ISOLATION OF MULTIPLE DIMERIC FORMS OF PHOSPHORIBULOKINASE FROM AN ALGA AND A HIGHER-PLANT

Dimeric phosphoribulokinase from either spinach (Spinacia oleracea) le af or from the green alga, Scenedesmus obliquus can be separated into three distinct forms by hydrophobic interaction chromatography. Variat ion of the redox conditions prior to and during chromatography resulte d in specific forms of phosphoribulokinase being eluted. It is suggest ed that three dimeric forms of phosphoribulokinase differ in the exten t of disulfide bond formation between Cys-16 and Cys-55 in each of the two subunits. Phosphoribulokinase-3, isolated under the most oxidisin g conditions and exhibiting unusual kinetics, has properties consisten t with those expected of an oxidised form of the enzyme in which Cys-1 6 and Cys-55 are completely oxidised to form a disulfide bond in each subunit. Phosphoribulokinase-1 is the completely reduced form predomin ating following incubation of extracts with dithiothreitol. Phosphorib ulokinase-2, the intermediate species in which only one subunit posses ses the disulfide, predominates only when extracts, previously reduced by high concentrations of 2-mercaptoethanol, are allowed to stand ove rnight in the presence of air prior to chromatography.