| Autor: |
Allegrini S; Department of Veterinary Medical Chemistry, Swedish University of Agricultural Science, The Biomedical Center, Box 575, S-751 23 Uppsala, Sweden., Pesi R, Tozzi MG, Fiol CJ, Johnson RB, Eriksson S |
| Jazyk: |
angličtina |
| Zdroj: |
The Biochemical journal [Biochem J] 1997 Dec 01; Vol. 328 ( Pt 2), pp. 483-7. |
| DOI: |
10.1042/bj3280483 |
| Abstrakt: |
A cDNA coding for bovine cytosolic IMP/GMP-specific 5'-nucleotidase endowed with phosphotransferase activity was cloned from calf thymus RNA, by 5' and 3' rapid amplification of cDNA ends protocols (5' and 3' RACE). Two products were isolated: a 5' RACE 1.6 kb fragment and a 3' RACE 2.0 kb fragment, with an overlapping region of 505 bp, leading to a total length of approx. 2951 bp. The similarity in the coding region to that of the human 5'-nucleotidase cDNA sequence [Oka, Matsumoto, Hosokawa and Inoue (1994) Biochem. Biophys. Res. Commun. 205, 917-922], indirectly identified as a 5'-nucleotidase, was 94% and the deduced amino acid sequences were 99.5% identical. The bovine cDNA sequence included the sequences codifying for six peptides obtained from 5'-nucleotidase/phosphotransferase purified from calf thymus. Northern blots of human mRNA species from different tissues showed a 3.6 kb mRNA expressed at equal levels in most tissues. The cDNA was cloned into a pET-28c expression vector and the protein obtained after induction had a molecular mass of 61 kDa under SDS/PAGE. It exhibited both 5'-nucleotidase and phosphotransferase activity, as well as immunological and kinetic properties similar to those of the enzyme purified from calf thymus. This is the first time that a fully active recombinant 5'nucleotidase has been described. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
|
