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.
OBJECTIVE
Succinyl-CoA synthetase catalyses the substrate level phosphorylation 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 various 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 cell, 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-T15] cells, we measured the formation of succinyl-CoA from succinate, CoA and ATP or GTP. To confirm the identity of these two enzymes, individual subunits of ATP-specific and GTP-specific to succinyl-CoA synthetase were identified by Western blot analysis.


RESULTS
Both ATP-and GTP activities of succinylCoA synthetase were observed in the mitochondrial fractions from these cells. The ratios of GTP to ATP activities of succinyl-CoA synthetase were near unity in both of the cell types studied. Using affinity-purified antisera directed specifically against individual (alpha and beta) subunits of succinyl-CoA synthetase, we also identified both ATP-specific and GTP-specific forms of succinyl-CoA synthetase in HIT and INS cell mitochondria. Furthermore, using [gamma-32P]ATP as a phosphoryl donor, we observed that the alpha subunit of succinyl-CoA synthetase undergoes autophosphorylation at a histidine residue; co-provision of exogenous succinate and CoA resulted in pronounced dephosphorylation of the phosphorylated alpha subunit of succinyl-CoA synthetase.


CONCLUSIONS
We provide evidence for the localization of two distinct activities of succinyl-CoA synthetase in the beta cell mitochondria. Whereas it is well established that ATP is critical for the beta cell mitochondrial metabolism, we propose that GTP generated by the activation of succinylCoA synthetase could promote key functional roles in the mitochondrial metabolism leading to insulin secretion.
Kowluru
A
A
Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, Wayne State University, Detroit, MI 48202, USA.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002460:Cell Line; D002999:Clone Cells / Q000201:enzymology / Q000648:ultrastructure; D003065:Coenzyme A / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000201:enzymology* / Q000648:ultrastructure; D008928:Mitochondria / Q000201:enzymology; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D013389:Succinate-CoA Ligases / Q000378:metabolism*; D019802:Succinic Acid / Q000378:metabolism
D007328:Insulin; D010710:Phosphates; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D019802:Succinic Acid; D013389:Succinate-CoA Ligases; D003065:Coenzyme A