Purification and properties of ornithine decarboxylase from Physarum polycephalum.
Ornithine decarboxylase (EC 4.1.1.17) has been purified 3,500-fold from the plasmodia of Physarum polycephalum. The purified material exhibited a Km for ornithine of 0.6 mM and Vmax of 20 mumol of CO2 formed per min/mg at 30 degrees C (62 mumol/min/mg at 37 degrees C). It migrated as a single protein and activity species on high pressure liquid chromatography (TSK-3000) in 0.15 M NaCl (Mr = 80,000) and in native gels containing 5, 6.5, 8, and 9.5% acrylamide. A single protein band (Mr = 43,000) was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Activity was lost upon incubation with alpha-difluoromethyl[5-14C] ornithine, and the inactivated material appeared as a single Mr = 43,000 14C-band on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels. The decarboxylase activity was specific for ornithine and was pyridoxal-P-dependent. The Km for pyridoxal-P (10 microM) was identical with the Kd for pyridoxal-P binding determined from the quenching of protein fluorescence (lambda ex = 282 nm, lambda em = 350 nm, maximal quenching 81%). Using specific antibody obtained from rabbit hyperimmune serum as a probe, an Mr = 43,000 immunoreactive species was detected on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels of plasmodial homogenates and all pooled purification fractions, but no higher molecular weight cross-reactive material was detected.
D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D009952:Ornithine / Q000378:metabolism; D009955:Ornithine Decarboxylase / Q000302:isolation & purification* / Q000378:metabolism; D010804:Physarum / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000378:metabolism
D011732:Pyridoxal Phosphate; D009952:Ornithine; D009955:Ornithine Decarboxylase