Immunochemical Identification of Coenzyme Q0-Dihydrolipoamide Adducts in the E2 Components of the α-Ketoglutarate and Pyruvate Dehydrogenase Complexes Partially Explains the Cellular Toxicity of Coenzyme Q0

Abstract

Coenzyme Q 0 (Q 0 ), a strong electrophile, is toxic to insulin-producing cells. Q 0 was incubated with rat and human pancreatic islets and INS-1 insulinoma cells, and its attachment to cellular proteins was studied with Western analysis using antiserum raised against the benzoquinone ring structure of ubiquinone (anti-Q). Q 0 covalently bonded to two proteins, one of 50 kDa and another of 70 kDa. Both proteins were found to be mitochondrial in human and rat islet cells and in many rat organs. Mitochondria were incubated with Q 0 , and affinity-purified anti-Q was used to immunoprecipitate the 50-kDa protein. Amino acid sequencing identified it as dihydrolipoamide succinyltransferase, the E2 component of the ␣-ketoglutarate dehydrogenase complex (KDC). Western analysis also showed that Q 0 bonds to the E2 components of the purified KDC and the pyruvate dehydrogenase complex (PDC). Dihydrolipoamide acetyltransferase, the E2 of the PDC, has a molecular mass of 70 kDa, and the 70-kDa protein was inferred to be this enzyme. Q 0 was found to bond only to proteins containing dihydrolipoate, and in preparations of mitochondria, thiol reducing agents facilitated the attachment of Q 0 , but oxidizing agents prevented it, suggesting that Q 0 bonds to thiols of dihydrolipoamide. Incubation of human or pig PDC with Q 0 followed by matrix-assisted laser desorption ionization time-of-flight and liquid chromatography/electrospray ionization mass spectrometry analyses of chymotrypsin-digested peptides of PDC E2 confirmed that Q 0 bonds to the dihydrolipoamide in these proteins. In mitochondria, coenzymes Q 1 and Q 2 did not bond to the 50-kDa protein but competed with the bonding of Q 0 to this protein. The prevention by Q 1 of the bonding of Q 0 to KDC E2, as well as other characteristics of the Q 0 effect, are reminiscent of the action of Q 0 on the mitochondrial permeability transition pore described previously (Fontaine, E., Ichas, F., and Bernardi, P. (1998) J. Biol. Chem. 273, 25734 -25740).Ubiquinone analogs (1, 2) and other quinones (3-6) stimulate insulin release from pancreatic islets, but they also kill the insulin-producing cell. The insulin release is not simply due to insulin leaked from dying cells because numerous inhibitors of cellular respiration that kill the cell, such as rotenone, antimycin A, cyanide, and dinitrophenol, inhibit insulin release rather than stimulate it (7, 8). The injurious effects of these compounds may be due, in part, to redox cycling that collapses the proton gradient that maintains the electrical potential of the inner mitochondrial membrane. However, many quinones, because of their electrophilicity, can induce various patterns of oxidative damage to cells. Also, some ubiquinone analogs influence the mitochondrial permeability transition pore (PTP) 1 (9 -11). To learn more about the roles of quinones in these processes, we raised antisera to the ring structure of ubiquinone (anti-Q antibodies) and used them in Western analysis and for immunoprecipitation. This enabled us to identify se...

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