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

254 Alkonyt et al.: Lag Period in the Pyruvate Dehydrogenase Action Materials and methods NAD + and CoASH were purchased from C. F. Boehringer (Mannheim, Germany). Thiamine pyrophosphate was supplied by Serva (Heidelberg, GFR). All other chemicals of the highest purity available were obtained from Reanal (Budapest). Highly purified pyruvate dehydrogenase complex was prepared from pig heart mitochondria as described by Cooper et al. (1974). However, the enzyme complex preparations obtained contained variable amounts of TPP: enzyme activities between 10 and 30% of the maximum velocity were observed in the absence of added TPP. Therefore the TPP was removed from the enzyme complex as described by Shepherd and Hammes (1976). After this treatment the enzyme complex completely lost its activity without the addition of TPP to the assay mixture. The time course of the overall reaction of PDC was followed by recording the formation of NADH at 340 nm with a Specord recording spectrophotometer at 25°C. The standard assay mixture contained, in a total volume of 1.0 ml, 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 amounts of TPP and enzyme complex specified in the figures. Results and discussion The overall reaction of the pig heart PDC was studied at low TPP concen­trations. Investigating the curve of the time-dependent product formation we observed that the steady-state velocity developed after a lag period (Fig. 1). г was employed for the quantitative characterization of this phenomenon. In order to obtain some information about the nature of the lag period it was necessary to investigate it as a function of the enzyme concentration. Figure 2 shows that an increase in enzyme concentration to 25 mU/ml caused a concomitant shortening of t, while above this value the duration of the lag period was inde­pendent of the enzyme concentration. Thus at lower concentrations of the enzyme complex both isomerization and aggregation-dissociation reactions may play an important role in the devel­opment of the lag period. Since at higher enzyme concentrations these aggregation­­dissociation reactions become fast, the isomerization processes are presumably the rate-limiting steps. The fact that the lag period did not disappear at infinite enzyme concentration (Fig. 2) seems to support this hypothesis. The slope of the curve (Fig. 3) shows that a lag period appeared when the enzyme reaction was started by either enzyme, pyruvate or TPP. However, the lag period disappeared when the enzyme reaction was started by CoASH or NAD + . That is, the preincubation of the enzyme complex in the assay mixture containing both pyruvate and TPP abolished the lag period because the processes responsible Acta Biochimica et Biophysica Academiae Scientiarum Hungaricae 13, 1978

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