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.
Oksidazy L-aminokislot (L-AAO (L-amino acid oxidases), KF 1.4.3.2) predstavliaiut gruppu flavoproteinov, kataliziruiushchikh okislitel'noe dezaminirovanie L-al'fa-aminokislot do sootvetstvuiushchikh al'fa-ketokislot, NH3 i N2O2. Chashche vsego éti fermenty predstavliaiut gomodimery s molekuliarnoĭ massoĭ 100-150 kDa, nadelennye protivovirusnoĭ, protivogribkovoĭ, antibakterial'noĭ i protivoopukholevoĭ aktivnost'iu. Osoboe mesto sredi fermentov étoĭ gruppy zanimaet L-lizin-al'fa-oksidaza (LO), biologicheskoe deĭstvie kotoroĭ mozhet otlichat'sia ot deĭstviia drugikh L-AAO, tak kak étot ferment preimushchestvenno rasshchepliaet nezamenimuiu dlia chelovecheskogo organizma aminokislotu L-lizin, prakticheski ne deĭstvuia na drugie aminokisloty. Poskol'ku molekuliarnye mekhanizmy tsitotoksicheskogo deĭstviia LO k nastoiashchemu vremeni izucheny nedostatochno, tsel'iu raboty iavlialos' izuchenie vozmozhnogo mekhanizma deĭstviia LO iz Trichoderma cf. aureoviride Rifai VKMF-4268D. V kachestve modeli ispol'zovali kul'turu kletok feokhromotsitomy krysy RS12. Metodom protochnoĭ tsitometrii pokazana dozo-zavisimaia gibel' kletok pod deĭstviem LO. Uvelichenie vnutrikletochnogo soderzhaniia aktivnykh form kisloroda, pokazannoe s pomoshch'iu 2,7-dikhlordigidrofluorestseina, pozvoliaet rassmatrivat' okislitel'nyĭ put', kak odin iz mekhanizmov proiavleniia tsitotoksicheskogo deĭstviia fermenta, chto ne iskliuchaiut drugikh ranee dokazannykh mekhanizmov vliianiia LO na gibel' kletok.
Keywords: L-amino acid oxidase; L-lysine alpha-oxidase; PC12 cell line; mechanism of cytotoxicity; reactive oxygen species (ROS).