Interaction between kinesin, microtubules, and microtubule-associated protein 2.
Kinesin from porcine brain was prepared by a procedure based on the strong binding of the protein to microtubules in the presence of sodium fluoride and ATP. The protocol reduces the requirement for taxol and AMP-PNP. The kinesin is active in terms of its ability to move microtubules on glass slides and its ATPase. The ATPase of this kinesin is about 8 nmol/min/mg; it is activated to 19 nmol/min/mg in the presence of microtubules. The relationship between gliding velocity and ATP concentration follows Michaelis-Menten kinetics. Using the motility assay, the maximal velocity is 0.78 micron/sec, and the Km value is 150 microM for ATP. For GTP the corresponding values are 0.38 micron/sec and 1.7 mM. ADP is a competitive inhibitor (Ki = 0.29 mM). Crude preparations of kinesin do not support motility on glass slides, whereas gel-filtered kinesin does. A search for potential inhibitory factors showed that one of them is MAP2; however, its inhibitory effect becomes visible only in certain conditions. MAP2 bound to microtubules does not inhibit kinesin-induced motility. However, when MAP2 and kinesin are preadsorbed to the glass surface independently of microtubules, MAP2 prevents the interaction of kinesin with microtubules, as if it formed a "lawn" that acted as a spacer and thus repelled the MAP-free microtubules or crosslinked the MAP-containing ones. The repelling effect of MAP2 domains (projection or assembly fragments obtained by chymotryptic cleavage) added separately is less pronounced and can be overcome by kinesin. These results reinforce the view of MAP2 as a spacer molecule.
von Massow
A
A
Max-Planck-Unit for Structural Molecular Biology, Hamburg, Federal Republic of Germany.
Mandelkow
E M
EM
Mandelkow
E
E
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006651:Histocytochemistry; D016547:Kinesins; D007700:Kinetics; D008868:Microtubule Proteins / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism* / Q000502:physiology; D009068:Movement; D012969:Sodium Fluoride / Q000494:pharmacology; D013552:Swine
D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D012969:Sodium Fluoride; D000251:Adenosine Triphosphatases; D016547:Kinesins