Analysis of the binding of glyceraldehyde-3-phosphate dehydrogenase to microtubules, the mechanism of bundle formation and the linkage effect.
The binding of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to microtubules was analysed by the determination of the concentration of the free enzyme in equilibrium with the complex. At low ionic strength (0.03 M) the binding data are best described by a sum of high (Kd = 0.28 microM) and low affinity (Kd = 7.14 microM) sites, showing positive cooperativity. Addition of 1 mM adenosine 5'-[beta,gamma-imido]triphosphate (p[NH]ppA) or increasing the ionic strength to 0.1 M reduces the binding constant of the high-affinity sites considerably. Adding microtubule-associated proteins (at I = 0.1 M) does not appreciably influence the affinities. Total stoichiometries vary over 2.1-1.2 tubulin dimers involved in a binding site for GAPDH. Bundling is reduced concomitantly with the reduction of the affinities and the increase of the stoichiometry to close to 1 mol GAPDH/mol tubulin dimer. The critical concentration of tubulin is practically not influenced by the binding of the enzyme. This behaviour is discussed in terms of the concept of linked functions. p[NH]ppA dissociates the bundles very rapidly. Analytical sedimentation studies showed that the dissociation of the bundles by p[NH]ppA is not due to the dissociation of the tetrameric enzyme. Bundling slows down association and dissociation of microtubules. The rate of bundle formation, after addition of GAPDH to preformed microtubules, is not dependent on the GAPDH concentration.
Somers
M
M
Laboratory of Chemical and Biological Dynamics, Katholieke Universiteit Leuven, Belgium.
Engelborghs
Y
Y
Baert
J
J
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001923:Brain Chemistry; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D008868:Microtubule Proteins / Q000378:metabolism / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D009132:Muscles / Q000201:enzymology; D009994:Osmolar Concentration; D011108:Polymers; D011485:Protein Binding; D011817:Rabbits; D013552:Swine
D008868:Microtubule Proteins; D011108:Polymers; D000255:Adenosine Triphosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases