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

Мұқаба

Дәйексөз келтіру

Толық мәтін

Ашық рұқсат Ашық рұқсат
Рұқсат жабық Рұқсат берілді
Рұқсат жабық Рұқсат ақылы немесе тек жазылушылар үшін

Аннотация

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.

Толық мәтін

Рұқсат жабық

Авторлар туралы

G. Anokhina

Voronezh State University

Email: bc366@bio.vsu.ru
Ресей, Voronezh, 394006

E. Plotnikova

Voronezh State University

Email: bc366@bio.vsu.ru
Ресей, Voronezh, 394006

A. Eprintrsev

Voronezh State University

Хат алмасуға жауапты Автор.
Email: bc366@bio.vsu.ru
Ресей, Voronezh, 394006

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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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