work the methods of conjugation of antineoplastic enzyme L-lysine alpha-oxidase with antibodies were elaborated. Conjugates were worked out through the attachment of amino groups on the antibody surface either with the aldehyde groups which were created in L-lysine alpha-oxidase molecule (0.2% of initial enzymatic activity) or with the aldehyde groups of cross-linking molecules. Maximal (78%) L-lysine alpha-oxidase activity in conjugates was observed when oxidized peroxidase which contained the aldehyde groups was used as crosslinking agent. The glutaraldehyde method yielded 70% of initial enzyme activity.

The goal of the present study was the development of the optimal method of co-immobilization of two enzymes: L-lysine alpha-oxidase from Trichoderma sp. and horseradish peroxidase. Commercial nitrocellulose, nylon and N+ nylon membranes were used as carriers. The immobilization was carried out either by absorbtion or by covalent binding with aldehyde groups. The aldehyde groups were attached to the surface of the carriers by UV-irradiation of membranes in the presence of p-azidotetrafluorobenzaldehyde. The optimal concentrations of reagents, enzymes and reaction conditions were found. The membranes with the co-immobilised L-lysine alpha-oxidase and peroxidase were shown to be useful for the determination of L-lysine concentrations.

Homogeneous preparations of L-lysine-alpha-oxidase were obtained from Trichoderma sp cultivated by using a surface technique and a fermenter set. The homogeneous enzyme preparations were similar in molecular mass, isoelectric point and substrate specificity. There was the single difference in the absorbance spectra, which may occur due to the presence of cofactor FAD in various oxidation states. The findings suggest that cultivation of Trichoderma sp in the fermenter set did not alter properties of L-lysine-alpha-oxidase produced.

Some physicochemical properties of L-lysine oxidase from two strains of Trichoderma were studied. Content of metals and cofactors (Se, Zn, Cu, Fe, Co, Mn, Mo), amino acid analysis, secondary structure were estimated. The enzyme molecule from Trichoderma sp was found to contain both FAD and PQQ cofactors.

A simple and relatively sensitive procedure was developed for determination of L-lysine at 3-30 mmole/L concentration. The procedure does not involve the carcinogenic compound o-dianisidine. L-lysine alpha-oxidase catalyzed oxidative deamination of L-lysine with O2 consumption and formation of H2O2, NH3 and alpha-keto-epsilon-aminocaproic acid. Horseradish peroxidase and a non-carcinogenic compound 3,3,5,5'-tetramethylbenzidine dihydrochloride as an electron donor were used in determination of H2O2 formed. The procedure developed enabled also to measure the L-lysine alpha-oxidase activity at the enzyme concentrations of 10-500 ng/ml. The only limitation of the procedure is relatively low pH-values of the reaction medium.

A method of microencapsulating of the proteolytic enzyme alpha-chymotrypsin into semi-permeable nylon membranes is worked out. The membrane is a polimer of 1,6-hexamethylenediamine and sebacoyl chloride. alpha-Chymotrypsin is enclosed into the capsule together with polyethyleneimine, capable of joining the walls of microcapsules and making the membrane more stable. The optimal concentrations of polyenthyleneimine and alpha-chymotrypsin are 5% and 1% correspondingly. The highest yield of microencapsulated enzyme was obtained for completely acetylated delta-chymotrypsin. The kinetic properties of microencapsulated alpha-chymotrypsin change very slightly as compared to those of the native one.

L-lysine-alpha-oxidase from Trichoderma sp. fungi inhibits the synthesis of DNA, RNA and protein in cultivated cells of the Burkitt lymphoma (strain P3HRj) in vitro, which results in the blocking of the cells' transfer from phase S to phase G2/M of the mitotic cycle.

Activity of L-amino acid oxidases was studied using several procedures. Optimal concentrations of L-lysine-alpha-oxidase, suitable for each procedure, were established involving highly purified preparations of the enzyme from Trichoderma sp. Estimation of the enzymatic activity carried out by means of calculation of the reduced cofactor accumulated led to two-fold exceeding of the results. The most sensitive procedure was based on evaluation of ammonium content in the reaction catalyzed by glutamate dehydrogenase and the procedure where peroxidase and o-dianizidine were used.

L-lysine-alpha-oxidase, isolated from a strain of Trichoderma sp. exhibited enzymological properties suitable for a chemotherapeutic drug used in experimental oncology.

The microgranules containing covalently bound cis-cinnamoylchymotrypsin were obtained by emulsion polymerization of acrylamide and the cross-linking agent in the presence of a light-sensitive enzyme premodified by acryloylchloride. The kinetics of light reactivation of microgranulated cis-cinnamoylchymotrypsin was studied. It was proposed to develop the latent enzyme image by the reaction of N-acetyl-L-leucine indoxyl ester hydrolysis. The product of this reaction, indoxyl, forms indigo in the presence of oxygen. The intensity of staining was shown to depend on the exposition time. This system can be used for production of half-tone enzyme photomaterials.

alpha-Chymotrypsin and its light-sensitive derivative, cis-cinnamoylchymotrypsin, free and immobilysed (by means of microcapsulation, covalent binding with sepharose 4B and p-aminobenzyl cellulose) are studied. Effect of pH on the hydrolysis rate of N-alpha-acetyl-L-tyrosine ethyl ester by different immobilized enzyme preparations, their acylation with isomeric mixture of cis- and trans-cinnamoylimidazole, reactivation of stable cis-derivatives of different enzyme samples and thermal stability of native and microcapsulated enzyme preparations are investigated.

An improved and relatively rapid procedure is developed for isolation and purification of a new antitumor enzyme L-lysyl-alpha-oxidase from Trichoderma sp. The method involves four steps, instead of six steps described previously, with a yield of 22.4%. The purified enzyme preparation was homogeneous as shown by polyacrylamide gel disc electrophoresis and ultracentrifugation. Physico-chemical and antitumor properties of the enzyme are under study.