Identification, separation and characterisation of two forms of cytosolic 5'-nucleotidase/nucleoside phosphotransferase in calf thymus.
Cytosolic 5'-nucleotidase, acting preferentially on IMP, GMP and their deoxyderivatives, endowed with phosphotransferase activity, is a widespread enzyme responsible for the regulation of intracellular IMP and GMP concentrations and the phosphorylation of purine nucleoside pro-drugs. The enzyme activity is stimulated by ATP, ADP and 2,3-bisphosphoglycerate (BPG), and is inhibited by phosphate. Calf thymus possesses two active proteins with a different electrophoretic mobility. In this report we show that the two forms can be separated by ADP-agarose affinity chromatography. Whereas form A binds weakly to the column, form B is tightly bound and is released by the addition of ADP into the elution buffer. The two enzyme forms differ in terms of electrophoretic, chromatographic behaviour and regulatory characteristics. Form B, as already described for the enzyme purified from the same source (Pesi et al., 1996, Biochim Biophys Acta 294, 191-194), exhibits three different sites for the three activators with a synergistic effect between ADP and BPG. Form A has a high affinity regulatory site for BPG, while ADP and ATP appear to share the same low affinity site and no synergistic effect is observed.
Pesi
R
R
Dipartimento di Fisiologia e Biochimica, Università di Pisa, Italy.
Baiocchi
C
C
Allegrini
S
S
Moretti
E
E
Sgarrella
F
F
Camici
M
M
Tozzi
M G
MG
D015720:5'-Nucleotidase / Q000737:chemistry* / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D015151:Immunoblotting; D010770:Phosphotransferases / Q000378:metabolism*; D013950:Thymus Gland / Q000378:metabolism*
D000244:Adenosine Diphosphate; D010770:Phosphotransferases; C021892:nucleoside phosphotransferase; D015720:5'-Nucleotidase