Purification and Properties of Nitrate Reductase from Escherichia coli K12

Abstract

Abstract The membrane-bound enzyme nitrate reductase from Escherichia coli has been solubilized and purified 112-fold. The purified enzyme appeared homogeneous by polyacrylamide gel electrophoresis and in the analytical ultracentrifuge. The molecular weight of the enzyme, as measured on an agarose column, was 720,000 and, as measured in the ultracentrifuge, was 773,600. The s0 value was determined by sedimentation analysis to be 23.0 S. Electron micrographs showed the enzyme to be spherical which was in agreement with the intrinsic viscosity as measured in the magnetic visco-densimeter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the enzyme is composed of two different subunits with molecular weights of 142,000 and 58,000 and that these are present in roughly equal amounts. As determined by activation analysis, the enzyme contained 3.2 ± 0.5 moles of molybdenum per mole of enzyme. These data are consistent with a structure composed of four large polypeptides, four small polypeptides, and four molecules of molybdenum per molecule of enzyme.