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, Chromatographie
behaviour and regulatory characteristics.
Form B, as already described for the enzyme purified
from the same source (Pesi ef a/., 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.