Acta Biochimica et Biophysica 13. (1978)
1978 / 4. szám - Alkonyi, I.-Gyócsi, L.-Sümegi, B.: Demonstration of a Lag Period in the Time-Course of the Reaction Catalyzed by Pyruvate Dehydrogenase Complex
AIkonyi et al.: Lag Period in the Pyruvate Dehydrogenase Action 255 for it had taken place by the end of the preincubation. However, preincubation of the enzyme complex in the assay mixture containing only one of the two substrates of Ej (pyruvate, TPP) did not abolish the lag period (Fig. 3, curves 2 and 3). Since both pyruvate and TPP bind to the Els the reactions producing the lag period take place on the Er Fig. 2. Effect of enzyme concentration on the lag period. The enzyme complex was preincubated for 5 minutes in the assay mixture containing 50 mM potassium phosphate buffer, pH 8.0, 2 mM MgCl2, 1 mM NAD+, 0.25 mM CoASH, 1 mM 2-mercaptoethanol and 0.12 /tM TPP in a total volume of 1.0 ml. The reaction was initiated by addition of 2 /«moles of pyruvate Time (min) Fig. 1. Time-course of the reaction catalyzed by PDC. The assay mixture contained 50 mM potassium phosphate buffer, pH 8.0, 2 mM MgCl2, 1 mM NAD+, 2 mM pyruvate, 0.25 mM CoASH, 1 mM 2-mercaptoethanol and 0.5 /iM TPP in a total volume of 1.0 ml. The reaction was initiated by addition of 15 mU of enzyme complex, r was obtained from the intercept of the linear part of the kinetic curve with the time axis [E] 'ml' 10 50 100 150 Acta Biochimica et Biophysica Academiae Scientiarum Hungaricae 13, 1978