Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain.
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)
Shimizu
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T
Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA.
Sablin
E
E
Vale
R D
RD
Fletterick
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R
Pechatnikova
E
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Taylor
E W
EW
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D004789:Enzyme Activation; D016547:Kinesins / Q000378:metabolism; D007700:Kinetics; D008868:Microtubule Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D029721:Drosophila Proteins; D008868:Microtubule Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C066696:ncd protein, Drosophila; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins