Endogenous protein phosphorylation in rat brain mitochondria: occurrence of a novel ATP-dependent form of the autophosphorylated enzyme succinyl-CoA synthetase.
When rat brain mitochondria are incubated with [gamma-32P]ATP, there is a rapid (10 s) phosphorylation of proteins designated E1 and F of M.W. 42,000 and 32,000, respectively. Although [gamma-32P]ATP was the preferred substrate for protein F, a small amount of labeling did occur with [gamma-32P]GTP. Phosphorylation of E1 was absolutely ATP-dependent. On the other hand, a 32,000 M.W. protein from rat liver mitoplasts (mitochondria devoid of an outer membrane) was highly phosphorylated when [gamma-32P]GTP was used but not at all phosphorylated within short time periods with [gamma-32P]ATP. Both the ATP-labeled brain phosphoprotein F and GTP-labeled liver protein migrated to identical positions on high-resolution two-dimensional polyacrylamide gels, and both contained acid-labile phosphoryl groups. Furthermore, both phosphoproteins were identified as the autophosphorylated subunit of succinyl-CoA synthetase (SCS, EC 6.2.1.4) by using antibody directed against purified GTP-dependent porcine SCS. However, immunotitration experiments with anti-porcine SCS revealed that ATP- and GTP-labeled protein F in brain differed in their interactions with antibody, suggesting that in rat brain mitochondria two different forms of the enzyme exist that are immunologically distinct and differ in substrate specificity. When mitochondrial preparations enriched in particular brain cell or subcellular types were examined, an unequal distribution of E1 and the two forms of protein F were observed. A brain subfraction containing neuronal cell body and glial mitochondria (CM) was found to contain E1 and approximately equal amounts of the ATP- and GTP-dependent forms of protein F. Light synaptic mitochondria (SM1) contained ATP-dependent protein F almost exclusively and were depleted in E1. Dense synaptic mitochondria (SM2) are rich in the ATP form of SCS but also contain low amounts of the GTP enzyme.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D003066:Coenzyme A Ligases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006160:Guanosine Triphosphate / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D013389:Succinate-CoA Ligases / Q000378:metabolism*
D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; D013389:Succinate-CoA Ligases