Phosphorylation of rubisco in Cicer rietinum: Non-phosphoprotein nature of rubisco in Nicotiana tabacun

Abstract

Leaf discs from Cicer and Nicotiana were incubated in a solution of [ 32P]-orthophosphate (24 hr). RUBISCO was purified from these and electrophoresed on native PAGE. A single protein stained band was observed on gels, thus proving the electrophoretic homogeneity of RUBISCO both in Cicer and Nicotiana. Autoradiography of gels revealed a radioactive band in the zone of protein stained band of Cicer RUBISCO, whereas no radioactivity was detected in the region of the protein stained band of Nicotiana RUBISCO. This suggested that Cicer RUBISCO unlike that of Nicotiana is a phosphoprotein. Further analysis of [ 32P]-labelled purified Cicer RUBISCO on SDS—PAGE showed two protein stained bands corresponding to the large and small subunits of the enzyme. Autoradiography of the denaturing gel showed a radioactive band exclusively in the zone of the small subunit. Chemical characterization of [ 32P]-labelled Cicer RUBISCO by acid hydrolysis showed radioactivity in phosphoserine and phosphotyrosine. Treatment of [ 32P]-labelled Cicer RUBISCO with purified alkaline phosphatase brought about a significant dephosphorylation (∼70%) of the enzyme. The in vitro dephosphorylation of Cicer RUBISCO resulted in the dissociation of the small subunits from the catalytic octameric large subunits. Concomitantly, there was a marked decrease in the catalytic activity of the dephosphorylated Cicer RUBISCO in comparison with the phosphorylated form of RUBISCO. Phosphorylation of the small subunit seems crucial for the assembly of large and small subunits and thus contributes to the regulation of Cicer RUBISCO. By contrast, Nicotiana RUBISCO showed no loss of catalytic activity following its treatment with alkaline phosphatase.