Binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase from bovine pancreas under pH conditions of maximum activity. Analysis by ultracentrifugation, fluorescence quenching and chemical modification.
The binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase (EC 6.1.1.2) from beef is examined by three approaches, under pH conditions of maximum activity (pH 8.0). (1) Analytical ultracentrifugation evidences the binding of a single mol of tRNATrp in a 2.5-10 microM concentration range. (2) tRNATrp quenches the fluorescence of the enzyme. The dependence of this fluorescence quenching on the tRNATrp concentration (0.1-4 microM) reflects also the binding of 1 mol of tRNA per mol of enzyme, with a Kd value of 0.19 +/- 0.02 microM. (3) tRNATrp protects the enzyme against derivatization by oxidized ATP. Out of the two fast-reacting lysine residues of the native enzyme, only one is prevented from reacting by tRNATrp in the 0.5-110 microM concentration range. This protection can be significantly analyzed only by assuming a one-to-one complex between the enzyme and tRNA. These results, obtained at pH 8.0 and 25 degrees C, are in contrast with the stoichiometry of 2 mol of tRNA to 1 mol of enzyme, previously observed at pH 6.0 and 4 degrees C.
Institut de Biochimie Cellulaire et Neurochimie, CNRS Bordeaux, France.
M
M
Fournier
C
C
Plantard
B
B
Labouesse
J
J
Labouesse
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000032:analysis; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D012364:RNA, Transfer, Trp / Q000378:metabolism*; D014369:Tryptophan-tRNA Ligase / Q000378:metabolism*; D014461:Ultracentrifugation
D012345:RNA, Transfer, Amino Acid-Specific; D012364:RNA, Transfer, Trp; D000604:Amino Acyl-tRNA Synthetases; D014369:Tryptophan-tRNA Ligase