Acta Biochimica et Biophysica 13. (1978)

1978 / 3. szám - Alkonyi, I.-Bolygó, E.-Gyócsi, L.-Sümegi, B.: Kinetic Studies of the Inhibitory Effect of Acetaldehyde and D(-)Acetion on the Pyruvate Dehydrogenase Complex from Pig Heart

144 Alkonyi et al. : Inhibition of Pyruvate Dehydrogenase Complex Materials and methods NAD+ and CoA were purchased from Boehringer (Mannheim). Thiamine pyrophosphate was supplied by Serva (Heidelberg). Potassium pyruvate and acetaldehyde were obtained from Merck (Darmstadt). Acetaldehyde was redis­tilled before use. D(-)acetoin was synthesized according to Tankó et al. (1940). The optical rotation of the acetoin obtained was aD20 = —108.4°. All other chemicals were of analytical grade available commercially. Highly purified preparations of the pyruvate dehydrogenase complex were prepared from pig heart mitochondria as described by Cooper et al. (1974). The specific activity was in the range of 6 — 8 units/mg. Pyruvate dehydrogenase activity was tested by a modification of the method described by Schwartz et al. (1968). In a volume of 1.0 ml the reaction medium contained 80 /(moles of tricine buffer, 4.0 /(moles of MgCl2, 0.12 /(mole of thia­mine pyrophosphate, 25 /(moles of potassium pyruvate and 0.6 /(mole of potassium ferricyanide. The pH of the reaction mixture was adjusted to 7.5. After tempera­ture equilibration the reaction was started by the addition of 500 /(g of the enzyme complex. Reduction of ferricyanide was monitored at 420 nm with a Specord UY VIS spectrophotometer at 25 °C. The initial rate of the overall reaction (1) was determined by monitoring the formation of NADH at 340 nm and 25 °C (Linn et ah, 1972). The standard assay mixture contained, in a total volume of 1.0 ml, 50 /(moles of potassium phosphate buffer, pH 8.0, 0.2 /(mole of thiamine pyrophosphate, 1.0 gmole of MgCl2 and amounts of pyruvate, CoA and NAD+ as specified in the figures. After temperature equilibration the reaction was started by the addition of 10 jug of the enzyme complex. When inhibition by acetaldehyde was studied the inhibi­tor was added to the reaction mixture just before the starting of the reaction, and the cuvette was covered with a lid to prevent the acetaldehyde from evaporating. Initial reaction velocities were expressed as absorbancy differences at 340 nm (dA340) per min. In the assay of lipoate acetyltransferase activity the formation of thioester was measured as an increase in absorbancy at 240 nm on the basis of principles adopted earlier (Alkonyi et ah, 1976). Lipoamide dehydrogenase activity was measured as described by Sakurai et ah (1970). The kinetic nomenclature used in the equations is that of Cleland (1963). A, В and C are substrate concentrations (pyruvate, CoA and NAD+, respectively) I4 and I2 are inhibitor concentrations (acetaldehyde and D(-)acetoin, respectively) KA, KB and Kc are Michaelis constants, Kis is a graphical slope inhibition con­stant, K;i is a graphical intercept inhibition constant, v and V indicate initial and maximum velocities, respectively. Acta Biochimica et Biophysica Academiae Scientiarum Hungaricae 13, 1978

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