Characterization of molecularly cloned human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase.
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Sugita T, Aya H, Ueno M, Ishizuka T, Kawashima K
Characterization of molecularly cloned human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase.
J Biochem. 1997 Aug;122(2):309-13.
- PubMed ID
- 9378707 [ View in PubMed]
- Abstract
The cDNA encoding human 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase has been cloned from a placenta cDNA library, utilizing a PCR-derived probe. It encodes a peptide of 592 amino acids. The amino (N)-terminal sequence of this enzyme, purified from HeLa cells and CCRF-CEM cells, was found to be APGQLALF-. Both sequencing results revealed a difference of six N-terminal residues when compared to the reported sequence of cloned cDNA from a hepatoma cDNA library. Northern-blot analysis of human AICAR transformylase mRNA showed the expression of a single 2.0 kb mRNA in all tissues examined. With the cloned cDNA fragment, we constructed expression vectors for mature and GST-fused AICAR transformylase. Both recombinant molecules possessing AICAR transformylase activity were overproduced in Escherichia coli. GST-AICAR transformylase can be purified to homogeneity by a single-step affinity procedure with glutathione Sepharose. Mutational analysis, utilizing this expression system, showed that His213 and His267 were essential for AICAR transformylase activity.