Physicochemical and catalytic properties of homogeneous isoforms of γ-hydroxybutyrate dehydrogenase from maize (Zea mays L.)

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Abstract

γ-Hydroxybutyrate dehydrogenase (GBDH) is an enzyme belonging to the oxidoreductase class, catalyzing the reversible conversion of succinic semialdehyde (SSA) to γ-hydroxybutyric acid (GHB). It has been established that in maize seedlings, GBDH has mitochondrial (73.7%) and cytoplasmic localization (26.3%). Two homogeneous preparations of GBDH isoforms were obtained from 7-day-old maize seedlings. The purified GBDH1 preparation had a native molecular mass of 60.3 kDa (Mr of individual subunits ~15 kDa). GBDH2, a heteromer with a molecular mass of ~286 kDa, consisted of subunits with Mr ranging from 52 to 66 kDa. The optimal pH values for the obtained enzymes differed: for GBDH1, the optimum pH for the oxidation reaction of γ-hydroxybutyrate was 9.0, while for GBDH2, the optimum pH was 7.0. The kinetics of the enzymatic reaction of GHB conversion to succinic semialdehyde follows the Michaelis-Menten equation. The Km value for GBDH1 with γ-hydroxybutyric acid was 0.31 ± 0.01 mM, and for NAD+ it was 0.47 mM ±0.02. For GBDH2, the Km value with the substrate GHB was 0.7 ± 0.03 mM, and the Km for NAD+ was 0.19 ± 0.01 mM. It was shown that CaCl2 and KCl increased the activity of GBDH1, while MgCl2 had a minor inhibitory effect. The catalytic activity of GBDH2 increased in the presence of CaCl2, KCl, and MgCl2. The study has both fundamental significance, expanding knowledge about the properties of GBDH and its role in plant cell metabolism, and applied significance — data on the mechanisms of regulation of GBDH work can be used to develop methods for increasing the productivity and resistance of plants to unfavorable environmental factors.

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About the authors

G. B. Anokhina

Voronezh State University

Email: bc366@bio.vsu.ru
Russian Federation, Voronezh, 394006

E. V. Plotnikova

Voronezh State University

Email: bc366@bio.vsu.ru
Russian Federation, Voronezh, 394006

A. T. Eprintrsev

Voronezh State University

Author for correspondence.
Email: bc366@bio.vsu.ru
Russian Federation, Voronezh, 394006

