Alternative Binding of Two Sequential Glycolytic Enzymes to Microtubules. MOLECULAR STUDIES IN THE PHOSPHOFRUCTOKINASE/ALDOLASE/MICROTUBULE SYSTEM (original) (raw)
1997, Journal of Biological Chemistry
Simultaneous binding of two sequential glycolytic enzymes, phosphofructokinase and aldolase, to a microtubular network was investigated. The binding of the phosphofructokinase to microtubules and its bundling activity has been previously characterized (Lehotzky, A., Telegdi, M., Liliom, K., and Ová di, J. (1993) J. Biol. Chem. 268, 10888 -10894). Aldolase binding to microtubules at near physiological ionic strength is weak (K d ؍ 20 M) as compared with that of the kinase (K d ؍ 1 M). The interactions of both enzymes with microtubules are modulated by their common intermediate, fructose-1,6bisphosphate. Pelleting and electron microscopic measurements have revealed that the aldolase binding interferes with that of phosphofructokinase, although they have distinct binding domains on microtubules. The underlying molecular mechanism responsible for this finding is that in the solution phase aldolase and phosphofructokinase form a bienzyme complex that does not bind to the microtubule. The bienzyme complex formation does not influence the catalytic activity of aldolase, however, it inhibits the dissociation-induced inactivation of the kinase by stabilizing a catalytically active molecular form. The present data suggest the first experimental evidence that two sequential glycolytic enzymes do not associate simultaneously to microtubules, but their complexation in solution provides kinetic advantage for glycolysis.
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Journal of Biological Chemistry
The linked equilibria involved in the binding of phosphofructokinase (EC 2.7.1.11, ATP:D-fructose-6-phosphate 1-phosphotransferase) to tubulin and microtubules were studied at high ionic strength in vitro. The concentration-dependent dissociation of phosphofructokinase was analyzed in the absence and presence of tubulin or microtubules, and the binding of kinase to the tubulin dimer and microtubules was compared. Enzyme activity of phosphofructokinase was inhibited by both tubulin and microtubules: the relative inhibition increased with decreasing enzyme concentration. The complex formation between phosphofructokinase and tubulin was demonstrated by means of fluorescent anisotropy. Concentration-dependent copelleting of the kinase with taxol-stabilized microtubules revealed binding of the enzyme to microtubules as well as phosphofructokinase-enhanced pelleting of microtubules. The binding data agree with the enzyme kinetic findings that the inactive dissociated forms of phosphofruct...
Biochemistry, 1997
Phosphofructokinase interacts with both microtubules and microtubules containing microtubuleassociated proteins to produce bundling and periodical cross-bridging of tubules. Immunoelectron microscopy using anti-phosphofructokinase antibodies provided direct evidence that the kinase molecules are responsible for the cross-bridging of microtubules. Limited proteolysis by subtilisin, a procedure that cleaves the N-terminal segment of the free enzyme as well as the C-terminal "tails" of tubulin subunits exposed on microtubules, showed that while phosphofructokinase becomes resistant, tubulin retains sensitivity against proteolysis within the heterologous complex. These data suggest that the N-terminal segment of the enzyme, but not the C-terminal "tail" of tubulin subunits, is involved in the interaction between the microtubule and the kinase. The phosphorylation of phosphofructokinase or microtubules containing microtubule-associated proteins by the cAMP-dependent protein kinase did not interfere with the heterologous complex formation. MgATP prevents phosphofructokinase binding to the microtubules, and it can displace the enzyme from the single microtubules. However, the bundled microtubules are apparently resistant to the MgATP dissociation effect. Modelling of the assembly process suggests that the tubulin-kinase complex is able to polymerize as the free tubulin. Vinblastine, an anti-mitotic agent, inhibits tubulin assembly; however, its inhibitory effect is partially suppressed in the presence of phosphofructokinase. Fluorescence anisotropy measurements indicated that kinase and vinblastine compete for tubulin binding with no evidence for ternary complex formation. This competitive mechanism and the ability of the tubulin-enzyme complex to polymerize into microtubules may result in the resistance of the tubulin-enzyme complex against the inhibition of assembly induced by vinblastine. Microtubules formed in the presence of vinblastine plus phosphofructokinase can be visualized by electron microscopy. A molecular model is suggested that summarizes the effects of MgATP and vinblastine on the multiple equilibria in the tubulin/microtubules/phosphofructokinase system.
European journal of biochemistry / FEBS, 1990
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. ...
The conversion of tubulin carboxyl groups to amides has a stabilizing effect on microtubules
Biochemistry, 1992
In a recent study, we demonstrated that the conversion of carboxyl residues in the C-termini of tubulin to neutral amides with glycine ethyl ester enhanced the ability of the protein to assemble into microtubules and decreased its interaction with microtubule-associated proteins (MAPs). In this work, we investigated the effects of carboxyl modification on the dynamic behavior of microtubules at polymer mass steady state. After steady state, microtubules assembled from unmodified tubulin were sheared, and the mean polymer lengths decreased to 5 microns and then increased to 29 microns within 130 min. In contrast, lengths of sheared microtubules polymerized from tubulin containing 23 modified carboxyl groups increased by only 2-fold. Stabilization of polymer lengths was also observed directly by video-enhanced light microscopy of microtubules grown off of axonemes. Rapid shortening was seen in microtubules composed of unmodified but not modified tubulin. Further evidence for the less dynamic behavior of microtubules as a result of carboxyl modification was obtained from kinetic studies of the elongation phase during assembly which showed a 3-fold lower off-rate constant, k-, for modified microtubules. Another effect of the modification was a 12-fold reduction in the steady-state rate constant for GTP hydrolysis (165 s-1 for unmodified and 14 s-1 for modified). These results suggest that reduction of the negative charges in the C-termini by modification of the acidic residues stabilizes microtubules against depolymerization. MAPs may stabilize microtubules in an analogous manner.
Mechanism for nucleotide incorporation into steady-state microtubules
Biochemistry, 1984
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Biochemical Studies on the in Vitro Assembly and Disassembly of Microtubules
Annals of the New York Academy of Sciences, 1975
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