[18] Tryptophanyl-tRNA synthetase from beef pancreas

Affinity labelling of tryptophanyl‐tRNA synthetase with mesitoyl‐AMP

“…The capabilities of the enzyme to catalyse sponds to the saturation of the enzyme with the inhibitor and indicates (see [ 131 and references therein) that intermediate reversible enzyme . inhibitor complex is formed prior to the chemical reaction according to the scheme: At the same time, mesitoyl-AMP is characterized with a relatively high affinity, for an ATP derivative, for the enzyme: Ki is 0.34 mM (table 1) as compared with a IL',, of 2 mM for ATP and with a k'i of 3 mM for adenosine (see [9]). The value of Ki for mesitoyl-AMP as well as the protective action of both ATP and tryptophan against inactivation of tryptophanyltRNA synthetase (see fig.3A) allow one to suggest that mesitoyl-AMP being bound to the active centre of the enzyme behaves as an analog of tryptophanyl adenylate.…”

“…Electrophoretically homogeneous tryptophanyltRNA synthetase was isolated from beef pancreas as in [9]. Prior to use, the preparation of the enzyme was passed through a charcoal filter [ 121 (Ederol) or was precipitated at pH 5 .O and redissolved.…”

“…The preparations of mesitoyl-AMP were synthesized as described [ 111. The enzyme (1.6 PM) was incubated with mesitoyl-AMP (1 mM, unless specified) at 37°C in 0.05 M cacodylate buffer (pH 7.5). The activity of the enzyme was assayed in ATP-p2P]pyrophosphate exchange and tRNATrP aminoacylation reactions as in [9].…”

“…In this work, affinity labelling of beef pancreas tryptophanyl-tRNA synthetase (EC 6.1 .I .2) (M, 108 000-120 000, ff2 type (see [9]) was accomplished using a mixed anhydride of AMP with mesitylenecarboxylic acid [ lo]:…”

P1, P3‐bis(5′‐adenosyl)triphosphate (Ap3A) as a substrate and a product of mammalian tryptophanyl‐tRNA synthetase

“…TrpRS was purified from the bovine pancreas as described earlier [9,10]. Two types of enzyme preparations were used: (i) the preparations containing one Zn 2+ atom per dimeric molecule isolated in the absence of EDTA [9] and (ii) Zn:+-deprived preparations isolated either after addition of EDTA or after prolonged storage [10].…”