References

  1. Breitkreuz K.E., Allan W.L., Van Cauwenberghe O.R., Jakobs C., Talibi D., Andre B., Shelp B.J. //J. Biol. Chem. 2003. V. 278. № 42. P. 41552−41556. https://doi.org/10.1074/jbc.M305717200
  2. Eprintsev A.T., Anokhina G.B., Selivanova P.S., Moskvina P.P., Igamberdiev A.U. // Plants. 2024. V.13. № 18. P. 2651. https://doi.org/10.3390/plants13182651
  3. Busch K. B., Fromm H. // Plant Physiol. 1999. V. 121. № 2. P. 589−598. https://doi.org/10.1104/pp.121.2.589
  4. Weber H., Chételat A., Reymond P., Farmer E.E. // Plant J. 2004. V. 37. № 6. P. 877−888. https://doi.org/10.1111/j.1365-313X.2003.02013.x
  5. Ji J., Yue J., Xie T., Chen W., Du C., Chang E. et al. // Planta. 2018. V. 248. P. 675−690. https://doi.org/10.1007/s00425-018-2915-9
  6. Andriamampandry C., Siffert J.C., Schmitt M., Garnier J.M., Staub A., Muller C. et al. // Biochem. J. 1998. V. 334. № 1. P. 43−50. https://doi.org/doi: 10.1042/bj3340043
  7. Schaller, M., Schaffhauser, M., Sans, N. and Wermuth, B. // Eur. J. Biochem. 1999. V. 265. № 3. P. 1056−1060. https://doi.org/10.1046/j.1432-1327.1999.00826.x
  8. Hoover G.J., Prentice G.A., Merrill A.R., Shelp B.J. // Botany. 2007. V. 85. № 9. P. 896−902. https://doi.org/10.1139/B07-082
  9. Simpson J.P., Di Leo R., Dhanoa P.K., Allan W.L., Makhmoudova A., Clark S.M., et al. // J. Exp. Bot 2008. V. 59. № 9. P. 2545−2554. https://doi.org/10.1093/jxb/ern123
  10. Allan W.L., Simpson J.P., Clark S.M., Shelp B.J. // J. Exp. Bot. 2008. V. 59. № 9. P. 2555−2564. https://doi.org/10.1093/jxb/ern122
  11. Allan L.W., Peiris C., Bown A.W., Shelp B.J. // Canadian Journal of Plant Science. 2003. V. 83. № 4. P. 951−953. https://doi.org/10.4141/P03-085
  12. Fait A., Yellin A., Fromm H. // Communication in Plants: Neuronal Aspects of Plant Life. 2006. P. 171−185. https://doi.org/10.1007/978-3-540-28516-8_12
  13. Tay E., Lo W. K. W., Murnion B. // Subst. Abuse Rehabil. 2022. V. 13. P. 13−23. https://doi.org/10.2147/SAR.S315720
  14. Trainer M.A., Charles T.C. // Appl. Microbiol. Biotechnol. 2006. V. 71. № 4. P. 377−386. https://doi.org/10.1007/s00253-006-0354-1
  15. Плотникова Е.В., Анохина Г.Б., Епринцев А.Т., Вандышев Д.Ю. // Вестник Воронежского государственного университета Серия: Химия. Биология. Фармация. 2023. № 3 С. 25−30.
  16. Kaufman E.E., Nelson T. // Neurochem. Res. 1991. V. 16. P. 965−974. https://doi.org/10.1007/BF00965839
  17. Taxon E.S., Halbers L.P., Parsons S.M. // BBA-Proteins and Proteomics. 2020. V. 1868. № 5. P. 140376. https://doi.org/10.1016/j.bbapap.2020.140376
  18. Остерман Л.А. Методы исследования белков и нуклеиновых кислот: Электрофорез и ультрацентрифугирование, М.: Наука, 1981. 288 с.
  19. Pathuri I.P., Reitberger I.E., Hückelhoven R., Proels R.K. // J. Exp. Bot. 2011. V. 10. P. 3449–3457. https://doi.org/10.1093/jxb/err017
  20. Попов В.Н., Епринцев А.Т., Федорин Д.Н. // Физиология растений. 2007. Т. 54. № 3. С. 409‒415.
  21. Епринцев А.Т., Федорин Д.Н., Селиванова Н.В., Ву Т.Л., Махмуд А.С., Попов В.Н. // Физиология растений. 2012. Т. 59. № 3. С. 332–340.
  22. Jelski W., Laniewska-Dunaj M., Orywal K., Kochanowicz J., Rutkowski R., Szmitkowski M. // Neurochem Res. 2014. V. 39. P. 2313–2318.
  23. Детерман Г. Гель-хроматография. М.: Мир, 1970. C. 252.
  24. Tulchin N., Ornstein L., Davis B.J. // Anal. Biochem. 1976. V. 72. № 1−2. P. 485−490. https://doi.org/10.1016/0003-2697(76)90558-3
  25. Молекулярно-генетические и биохимические методы в современной биологии растений / Под.ред. Вл.В. Кузнецова, В.В. Кузнецова, Г.А. Романова. Эл. Издание. М.: БИНОМ. Лаборатория знаний, 2012. С. 487
  26. Маурер Г. Диск-электрофорез: Теория и практика электрофореза в полиакриламидном геле. Пер. с нем. М.: Мир, 1971. С. 248.
  27. Гааль Э., Медьеши Г., Верецкеи Л. Электрофорез в разделении биологических макромолекул. М.: Мир, 1982. C. 446.
  28. Laemmly U.K. // Nature. 1970. V. 77. № 4. P. 680−683. https://doi.org/10.1038/227680a0
  29. Shevchenko A., Wilm M., Vorm O., Mann M. // Anal. Chem. 1996. V. 68. № 5. P. 850−858. https://doi.org/10.1021/ac950914h
  30. Zarei A., Brikis C.J., Bajwa V.S., Chiu G.Z., Simpson J.P. et al. // Frontiers in Plant Science. 2017. V. 8. P. 1399. https://doi.org/10.3389/fpls.2017.01399
  31. Bouché N., Fait A., Bouchez D., Moller S.G., Fromm H. // PNAS. 2003. V. 100. № 11. P. 6843−6848. https://doi.org/10.1073/pnas.1037532100
  32. Shelp B.J., Allan W.L., Faure D. //Plant-Environment Interactions From Sensory Plant Biology to Active Plant Behavior. / Ed. F. Baluska. Springer, 2009. P. 73−84. https://doi.org/10.1007/978-3-540-89230-4_4
  33. Bravo D.T., Harris D.O., Parsons S.M. // Journal of Forensic Sciences. 2004. V. 49. № 2. P. 379−387.

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2. Fig. 1. Scheme of the transformation of succinyl semialdehyde into γ-hydroxybutyric acid.

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3. Fig. 2. Electropherograms of cytoplasmic (1) and mitochondrial (2) fractions from maize leaves. Gels were stained using the tetrazolium method. HBDGcyt — HBDG in the cytoplasmic fraction, HBDGmth — mitochondrial fraction, F — dye front.

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4. Fig. 3. Electropherograms of purified preparations of HBDG1 (a) and HBDG2 (b) from corn sprouts: 1, 3 — staining of HBDG using AgNO3, 2, 4 — specific manifestation of HBDG, protein band of HBDG1 and HBDG2, F — dye front.

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5. Fig. 4. Electropherogram of the preparations GDBG1 (a) and GDBG2 (b), obtained as a result of SDS-electrophoresis: staining using colloidal green Coomassie G-250, M — markers, P — protein band.

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6. Fig. 5. Dependence of the activity of the obtained preparation GBDG1 (1) and GBDG2 (2) from corn leaves on pH.

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7. Fig. 6. Determination of the Michaelis constant of the preparations HBDG1 (a, b) and HBDG2 (c, d) obtained from corn leaves: a, c − for γ-hydroxybutyrate, b, d − for NAD+.

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8. Fig. 7. The effect of CaCl2 (a, g), MgCl2 (b, d), KCl (c, e) on the activity of HBDG1 (a, b, c) and HBDG2 (g, d, f).

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