Cloning, characterization, and expression of the beta subunit of pig heart succinyl-CoA synthetase.
The form of succinyl-CoA synthetase found in mammalian mitochondria is known to be an alpha beta dimer. Both GTP- and ATP-specific isozymes are present in various tissues. We have isolated essentially identical complementary DNA clones encoding the beta subunit of pig heart succinyl-CoA synthetase from both newborn and adult tissues. These cDNAs include a 1.4-kb sequence encoding the cytoplasmic precursor to the beta subunit comprised of 417 amino acid residues including a 22-residue mitochondrial targeting sequence. The cDNA encoding the 395-amino acid, 42,502-Da mature protein was confirmed to be the succinyl-CoA synthetase beta subunit by agreement with the N-terminal protein sequence and by high homology to prokaryotic forms of the beta subunit that were previously cloned (about 45% identical to beta from Escherichia coli). In contrast to a previous report (Nishimura, J.S., Ybarra, J., Mitchell, T., & Horowitz, P.M., 1988, Biochem. J. 250, 429-434), we found no tryptophan residue to be encoded in the sequence for the mature beta subunit, and this finding is corroborated by the fact that highly purified pig heart succinyl-CoA synthetase shows no tryptophan fluorescence or tryptophan content in amino acid compositional analysis. The cDNA clones encoding the mature pig heart beta subunit and its counterpart alpha subunit were coexpressed in a deletion mutant strain of E. coli. Recovery of succinyl-CoA synthetase activity demonstrated that this combination of subunits forms a productive enzymatic complex having GTP specificity.
Bailey
D L
DL
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Wolodko
W T
WT
Bridger
W A
WA
D000375:Aging / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013389:Succinate-CoA Ligases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine / Q000235:genetics*
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013389:Succinate-CoA Ligases