title: Studies on the dark/light regulation of maize leaf pyruvate, orthophosphate dikinase by reversible phosphorylation.
abstract: In maize leaves, pyruvate, orthophosphate dikinase (PPDK) is deactivated in the dark and reactivated in the light. Studies in vitro using purified PPDK and a partially purified regulatory protein from maize confirmed previous reports correlating deactivation/reactivation with the reversible phosphorylation/dephosphorylation of a threonyl residue. By monitoring the stability of the exogenous 32P-labeled adenylate substrates during deactivation, we have firmly established ADP as the specific phosphate donor. In isolated maize leaf mesophyll protoplasts preilluminated with 32Pi, we observed a three- to fivefold higher PPDK activity in situ in the light, and a corresponding three- to fivefold higher level of phosphorylation of the 94-kDa PPDK protomer in the dark. HPLC-based phosphoamino acid analysis of PPDK purified from maize leaves of both light- and dark-adapted plants revealed the presence of P-serine. The inactive enzyme from dark-adapted plants (inactivated in vivo) also contained P-threonine. Total phosphate content of PPDK purified from leaves of light-adapted plants was approximately 0.5 mol/mol protomer, and 1.5 mol/mol protomer from leaves of dark-adapted plants. Since the difference between enzyme purified from light-adapted (active PPDK) and dark-adapted (inactive PPDK) plants is the presence of P-threonine in the latter, this suggests an inactivation stoichiometry in vivo of 1 mol P-threonine/mol 94-kDa protomer. These complementary studies with maize leaf PPDK in vitro, in situ, and in vivo provide convincing evidence for the dark/light regulation of this key C4-photosynthesis enzyme by reversible phosphorylation.
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003624:Darkness; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008027:Light; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D011772:Pyruvate, Orthophosphate Dikinase / Q000378:metabolism*; D003313:Zea mays / Q000201:enzymology
chemical_list: D000244:Adenosine Diphosphate; D010770:Phosphotransferases; D011772:Pyruvate, Orthophosphate Dikinase