Cloning and sequencing of cDNAs encoding. cap alpha. and. beta. subunits of human pyruvate dehydrogenase
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), Vol. 85:1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
OXIDOREDUCTASES
AMINO ACID SEQUENCE
RECOMBINANT DNA
DNA SEQUENCING
DNA-CLONING
HELA CELLS
IMMUNOLOGY
IODINE 125
MITOCHONDRIA
MOLECULAR STRUCTURE
PHOSPHORUS 32
PURIFICATION
SULFUR 35
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
CLONING
DAYS LIVING RADIOISOTOPES
DNA
DNA HYBRIDIZATION
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
EVEN-ODD NUCLEI
HYBRIDIZATION
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPES
LIGHT NUCLEI
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
PHOSPHORUS ISOTOPES
RADIOISOTOPES
STRUCTURAL CHEMICAL ANALYSIS
SULFUR ISOTOPES
550201* - Biochemistry- Tracer Techniques