Determination of the molecular weight of native molecules of γ-hydroxybutyrate dehydrogenase isoforms obtained from maize (Zea mays L.) seedlings by gel chromatography

Abstract

It is known that under oxygen deficiency the functioning of the tricarboxylic acid cycle is impaired, resulting in the activation of an alternative pathway, the GABA shunt. It maintains the functioning of the citric acid cycle by providing succinic acid. The key phase of this bypass is a reaction catalysed by succinate semialdehyde dehydrogenase (SSADH, EC 1.1.1.16). Under oxygen deficiency, SSADH ceases to function efficiently. This leads to the accumulation of succinic acid semialdehyde in the mitochondrial matrix and its high level adversely affects the plant cell metabolism. γ–hydroxybutyrate dehydrogenase (HBDH, EC 1.1.1.61) is an enzyme belonging to the group of oxidoreductases. It transforms γ–hydroxybutyrate into succinic acid semialdehyde, participating in the process of its detoxification, which is important in the maintenance of plant metabolism under oxygen deficiency. To date, unfortunately, there are no data on the biochemical and kinetic features of γ–hydroxybutyrate dehydrogenase. In this regard, our laboratory developed a method for purification of HBDH from green maize leaves, which makes it possible to study the physicochemical properties of this enzyme. During the study, we obtained γ-hydroxybutyrate dehydrogenase from 7-day-old seedlings of Zea mays L. by a five-stage purification. Homogenised plant material with extracted proteins was subjected to two-stage ammonium sulphate fractionation. The catalytic activity was determined spectrophotometrically at λ=340 nm by the amount of reduced NAD+. To remove ammonium salts, we used gel filtration through Sephadex G-25. Proteins were separated according to their charge by DEAE-Sephacel ion exchange chromatography. The enzyme was desorbed using a linear sodium chloride gradient (100-300 mM). The use of gel chromatography through Sephadex G-200 made it possible to determine the molecular mass of the purified enzyme isoforms. The homogeneity of enzyme preparations was confirmed by electrophoresis in polyacrylamide gel with universal AgNO3 staining. We used the tetrazolium method to confirm that the obtained protein preparations were γ-hydroxybutyrate dehydrogenase. As a result, homogeneous preparations of two isoforms of the enzyme (HBDH1 and HBDH2) were obtained. The first isoform of γ-hydroxybutyrate dehydrogenase was 185.7 times purified with a yield of 10% and had a specific activity of 343.6 U/mg of protein. The purification rate of the second isoform was 209 times with a yield of 7.74%. The specific activity of the obtained preparation was 386.7 U/mg of protein. Using gel chromatography through Sephadex G-200, we determined the molecular mass of native molecules of hydroxybutyrate dehydrogenase. We found that in 7-day-old maize seedlings the investigated enzyme was presented in low-molecular and high-molecular forms: for HBDH1 the Mr value was ⁓60.3 kDa, while for HBDH2 the molecular mass of the enzyme was 286 kDa.