Effect of bivalent metal cations on the functioning of NADPH+-dependent malate dehydrogenase from corn leaves
Abstract
Malate dehydrogenase (MDH, NADPH-denpendent oxidoreductase, EC 1.1.1.82) - NADPH+- MDH (EC 1.1.1.82) is an important enzyme responsible for the metabolization of organic plant acids. The aim of the study was to determine the regulatory properties of the enzyme such as the effect of bivalent metals on its activity. 10-day-old corn seedlings were chosen as the subject for the study. The Klechkowski method was used for the separation of tissues. Enzyme purification was carried out by a traditional method. Chlorides of such bivalent metal cations as magnesium, calcium, barium, and manganese were used as the studied ions. Thus, four-stage purification, including homogenisation, fractionation with ammonium sulfate, elfiltration on Sephadex G-25, and ion-exchange chromatography on DEAE-Sephacel made it possible to obtain high purity NADPH+ malate dehydrogenase from mesophyll of corn leaves. The degree of purification increased by 57 times and the yield of the studied protein was 6% of the total. The specific activity of the purified NADPH+-dependent malate dehydrogenase in terms of protein milligrams was 91 enzyme units. Universal staining with silver nitrate and specific staining by tetrazolium method, polyacrylamide gel obtained as a result of electrophoresis of purified MDH revealed only one protein band which means that the purified malate dehydrogenase preparation was homogenous. The study of the effect of cations of different metals allowed us to identify their effect on the functioning of malate dehydrogenase. The effect of chlorides of Mg2+, Мn2+, Ва2+, and Са2+ ions on the activity of the purified preparation was studied. It was demonstrated that small concentrations of magnesium (within 3 mM) activates NADPH-denpendent MDH. Barium and manganese inhibit the enzyme. Calcium ions did not affect the MDH activity. The analysis of the obtained data by means of Lineweaver–Burk plots revealed different types of inhibition. Competitive inhibition is characteristic of manganese cations. Barium cations demonstrate incompetitive enzyme inhibition
Downloads
References
Komov V.P., Shvedova V.N., Biokhimija, M., Uright, 2015, 640 p. 2. Mamushina N.S., Zubkova E.K., Botanicheskij zhurnal, 2005, Vol. 90, No 11, pp. 16411650. 3. Marri R., Grenner D., Mejes P., Rodujell V. Biokhimija cheloveka, V 2-h tomah. Vol. 1, M., Mir, 1993, 384 p. 4. Minkina T.M., Motuzova G.V., Mandzhieva S.S., Nazarenko O.G. et al., Nauchnyj zhurnal Rossijskogo NII problem melioracii, 2011, No 4. 5. Bednjakov D.A., Nevaljonnaja L.A., Novinskij V.Ju., Vestnik Astrahanskogo gosudarstvennogo tehnicheskogo universiteta. Serija: Rybnoe hozjajstvo, 2011, No 2, pp. 74-77. 6. Gataullina M.O., Eprintsev A.T., Sorbtsionnye i khromatograficheskie protsessy, 2018, Vol. 18, No 1, pp. 111-117.
Eprincev A.T., Gataullina M.O., Ljashhenko M.S., Prikladnaja biohimija i mikrobiologija, 2016, Vol. 52, No 4, pp. 365-369. 8. Davis B.J., Ornstein L. Society for the study at the New York Academy of medicine. 1959. pp. 112-118. 9. Lowry O.H., Rosebrough H.J., Papp A.H. et al., J. Biologi, 1951, Vol. 193, pp. 265-275. 10. Selemenev V.F., Slavinskaya, G.V., Khokhlov V.Yu., Ivanov V.A. et al., Praktikum po ionnomu obmenu, Voronezh, VGU, 2004, 160 p. 11. Rudakov O.B., Selemenev V.F. Fizikohimicheskie sistemy sorbat-sorbent-jeljuent v zhidkostnoj hromatografii, Voronezh, VGU, 2003, 300 p. 12. Smirnov V.A., Klimochkin Ju.N. Vitaminy i kofermenty, Samara, Samar. gos. tehn. un-t, 2008, 92 p. 13. Shlejkin A.G., Skvorcova N.N., Blandov N.N. Prikladnaya enzymologia: uchebnoeposobie. SPb, Universitet ITMO, 2019. 160 p. 14. Peers M.K. Toogood H.S., Heye, D.J., Mansell D. et al., Catalysis science & technology, 2016, Vol.6, No 1, pp.169-177. 15. Neneng L., Gunawan Y.E., Journal of Tropical Life Science, 2018, Vol. 8, No 1, pp. 228158.
Ahmed K.B.A., Raman T., Veerappan A., Materials Science and Engineering: C, 2016, Vol. 68, pp. 939-947. 17. Parfenova I.V. avtoref. dis. kand. biolog.nauk. Voronezh, VGU, 2011, 23 p. 18. Plakunov V.K., Nikolaev Ju.A., Osnovy dinamicheskoj biohimii, M., Logos, 2010, 216p.
