Amino acid activation in mammalian brain. Purification and characterization of tryptophan-activating enzyme from buffalo brain.
l-Tryptophan-activating enzyme [l-tryptophan-tRNA ligase (AMP), EC 6.1.1.2] of water-buffalo brain was purified to near homogeneity by heat and pH treatments, ammonium sulphate fractionation, column chromatography on DEAE-cellulose, hydroxyapatite and Amberlite CG-50, and gel filtration on Sephadex G-200. The purified enzyme catalyses tryptophanyl-tRNA formation with yeast tRNA, but not with Escherichia coli tRNA. The enzyme exhibits multiple peaks of activity in Sephadex gel filtration with molecular weights corresponding to 155000, 105000 and 50000. However, only one peak of activity with molecular weight of 155000 can be detected when the enzyme is subjected to gel filtration at high concentration. Disc gel electrophoresis in the presence of sodium dodecyl sulphate reveals a single band with molecular weight of 55000. The activity of the enzyme is concentration dependent. Different K(m) and V(max.) values are obtained at different enzyme concentrations. These data suggest that this enzyme may exist in different quaternary structures, each with its own kinetic constants. The enzyme activity is inhibited by p-chloromercuribenzoate, and is not protected by the presence of the substrates, l-tryptophan, Mg(2+), ATP, in any combination.
D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000378:metabolism*; D002020:Buffaloes; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004589:Electrophoresis, Disc; D004926:Escherichia coli; D007700:Kinetics; D008274:Magnesium; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D012343:RNA, Transfer; D046249:Transfer RNA Aminoacylation; D014364:Tryptophan
D002729:Chloromercuribenzoates; D010761:Phosphorus Radioisotopes; D014364:Tryptophan; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D008274:Magnesium