FIG. 7.

Effect of CoA and organic acids on the synthesis of p₄A catalyzed by acyl-CoA synthetase. (A) Effect of CoA on the synthesis of p₄A. The reaction mixture (50 μl) contained 50 mM MES-KOH (pH 6.3), 0.1 mM dithiothreitol, 11 mM MgCl₂, 1 mM [2,8-³H]ATP, 10 mM P₃, and the indicated concentrations of CoA and acyl-CoA synthetase (8.3 μg of protein). (B) Effect of organic acids on the inhibitory effect of CoA on the synthesis of p₄A. Reaction mixtures (28 μl) containing 72 mM MES-KOH (pH 6.3), 0.14 mM dithiothreitol, 7.9 mM MgCl₂, 0.76 mM [α-³²P]ATP, 7.2 mM P₃, 0.14 mM CoA, 0.7 μl of desalted inorganic pyrophosphatase, and acyl-CoA synthetase (5.2 μg of protein) were preincubated at 30°C for 20 min. Thereafter, they were supplemented with 12 μl of the following solutions: water (lane b), 1% Triton X-100–5% ethanol (solution C; lane c), 1 mM solutions of palmitic acid in solution C (lane d), octanoic acid in water (lane e), or other possible effectors (acetic acid, lysine, methionine, phenylalanine, tryptophan, and luciferin; lanes f to k, respectively). One hour after the addition of organic acids, aliquots of the reaction were analyzed by TLC. Control without acyl-CoA synthetase is shown in lane a.