A simple, single-tube radioisotopic assay for the phosphorylation/inactivation activity of the pyruvate,orthophosphate dikinase regulatory protein

Abstract

A simple, single-tube radiolsotopic method has been developed to assay the relative phosphorylation (inaetivation) activity of the bifunctional regulatory protein (RP) of C4-leaf pyruvate,orthophosphate dikinase (PPDK) in desalted leaf homogenates and partially purified preparations. RP catalyzes the inactivation of maize PPDK by phosphorylation of Thr-456, utilizing [β-P]ADP as the specific phosphoryl donor. Existing spectrophotometric and radioisotopic assays for the detection of RP activity are either relatively insensitive or labor-intensive and timeconsuming. We describe a modified radioisotopic assay that couples the synthesis of [β-(32)P]ADP by exogenous adenylate kinase with the subsequent RP-catalyzed [β-(32)P]ADP-dependent phosphorylation of exogenous maize PPDK. The incorporation of [β-(32)P] is dependent on the initial concentrations of ATP and PPDK, as well as the presence of active RP. Desalted leaf homogenates of C3 species fail to catalyze (32)P incorporation into exogenous maize PPDK. Conversely, heterologous systems containing the maize target enzyme and leaf homogenats of other C4 species result in PPDK-specific (32)P-incorporation. This simple radioisotopic assay is at least 40-times more sensitive than the routine spectrophotometric assay, and qualitatively exhibits comparable sensitivity and requires significantly less time than the currently available radioisotopic RP assay. The present assay reliably generates [β-(32)P]ADP and as such may be useful for studies of other systems requiring β-labeled ADP, which is not commercially available.