The activity of glutathione reductase under impaired liver function and regulation by the Krebs cycle intermediates of the catalytic action of the enzyme isolated by chromatographic methods
Abstract
The aim of the study was to determine the activity of glutathione reductase (GR, EC 1.6.4.2) in the blood serum of patients with alcoholic hepatitis (AH) and in the blood serum and livers of rats with experimental toxic hepatitis (ETH), as well as to develop a scheme for the purification of the enzyme from the liver of experimental animals using chromatographic methods. In the experiment, we used the blood serum of apparently healthy individuals with normal indices of general and biochemical blood tests (control group of patients), people diagnosed with alcoholic hepatitis (AH), as well as the serum and livers of rats in the control group and rats with ETG. The pathological state in experimental animals was modelled by oral administration of carbon tetrachloride, an organ-specific toxin with hepatotropic effect, as a 33% solution in paraffin oil at the rate of 64 µl of toxin per 100 g of animal weight. The animals were slaughtered on the 4th day after administration of the toxic agent. The control animals were injected with the corresponding aliquot of paraffin oil. The activity of GR was determined spectrophotometrically using a spectrophotometer SF-46 at a wavelength of 340 nm. The total amount of protein was determined by the Lowry method. To study the regulatory properties of the enzyme, it was purified from the livers of the control rats and those with induced toxic hepatitis using the protein separation by ammonium sulphate, as well as gel filtration through Sephadex G-25 and ion-exchange chromatography on DEAE-cellulose. As a result, we obtained 54.5 and 49.1 times purified GR enzyme preparations from the livers of the control rats and animals with ETG. We determined that in the process of ion-exchange chromatography using a column with DEAE-cellulose, the enzyme from the livers of the studied groups of animals was desorbed as a single peak at a KCl concentration of 100 mM. Using the obtained enzyme preparations, we detected differences in the regulation of GR activity under the action of Krebs cycle intermediates. They are obviously associated with conformational modifications of the enzyme molecules under oxidative stress developing during pathology.
Downloads
References
Rusyn I., Arzuaga X., Cattley R.C., Corton J.Ch., Ferguson S.S., Godoy P., Guyton K.Z., Kaplowitz N., Khetani S.R., Roberts R., Roth R.A., Smith M.T., Key Characteristics of Human Hepatotoxicants as a Basis for Identifi-cation and Characterization of the Causes of Liver Toxicity, Hepatology, 2021; 74(6): 3486-3496. https://doi.org/10.1002/hep.31999
You M., Arteel G. E., Effect of ethanol on lipid metabolism, Hepatology, 2019; 70: 237-248. https://doi.org/10.1016/j.jhep.2018.10.037
Sun H., Chen L., Zhou W., Hu L. Liang Li 1, Tu Q., Chang Y., Liu Q., Sun X., Wu M., Wang H., The protective role of hy-drogen-rich saline in experimental liver injury in mice, Journal of Hepatology, 2011; 54 (3): 471-480. https://doi.org/10.1016/j.jhep.2010.08.011.
Agarkov A.A. Diss. kand. biol. nauk. Voronezh, 2009, 222 р. (In Russ.)
Iskusnykh I.Y., Kryl’skii E.D., Bra-zhnikova D.A., Popova T.N., Shikhaliev K.S., Shulgin K.K., Matasova L.V., Popov S.S., Zhaglin D.A., Zakharova A.A., Popova N.R., Fattakhov N., Novel Antioxidant, Deethylated Ethoxyquin, Protects against Carbon Tetrachlo-ride Induced Hepatotoxicity in Rats by Inhibit-ing NLRP3 Inflammasome Activation and Apoptosis, Antioxidants, 2021; 10(1): 122. https://doi.org/10.3390/antiox10010122
Chen M., Zhong W., Xu W., Alcohol and the mechanisms of liver disease, Journal of Gastroenterology and Hepatology, 2023; 38: 1233-1240. https://doi.org/10.1111/jgh
Jakupova T.G., Karimov D.O., Bakirov A.B. Izmenenie jekspressii genov oksida-tivnogo stressa pri toksicheskih gepatitah raznoj jetiologii i ih korrekcija, Jeksperi-mental'naja i klinicheskaja gastrojenterologija, 2023; 216(8): 120-126. https://doi.org/10.31146/1682-8658-ecg-216-8-120-126 (In Russ.)
Babior B.M., Phagocytes and oxidative stress, Am. J. Med., 2000; 109 (1): 33-44.
Dudnik L.B., Viksna L.M., Majore A.Ja., Peroksidnoe okislenie lipidov i ego svjaz' s izmeneniem sostava i antiokislitel'nyh svojstv lipidov pri komatogennyh formah os-trogo virusnogo gepatita V, Voprosy medicinskoj himii, 2000; 6: 597-609. (In Russ.)
