Fig. 6.

Inhibition of PDHC activity by hydroxyl radical and peroxynitrite. Results obtained with exposure of purified porcine heart mitochondria for 10 min at 37°C to a Fenton reagent consisting of 0.25 mM FeSO₄ plus 0.5 mM H₂O₂ in the absence and presence of 2.5 mM diethylenetriaminepentacetic acid (DTPA). Reprinted from Free Radical Biology and Medicine, Vol 16, Bogaert et al., Postischemic inhibition of cerebral cortex pyruvate dehydrogenase, p 811–820, ^©1994 with permission from Elsevier. Additional results were obtained under similar conditions but using 0.6 mM of the peroxynitrite generator 3-morpholinosydnonomine (SIN-1) in the absence and presence of superoxide dismutase (SOD; 20 U/ml). Enzyme activity after the preincubations was measured either radioisotopically by determining the ¹⁴CO₂ production from [1-¹⁴C] pyruvate (Fenton reagent; Bogaert et al., 1994), or spectrophotometrically by measuring NADH production at 340 nm (SIN-1). Dithiothreitol (9 mM) was present during all preincubations to eliminate possible inactivation due to thiol oxidations or S-nitrosylation. Experimental conditions for experiments involving SIN-1 were modified from Hinman and Blass (1981) and consisted of 50 mM potassium phosphate buffer (37°C), 2.06 U/ml PDHC, 5 mM pyruvate, 0.3 mM thiamine pyrophosphate (TPP), 1 mM magnesium chloride, 0.01 mM calcium chloride, and 1 mM NAD⁺. The reaction was initiated by the addition of 0.12 mM coenzyme A whereas the reactions after exposure to the Fenton reagent were initiated with the addition of 2 mM pyruvate.