Article

Equilibrium binding studies of non-claret disjunctional protein (Ncd) reveal cooperative interactions between the motor domains.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Journal of Biological Chemistry (impact factor: 4.77). 01/1999; 273(52):35307-18. pp.35307-18
Source: PubMed

ABSTRACT Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis. We have pursued equilibrium binding experiments to examine the affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5'-adenylyl-beta, gamma-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP. Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules. The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparent Kd(Pi) for the two phosphate sites at 23.3 and 1.9 mM, respectively. The results indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release. Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt.MC1 complex at 14 s-1, yet AMP-PNP has no effect on the Mt.MC1 complex. These results are consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd.ADP.Pi intermediate.

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Keywords

ADP + Pi
 
affinity
 
AMP-PNP
 
apparent Kd(Pi)
 
ATP nonhydrolyzable analog 5'-adenylyl-beta
 
ATPase cycle
 
cooperatively stimulating release
 
dimeric MC1
 
Drosophila oogenesis
 
end-directed microtubule motor
 
equilibrium binding experiments
 
Escherichia coli
 
first phosphate site enhances binding
 
MgATP promotes dissociation
 
Mt.MC1 complex
 
Ncd.ADP.Pi intermediate
 
Non-claret disjunctional protein
 
normal spindle assembly
 
second site
 
Stopped-flow kinetics
 

K A Foster