Molecular cloning of rat amidophosphoribosyltransferase.
The cDNA of rat amidophosphoribosyltransferase (EC 2.4.2.14, ATase), which is the supposed regulatory allosteric enzyme of de novo purine nucleotide biosynthesis, has been cloned by polymerase chain reaction. The predicted open reading frame encodes a protein of 517 amino acids with a deduced molecular weight of 57,436 including a supposed 11-amino acid propeptide. The 16 amino acid residues next to the propeptide were identical to the N-terminal amino acid microsequence of a purified rat liver ATase, which is consistent with the cleavage of the propeptide from the proenzyme in rat liver. The derived amino acid sequence is the first sequence reported for a mammalian ATase and it exhibits 81, 41, 36, and 31% identity with the sequences of chicken, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae ATases, respectively. The molecular weight (M(r)) of 57,436 suggests a tetrameric structure of native ATase with a M(r) of 240,000-248,000. Southern blot analysis suggested that the ATase gene exists as a single copy in the rat genome. Northern blot analysis revealed that ATase is expressed at a high level in brain, heart, liver, and stomach. The ATase mRNA in brain, heart, and stomach was 3.5 kilobases (kb) and in liver the 3.5-kb band was observed as well as an additional band of 4.2 kb. Reverse transcription-polymerase chain reaction analysis showed that ATase is ubiquitously expressed in all tissues examined. Comparison with chicken ATase showed that 2 cysteine residues for an iron-sulfur cluster were conserved. Three conserved and two non-conserved consensus phosphorylation sites for cAMP-dependent protein kinase were found.
Iwahana
H
H
Otsuka Department of Clinical and Molecular Nutrition, University of Tokushima, Japan.
Yamaoka
T
T
Mizutani
M
M
Mizusawa
N
N
Ii
S
S
Yoshimoto
K
K
Itakura
M
M
D000582:Amidophosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004247:DNA; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D004247:DNA; D000582:Amidophosphoribosyltransferase