RESUMO
Enzymatic preparations of two isoforms of succinate dehydrogenase (SDG) with specific activity of 22.00 E/mg of protein were obtained from the colorless sulfur bacterium Sphaerotilus natans D-507 cultured organotrophically. Both SDG forms were shown to be heteromers with subunit molecular masses of 70.8, 35.0, 31.8, and 16.2 kDa. The K(m) values for the first and the second forms of SDG were evaluated as 0.615 and 0.531 mM, respectively, with an optimal pH value of 7.2. It was found that the Cl- ion has an activating effect on the SDG activity that can be explained by the specific chemical modification of the enzyme molecule. The results suggest that the isolated enzyme forms are included in different multienzyme complexes, which provide the functioning of the tricarboxylic acid cycle, and SDG preparations can be used for the investigation of other enzyme systems or in vitro modeling of supramolecular cellular structures.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Subunidades Proteicas/isolamento & purificação , Sphaerotilus/enzimologia , Succinato Desidrogenase/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cloretos/química , Cloretos/metabolismo , Cromatografia em Gel , Ciclo do Ácido Cítrico/fisiologia , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Sphaerotilus/química , Succinato Desidrogenase/química , Succinato Desidrogenase/metabolismoRESUMO
Electrophoretically homogenous preparations of malate dehydrogenase (MDH) isoforms of the bacteria Sphaerotilus natans D-507 with specific activity 7.46 U/mg and 5.74 U/mg with respect to protein concentration have been obtained. The dimeric isoform of the enzyme was shown to function under organotrophic growth conditions, whereas the tetrameric isoform was induced under mixotrophic cultivation conditions. PCR-analysis revealed a single gene encoding the malate dehydrogenase molecule. The topography of the MDH isoform surface was studied by atomic-force microscopy, and a 3D-structure of the enzyme was obtained. Spectraphotometric analysis data allowed us to suggest that stabilization of the tetrameric form of MDH is due to additional bounds implicated in the quaternary structure formation.
Assuntos
Malato Desidrogenase/química , Isoformas de Proteínas/química , Sphaerotilus/enzimologia , Sphaerotilus/crescimento & desenvolvimento , Técnicas de Cultura de Células , Malato Desidrogenase/isolamento & purificação , Isoformas de Proteínas/isolamento & purificação , Estrutura Quaternária de ProteínaRESUMO
High-purity preparations of malate dehydrogenase (EC 1.1.1.37) were obtained by multistage purification from the bacterium Sphaerotilus sp. strain D-507 growing under different conditions. Under organotrophic conditions, the enzyme was dimeric; under mixotrophic conditions, dimeric and trimeric. On the basis of studied properties of the enzyme preparations, data on the activity of enzymes of the glyoxylate and tricarboxylic-acid cycles, and analysis of published data, it can be concluded that malate dehydrogenase isoforms are implicated in the adaptive response of bacteria to changing culturing conditions.