Kryl'skii E.D., Sinitsyna D.A., Popova T.N., Shikhaliev K.S., Medvedeva S.M., Matasova L.V., Mittova V.O., The new antiox-idant 1-benzoyl-6-hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline has a protective effect against carbon tetrachloride-induced hepatic injury in rats, The Journal of Biomedical Research, 2022; 36(6): 423-434. https://doi.org/10.7555/JBR.36.20220098
Blinova T.V., Strahova L.A., Troshin V.V., Kolesov S.A., Umnjagina I.A., Ivanova Ju.V., Glutation kak prognosticheskij faktor riska narushenija zdorov'ja rabotajushhih lic, Analiz riska zdorov'ju, 2023; 2: 140-148. https://doi.org/10.21668/health.risk/2023.2.13 (In Russ.)
Kryl’skii, E.D.; Kravtsova, S.E.; Popova, T.N.; Matasova, L.V.; Shikhaliev, K.S.; Medvedeva, S.M., 6-Hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline Demonstrates Anti-Inflammatory Properties and Reduces Ox-idative Stress in Acetaminophen-Induced Liver Injury in Rats, Curr. Issues Mol. Biol., 2023; 45: 8321-8336. https://doi.org/10.3390/cimb45100525
Fedorova N.Ju. Avtoref. dis kand. biol. nauk. Voronezh, 1999, 24 p. (In Russ.)
Popova T.N., Shul'gin K.K., Shihaliev H.S., Kryl'skij E.D., Matasova L.V., Medvedeva S.M., Popov S.S., Verevkin A.N. Zajavka na patent. № 2017136371. 2019. (In Russ.)
Selemenev V.F., Rudakov O.B., Slav-inskaja G.V., Drozdova N.V. Pigmenty pish-hevyh proizvodstv (melanoidy). Moskva, DeLi print., 2008, 246 р. (In Russ.)
Kramarenko V.F. Toksikologicheskaja himija. Moskva, Kniga po Trebovaniju, 2013, 445 р. (In Russ.)
Glants S. Mediko-biologicheskaya statistika. Moskva, Praktika, 1998, 459 p. (In Russ.)
Lazebnik L. B., Golovanova E. V., Tarasova L. V., Krivosheev A. B., Sas E. I., Eremina E. Ju., Truhan D. I., Hlynova O. V., Cyganova Ju. V., Alkogol'naja bolezn' pecheni (ABP) u vzroslyh, Jeksperimental'naja i klinicheskaja gastrojenterologija, 2020; 174(2): 4-28. (In Russ.)
Voronina T.A., Rol' oksidativnogo stressa i antioksidantov pri dezadaptacii razlichnogo geneza, Farmacija i farmakologija, 2015; 3(5): 8-17. https://doi.org/10.19163/2307-9266-2015-3-5s (In Russ.)
Agarkov A.A., Popova T.N., Se-menihina A.V., Kataliticheskie svojstva gluta-tionreduktazy iz pecheni krysy v norme i pri toksicheskom gepatite, Biomedicinskaja himija, 2009; 55(2): 169-176. (In Russ.)
Pashkov A.N., Popov S.S., Semenihina A.V., Rahmanova T.I. Sostojanie sistemy glu-tationa i aktivnost' nekotoryh NADPH-generirujushhih fermentov v pecheni krys pri dejstvii melatonina v norme i pri toksicheskom gepatite, Bjulleten' jeksperimental'noj biologii i mediciny, 2005;139(5): 520-524. (In Russ.)
Jur'eva Je.A., Novikova N.N., Dlin V.V., Vozdvizhenskaja E.S., Molekuljarnyj stress i hronicheskie narushenija obmena vesh-hestv, Rossijskij vestnik perinatologii i pedi-atrii, 2020; 65(5): 12-22. (In Russ.)
Novikov V.E., Levchenkova O.S., Pozhilova E.V., Rol' aktivnyh form kisloroda v fiziologii i patologii kletki i ih farma-kologicheskaja reguljacija, Obzory po klinich-eskoj farmakologii i lekarstvennoj terapii, 2014; 12: 13-21. (In Russ.)
Luk'janova L.D., Sovremennye prob-lemy adaptacii k gipoksii. Signal'nye me-hanizmy i ih rol' v sistemnoj reguljacii, Pat. fiziol. i jeksperim. Terapija, 2011; 1: 3-19. (In Russ.)
Sazontova T.G., Arhipenko Ju.V., Rol' svobodnoradikal'nyh processov i redoks-signalizacii v adaptacii organizma k izmeneniju urovnja kisloroda, Ros. fiziol. zhurn. im. I.I. Sechenova, 2005; 91(6): 636-655. (In Russ.)
