Adenine and guanine nucleotide-specific succinyl-CoA synthetases in the clonal beta-cell mitochondria: implications in the beta-cell high-energy phosphate metabolism in relation to physiological insulin secretion

Aims/hypothesis. Succinyl-CoA synthetase catalyses the substrate level phos phorylation of ADP or GDP. It also supplies succinyl-CoA for heme synthesis . Recently, two distinct mitochondrial succinyl-CoA synthetase activities, one specific for ATP and the other for GTP, have been characterized in vari ous tissues of pigeon. Because of the relative importance of mitochondrial high-energy phosphate metabolism in physiological insulin secretion and the few data available on mitochondrial succinyl-Coa synthetase in the beta ce ll, this study examined whether ATP-specific and GTP-specific succinyl-CoA synthetase activities are localized in the clonal beta-cell mitochondria. Methods. Using the mitochondrial extracts from clonal beta [INS-1 and HIT-T 15] cells, we measured the formation of succinyl-CoA from succinate, CoA an d ATP or GTP. To confirm the identity of these two enzymes, individual subu nits of ATP-specific and GTP-specific to succinyl-CoA synthetase were ident ified by Western blot analysis. Results. Both ATP-and GTP activities of succinyl-CoA synthetase were observ ed in the mitochondrial fractions from these cells. The ratios of GTP to AT P activities of succinyl-CoA synthetase were near unity in both of the cell types studied. Using affinity-purified antisera directed specifically agai nst individual (alpha and beta) subunits of succinyl-CoA synthetase, we als o identified both ATP-specific and GTP-specific forms of succinyl-CoA synth etase in HIT and INS cell mitochondria. Furthermore, using [gamma-P-32]ATP as a phosphoryl donor, we observed that the alpha subunit of succinyl-CoA s ynthetase undergoes autophosphorylation at a histidine residue; co-provisio n of exogenous succinate and CoA resulted in pronounced dephosphorylation o f the phosphorylated alpha subunit of succinyl-CoA synthetase. Conclusion/interpretation. We provide evidence for the localization of two distinct activities of succinyl-CoA synthetase in the beta cell mitochondri a. Whereas it is well established that ATP is critical for the beta cell mi tochondrial metabolism, we propose that GTP generated by the activation of succinyl-CoA synthetase could promote key functional roles in the mitochond rial metabolism leading to insulin secretion.