Unlike mammalian cells, Drosophila melanogaster contains only a single member of the
diphosphoinositol polyphosphate phosphohydrolase subfamily of the Nudix hydrolases,
suggesting that functional specialisation has not occurred in this organism. In order
to evaluate its function, Aps was cloned and characterized. It hydrolyses a range
of (di)nucleoside polyphosphates, the most efficient being guanosine 5'-tetraphosphate
(K(m)=11 microM, k(cat)=0.79 s(-1)). However, it shows a 5-fold preference for the
hydrolysis of diphosphoinositol pentakisphosphate (PP-InsP(5), K(m)=0.07 microM, k(cat)=0.024
s(-1)). Assayed at 26 degrees C, Aps had an alkaline pH optimum and required a divalent
ion: Mg(2+) (10-20 mM) or Mn(2+) (1 mM) were preferred for nucleotide hydrolysis and
Mg(2+) (0.5-1 mM) or Co(2+) (1-100 microM) for PP-InsP(5) hydrolysis. GFP-fusions
showed that Aps was predominantly cytoplasmic, with some nuclear localization. In
the absence of dithiothreitol Aps was heat labile, rapidly losing activity even at
36 degrees C, while in the presence of dithiothreitol, Aps was heat stable, surviving
for 5 min at 76 degrees C. Heat lability was restored by H(2)O(2) and mass spectrometric
analysis suggested that this was due to reversible dimerisation involving two inter-molecular
disulphides between Cys23 and Cys25. Aps expression was highest in embryos and declined
throughout development. The ratio of PP-InsP(5) to inositol hexakisphosphate also
decreased throughout development, with the highest level of PP-InsP(5) found in embryos.
These data suggest that the redox state of Aps may play a role in controlling its
activity by altering its stability, something that could be important for regulating
PP-InsP(5) during development.