UDP-glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing.
We have isolated a cDNA encoding UDP-glucose pyrophosphorylase from a cDNA library of immature potato tuber using oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,758-base-pair insert including the complete message for UDP-glucose pyrophosphorylase with 1,431 base pairs. The amino acid sequence of the enzyme inferred from the nucleotide sequence consists of 477 amino acid residues. All the partial amino acid sequences determined protein-chemically [Nakano et al. (1989) J. Biochem. 106, 528-532] confirmed the primary structure of the enzyme. An N-terminal-blocked peptide was isolated from the proteolytic digest of the enzyme protein, and the blocking group was deduced to be an acetyl group by fast atom bombardment-mass spectrometry. On the basis of the predicted amino acid sequence (477 residues minus the N-terminal Met plus an acetyl group), the molecular weight of the enzyme monomer is calculated to be 51,783, which agrees well with the value determined by polyacrylamide gel electrophoresis. In the cDNA structure, the open-reading frame is preceded by a 125-base-pair noncoding region, which contains a sequence being homologous with the consensus sequence for plant genes, and is followed by a 174-base-pair noncoding sequence including a polyadenylation signal. Amino acid sequence comparisons revealed that the potato UDP-glucose pyrophosphorylase is homologous to the enzyme from slime mold, Dictyostelium discoideum, but not to ADP-glucose pyrophosphorylases from rice seed and Escherichia coli.
Katsube
T
T
Institute of Scientific and Industrial Research, Osaka University.
Kazuta
Y
Y
Mori
H
H
Nakano
K
K
Tanizawa
K
K
Fukui
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015183:Restriction Mapping; D011198:Solanum tuberosum / Q000201:enzymology*; D016339:Spectrometry, Mass, Fast Atom Bombardment; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000737:chemistry* / Q000235:genetics
D000596:Amino Acids; D004247:DNA; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D010450:Endopeptidases