Radiocopper in L-tryptophan 2,3-dioxygenase isolated from Pseudomonas acidovorans grown in the presence of 64Cu (II).
L-Tryptophan 2,3-dioxygenase (EC 1.13.11.11), isolated from L-tryptophan-induced Pseudomonas acidovorans, ATCC 11299b, which has been grown in a medium containing 64Cu(NO3)2, has been shown to contain radiocopper. At several stages of purification of the enzyme samples were taken, and these were subjected to disc acrylamide gel electrophoresis in the presence of 10 mM L-tryptophan. After electrophoresis the position of the yellow heme band, corresponding to tryptophan oxygenase, was visually located, and the gels were sliced and counted. A large peak of radioactivity was seen to occur at the location on the gel of tryptophan oxygenase no matter what the stage of purification. Treatment of each sample before electrophoresis for 30 min at 37 degrees with gamma-globulins prepared from rabbits sensitized to homogeneous pseudomonad tryptophan oxygenase greatly reduced this peak of radioactivity, whereas treatment of each sample with rabbit preimmune gamma-globulin did not. This direct demonstration of the presence of coper in pseudomonad tryptophan oxygenase, using 64-Cu, avoided the problems and artifacts inherent in the usual techniques of copper analysis and unequivocally refutes the recent contention of Ishimura and Hayaishi ((1973) J. Biol.Chem. 248, 8610-8612) "that copper is not an essential component of L-tryptophan 2,3-dioxygenase of Pseudomonas." The presence of copper in pseudomonad and rat liver tryptophan oxygenases, previously reported by us (Brady, F. O., Monaco, M. E., Forman, H. J., Schutz, G., and Feigelson, P. (P. (1972) J. Biol. Chem. 247, 7915-7922), is reaffirmed by the experiments reported herein.
D000818:Animals; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D002850:Chromatography, Gel; D003300:Copper / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D004589:Electrophoresis, Disc; D004790:Enzyme Induction / Q000187:drug effects; D006207:Half-Life; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D008970:Molecular Weight; D011485:Protein Binding; D011549:Pseudomonas / Q000187:drug effects / Q000201:enzymology*; D011817:Rabbits / Q000276:immunology; D011868:Radioisotopes; D013997:Time Factors; D014366:Tryptophan Oxygenase / Q000096:biosynthesis* / Q000302:isolation & purification; D005719:gamma-Globulins
D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D011868:Radioisotopes; D005719:gamma-Globulins; D003300:Copper; D014366:Tryptophan Oxygenase