A cDNA encoding a pea nuclear apyrase was previously cloned. Overexpression
s of a full-length and a truncated cDNA have been successfully expressed in
Escherichia coli BL21(DE3). The resulting fusion proteins, apyrase and the
C-terminus (residues 315-453) of apyrase, were used for calmodulin (CaM) b
inding and phosphorylation studies. Fusion protein apyrase but not the C-te
rminus of apyrase can be recognized by polyclonal antibody pc480. This sugg
ested that the motif recognized by pc480 was located in the N-terminal regi
on of apyrase. The recombinant apyrase protein also showed an activity 70 t
imes higher than that of endogenous apyrase using ATP as a substrate. The r
ecombinant apyrase has a preference for ATP more than other nucleoside trip
hosphate substrates. CaM can bind to recombinant apyrase, but not to the C-
terminus of apyrase. This implies that the CaM-binding domain must be in th
e first 315 amino acids of the N-terminal region of apyrase. We found that
one segment from residue 293 to 308 was a good candidate for the CaM-bindin
g domain. This segment 293 FNKCKNTIRKALKLNY 308 has a basic amphiphilic-hel
ical structure, which shows the predominance of basic residues on one side
and hydrophobic residues on the other when displayed on a helical wheel plo
t. Using the gel mobility shift binding assay, this synthetic peptide was s
hown to bind to CaM, indicating that it is the CaM-binding domain. Both rec
ombinant apyrase and the C-terminus of apyrase can be phosphorylated by a r
ecombinant human protein kinase CKII. Phosphorylation does not affect CaM b
inding to recombinant apyrase. However, CaM does inhibit CKII phosphorylati
on of recombinant apyrase and this inhibition can be blocked by 5 mM EGTA.
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