Cloning and sequencing of cDNAs encoding. cap alpha. and. beta. subunits of human pyruvate dehydrogenase

Journal Article · Thu Dec 31 23:00:00 EST 1987 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.85.1.41· OSTI ID:6464696

Koike, K; Ohta, S; Urata, Y; Kagawa, Y; Koike, M

The cDNAs encoding fragments of the ..cap alpha.. and ..beta.. subunits of human pyruvate dehydrogenase were isolated from a HeLa cell cDNA library in the lambda gt11 expression vector by immunoscreening. Phage cDNA fragments were subsequently used to screen a human foreskin fibroblast cDNA library by colony hybridization. Nucleotide sequence analyses of the positive plasmid clones (pHPDA and pHPDB) revealed an insert of 1.36 kilobases (kb) for PDH..cap 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. A putative leader sequence of 29 amino acid residues was identified in pHPDA, resulting in a precursor protein of 392 amino acid residues (M/sub r/ 43,414) and a mature protein of 363 residues (M/sub r/ 40,334). A similar leader sequence of 30 amino acid residues in pHPDB was also identified, resulting in a precursor protein of 359 amino acid residues (M/sub r/ 39,046) and a mature protein of 329 residues (M/sub r/ 35,911). The amino acid sequences of NH/sub 2/-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..cap alpha.. of bovine and porcine enzymes were also conserved in the human PDH..cap alpha... Blot analysis of HeLA cell poly(A)/sup +/ RNA showed a single mRNA of 1.8 kb for PDH..cap alpha.. and 1.7 kb for PDH..beta.., respectively. The precursor proteins of PDH..cap alpha.. and PDH..beta.. were detected by immunoprecipitation from an /sup 35/S-labeled cell-free translation system.

• Research Organization: Nagasaki Univ. School of Medicine (Japan)
• OSTI ID: 6464696
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 85:1; ISSN PNASA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
CLONING
DAYS LIVING RADIOISOTOPES
DNA
DNA HYBRIDIZATION
DNA SEQUENCING
DNA-CLONING
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
EVEN-ODD NUCLEI
HELA CELLS
HYBRIDIZATION
IMMUNOLOGY
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
LIGHT NUCLEI
MITOCHONDRIA
MOLECULAR STRUCTURE
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
OXIDOREDUCTASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PURIFICATION
RADIOISOTOPES
RECOMBINANT DNA
STRUCTURAL CHEMICAL ANALYSIS
SULFUR 35
SULFUR ISOTOPES