Molecular cloning of cDNAs for alpha and beta subunits of human pyruvate dehydrogenase.
The cDNAs encoding human PDH alpha and PDH beta were isolated from a HeLa cell cDNA library in the lambda gt11 expression vector by immunoscreening, followed by colony hybridization from a human foreskin fibroblast cDNA library. Nucleotide sequence analyses of the positive plasmid clones (pHPDA and pHPDB) revealed an insert of 1.36 kilobases (kb) for PDH alpha and one of 1.69 kb for PDH beta, respectively, allowing us to predict the complete amino acid sequences of the precursor and mature proteins of these two subunits. The amino acid sequences of the amino-terminal regions of the two subunits of human PDH were highly homologous with those of mature porcine PDH. The amino acid sequences of phosphorylation sites determined in PDH alpha of the bovine and porcine enzymes were also conserved in the human PDH alpha. Blot analysis of HeLa cell poly(A)+ RNA and the transcriptional product of the two cDNAs showed a single mRNA of 1.8 kb for PDH alpha and one of 1.7 kb for PDH beta. The precursor proteins of PDH alpha and PDH beta were detected by immunoprecipitation from an 35S-labeled, cell-free translation system. Our sequence of PDH alpha cDNA was compared with those of two other origins. The differences among these three PDH alpha cDNAs have been discussed.
Koike
K
K
Department of Pathological Biochemistry, Nagasaki University School of Medicine, Japan.
Urata
Y
Y
Koike
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D015723:Gene Library; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D046911:Macromolecular Substances; D011768:Pyruvate Dehydrogenase Complex; D004247:DNA