Application of ion-exchange chromatography for the purification of glycolatoxidase from the leaves of the pea (Pisum sativum L.) and sorgo (Sorghum sudanense J.), investigation of its physical and chemical and regulatory properties
Abstract
The enzyme glycolate oxidase (EC 1.1.3.15) is widely distributed and studied, mainly in mammals and plants. In green plants, glycolate oxidase is one of the key enzymes in photorespiration, where it oxidizes glycolate to glyoxylate. It was found that, in addition to glycolate, this enzyme can use lactate as a substrate. In our work, we assumed that GO, being constitutive for plants, functions in both pea leaves and sorghum. Inpea leaves (Pisum sativum L.) and sorghum (Sorghum sudanense J.), the activity of one of the glycolate oxidase isoforms (l-Lactate Cytochrome c Oxidoreductase-like glycolate oxidase, EC 1.1.3.15) was induced. Using multi-stage purification, including ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephacel and gel chromatography on sephadex G-200, the enzyme was purified to an electrophoretic
homogeneous state. The preparation LCO-like GO from pea leaves had a specific activity of 314.37 U / mg protein, while the degree of purification was 207 times, and the yield was 11.5%. Similar indicators of the enzyme from sorghum leaves were: 274.22 U / mg protein, purification degree - 143 times, and yield - 13.8%. The physicochemical properties of the enzyme from these objects were studied. The molecular weight of the native molecule was determined (182 kDa and 168 kDa for peas and sorghum, respectively); it was shown that the enzyme consists of four subunits, the molecular weight of which is determined by PAGelectrophoresis in the presence of DDS-Na is 45 kDa for GO from pea leaves and 42 kDa - from sorghum. The kinetic and regulatory properties were investigated: the values of the Michaelis constants, the effect of the concentration of hydrogen ions and the temperature on the catalyzed reaction.
Downloads
References
2. Hackenberg C., Kern R., Huge J., Lucas J.S. et al., The Plant Cell., 2011, Vol. 23, pp. 2978-2990.
3. Engqvist M.K.M., Schmitz J., Gertzmann A., Florian A. et al., Plant Physiol., 2015, Vol. 169, pp. 1042-1061.
4. Zhang Z., Li X., Cui L., Meng S. et al., BMC Plant. Biol., 2017, Vol. 17, Issue 1, pp. 1-10.
5. Zelitch I, Schultes N.P., Peterson R.B., Brown P. et al., Plant Physiol., 2009, Vol. 149, Issue 1, pp. 195-204.
6. Lindqvist Y., Branden L., Mathews FS, Lederer F., J Biol Chem., 1991, Vol. 266, Issue 5, pp. 3198-3207.
7. Selemenev V.F., Rudakov O.B., Slavinskaya G.V., Drozdova N.V. Pigmenty pishchnvyh proizvodstv (melanoidy), M., De Li PRINT, 2000, 246 p.
8. Jain V., Singla N.K., Jain S., Gupta K., Physiol Mol Biol Plants., 2010, Vol. 16, Issue 3, pp. 241-247.
9. Setsuko K., Takahiro M., Hiroshi Y., PLos One, 2013, Vol. 8, Issue 6. е65301
10. Shevchenko A., Wilm M., Vorm O., Anal. Chem., 1996, Vol. 68, Issue 5, pp. 850-858.
11. Davis B.J., Ornstein L., Ann. NY Acad. Sci., 1964, Vol. 121, pp. 404-427.
12. Karpov S.I., Roessner F., Selemenev V.F., Gul'bin S.S. et al., Sorbtsionnye i khromatograficheskie protsessy, 2013, Vol. 13, No 2, pp. 125-140.
13. Eprintsev A.T., Fedorin D.N., Sazonova O.V., Igamberdiev A.U., Plant Phisiol Biochem., 2018, Vol. 129, pp. 305-309.
14. Eprintsev A.T., Fedorin D.N., Dobychina M.A., Igamberdiev A.U., Plant Science, 2018, Vol. 272, pp. 157-163.
15. Eprincev A.T., Gataullina M.O., PBM, 2018, Vol. 54, No 3, pp. 299-303.
16. Eprincev A.T., Larchenkov V.M., Komarova N.R., Kovalyova E.V. et al., PBM, 2018, Vol. 54, No 4, pp. 357-361.
17. Nishimura M., Akhmedov Y. D., Strzalka K., Akazawa T., Arch. Biochem. Biophys., 1983, Vol. 222, pp. 397-402.
18. Eprincev A.T., Komarova N.R., Falaleeva M.I., Larchenkov V.M., Izvestiya AN. Ser. biol., 2018, No 5, pp. 469-475.
