ncd is a kinesin-related motor protein from Drosophila that moves in the opposite
direction along microtubules to kinesin. To learn more about the ncd mechanism, ncd
motor domain (R335-K700) was expressed in Escherichia coli and its enzymatic characteristics
were studied. The ncd motor domain was purified from the cell lysate by S-Sepharose
chromatography, and trace amounts of contaminants were removed by passing through
a MonoQ column. The yield was 20 mg from a 500 mL culture of E. coli. The purified
ncd motor domain exhibited an unusual UV spectrum with a broad peak around 272-275
nm, which was at least partly due to the bound nucleotide. Upon incubation with radioactive
ATP, 3H at adenine but not 32P at gamma-phosphate was retained by the protein on gel
filtration, indicating it bound ADP but not ATP. Thus, like kinesin, nucleotide binding
to the ncd motor domain is tight, although there is an equilibrium between the protein
and free nucleotide. We also used a fluorescent ATP analogue, mantATP, for the kinetic
study of ncd motor domain. MantATP was turned over by ncd motor domain slowly in the
absence of microtubules, but microtubules activated the turnover to a similar extent
to that of ATP. Upon incubation with ncd motor domain, the fluorescent intensity of
mantATP increased at 0.005 s-1, which is likely to reflect the release of endogenous
ADP and incorporation of mantATP into the protein. The fluorescence intensity of the
ncd motor domain having bound mantADP, likewise, decreased upon mixing with ATP, representing
the mantADP release.(ABSTRACT TRUNCATED AT 250 WORDS)