Isolation of multiple dimeric forms of phosphoribulokinase from an alga and a higher plant

Abstract

Dimeric phosphoribulokinase from either spinach ( Spinacia oleracea) leaf or from the green alga, Scenedesmus obliquus can be separated into three distinct forms by hydrophobic interaction chromatography. Variation of the redox conditions prior to and during chromatography resulted in specific forms of phosphoribulokinase being eluted. It is suggested that three dimeric forms of phosphoribulokinase differ in the extent of disulfide bond form ation between Cys-16 and Cys-55 in each of the two subunits. Phosphoribulokinase-3, isolated under the most oxidising conditions and exhibiting unusual kinetics, has properties consistent with those expected of an oxidised form of the enzyme in which Cys-16 and Cys-55 are completely oxidised to forma disulfide bond in each subunit. Phosphoribulokinase-1 is the completely reduced form predominating following incubation of extracts with dithiothreitol. Phosphoribulokinase-2, the intermediate species in which only one subunit possesses the disulfide predominates only when extracts, previously reduced by high concentrations of 2-mercaptoethanol, are allowed to stand overyight in the presence of air prior to chromatography.