Purification and properties of phenylalanine ammonia-lyase in cut-injured sweet potato.
L-Phenylalanine ammonia-lyase (PAL) activity was developed in response to cut injury in sweet potato root tissue. The enzyme was purified from tissue incubated for 1 day after slicing by ammonium sulfate fractionation, column chromatographies on L-phenylalanyl Sepharose 4B, phosphocellulose. Sephadex G-200 and Sepharose 6B and preparative polyacrylamide gel electrophoresis. The molecular weight and sedimentation coefficient were estimated to be 285,000 to 320,000 and 11.6 to 11.9 S, respectively. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gel yielded a single stained protein band which corresponded to a subunit weight of 80,000. Thus, the enzyme seems to be composed of four subunits of the same size. Neither L-tyrosine nor D-phenylalanine served as a substrate. Two Km values for the PAL were observed above and below 30 micrometers at various temperatures and were lower than those for PALs of other plants. The slope of the Arrhenius plot had a discontinuity at 17 degrees C. The values of activation energy were calculated to be 15,000 cal and 19,000 cal above and below 17 degrees C, respectively. Similar discontinuities were also observed in the effect of temperature on the Km values and the Hill coefficients. Negative cooperativity was observed at 10 degrees C (n = 0.83), but was not marked above 20 degrees C (n = 0.94).
D000642:Ammonia-Lyases / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002934:Cinnamates / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010649:Phenylalanine / Q000494:pharmacology; D010650:Phenylalanine Ammonia-Lyase / Q000302:isolation & purification / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D014443:Tyrosine / Q000494:pharmacology
D002413:Cations, Divalent; D002934:Cinnamates; D013439:Sulfhydryl Reagents; D014443:Tyrosine; D010649:Phenylalanine; D004492:Edetic Acid; D000642:Ammonia-Lyases; D010650:Phenylalanine Ammonia-Lyase