Affinity chromatography and affinity labeling of rat liver succinyl-CoA synthetase.
Succinyl-CoA synthetase has been purified to apparent homogeneity from rat liver. The key step in the purification procedure involved adsorption on a GDP dialdehyde (dial-GDP)-adipic dihydrazide-Sepharose 4B column and elution by GDP-Mg2+. Like the pig heart enzyme (Brownie, E. R., and Bridger, W. A. (1972) Can. J. Biochem. 50, 719--724), the rat liver enzyme was an alpha beta heterodimer and only the alpha subunit was phosphorylated by [gamma-32P]GTP. The A 280(0.1%) of the enzyme was determined to be 0.5. Amino acid analyses revealed significant similarities in 50% of the amino acid residues of rat liver and Escherichia coli succinyl-CoA synthetases. However, immunodiffusion analysis failed to reveal any antigenic identity between the two enzymes. Incubation with the affinity label, dial-GDP, in the presence of Mg2+ resulted in a biphasic inactivation of the enzyme. The extent of the rapid phase of inactivation appeared to be related to the extent of dephosphorylation of the enzyme and was prevented by preincubation of the enzyme with GTP-Mg2+. The presence of GDP-Mg2+ in the incubation medium prevented the slow phase of the inactivation and retarded the rapid phase. Dephosphorylated enzyme was approximately 2 orders of magnitude more susceptible to inactivation by dial-GDP than phosphorylated enzyme. Labeling of succinyl-CoA synthetase with [3H]dial-GDP gave a linear relationship between inactivation and incorporation of radioactivity with an extrapolated value of less than 1.2 mol of analog/mol of enzyme at 100% inactivation. The distribution of the label in enzyme that was inactivated 40% was approximately 60% in the alpha subunit and 40% in the beta subunit. Thus, while phosphorylation of the enzyme occurs exclusively in the alpha subunit, the nucleotide binding site appears to include components from both alpha and beta subunits.
D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity; D003066:Coenzyme A Ligases / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D051381:Rats; D013389:Succinate-CoA Ligases / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D000596:Amino Acids; D046911:Macromolecular Substances; D006160:Guanosine Triphosphate; D003066:Coenzyme A Ligases; D013389:Succinate-CoA Ligases; D008274:Magnesium