Solubilization and purification of a cytoplasmic membrane bound enzyme catalyzing tetrathionate and thiosulphate reduction in Proteus mirabilis.

Abstract

Solubilization of cytoplasmic membrane bound tetrathionate and thiosulphate reductase was accomplished without detergents by repeated extraction of a purified cytoplasmic membrane suspension with tert-amyl alcohol. On homokinetic sucrose gradients both solubilized reductase activities banded in single peaks in the same fractions. A sedimentation constant of 6.9 Svedbergs was calculated using catalase as indicator enzyme. Further purification was obtained on an electro focusing column. Again both reductase activities banded as a single peak in the same fractions. It can be concluded that both have an isoelectric point of 5.22. It was demonstrated on polyacrylamide gels that the peak fractions contained virtually one protein component, which catalyzes tetrathionate reduction as well as thiosulphate reduction. It was concluded that Proteus mirabilis has only one enzyme for both enzymatic functions. From polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulphate it appeared that this enzyme consists of two subunits with molecular weights of approx. 43 000 and 90 000 daltons. The complete enzyme has thus a molecular weight of approx. 133 000 daltons. Protein profiles of cytoplasmic membranes isolated after various growth conditions suggest that the smaller subunit of this enzyme is present after anaerobic growth in the presence of KNO3 in spite of repression of the complete enzyme under this growth condition.