Binding of guanine nucleotides and Mg2+ to tubulin with a nucleotide-depleted exchangeable site

Binding of GTP and GDP to tubulin in the presence or absence of Mg2+ was measured following depletion of the exchangeable site--(E-site) nucleotide. The E-site nucleotide was displaced with a large molar excess of the nonhydrolyzable GTP analogue, GMPPCP, followed by the removal of the analogue. Using a micropartition assay, the equilibrium constant measured in 0.1 M 1.4-piperazinediethanesulfonic acid (Pipes), pH 6.9, 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, 1 mM dithiothreitol, and 1 mM MgSO4 at 4 degrees C was 9.1 x 10(6) M-1 for GTP and 4.4 x 10(6) M-1 for GDP. Removal of Mg2+ reduced the binding affinity of GTP by 160-fold while the affinity of GDP remained essentially unchanged. Similar values were obtained if 0.1 M Tris, pH 7.0, was used instead of Pipes. Binding of Mg2+ to tubulin containing GTP, GDP, or no nucleotide at the E-site was also examined by the micropartition method. Tubulin-GTP contained one high affinity Mg2+ site (K alpha = 1.2 x 10(6) M-1) in addition to the one occupied by Mg2+ as tubulin is isolated, while only weak Mg2+ binding to tubulin-GDP and to tubulin with a vacant E-site (K alpha = 10(3) M-1) was observed. It is suggested that Mg2+ binds to the beta and gamma phosphates of GTP, and only to the beta phosphate of GDP, as shown for the H. ras p21 protein.     

Influence of the guanine nucleotide phosphorylation state and of Mg2+ ions on the interaction of vinzolidine/tubulin 6 S: a fluorescence quenching study

The binding of the new vincaalkaloid vinzolidine to tubulin 6 S was investigated by using fluorescence quenching methods. The value of the apparent equilibrium binding constant was found to depend on the phosphorylation state of the guanine nucleotide bound to the tubulin exchangeable site (E-site), with Ka values of 4.9 X 10(4) and 8.19 X 10(4) M-1 for GTP- and GDP-tubulin, respectively. The effect of Mg2+ ions on this binding was more important on GTP-tubulin than on GDP-tubulin, and might be related to the existence of Mg2+ site(s) independent of the nucleotide.     

Differential effects of magnesium on tubulin-nucleotide interactions

Magnesium-depleted 2-(N-morpholino)ethanesulfonate (Mes), glutamate, tubulin and microtubule-associated proteins were prepared and used to study the effects of exogenously added MgCl2 on tubulin-nucleotide interactions in 0.1 M Mes with microtubule-associated proteins and in 1.0 M glutamate. Endogenous levels of Mg2+ in the systems studied were approximately stoichiometric with the tubulin concentrations and largely derived from the tubulin. We examined the effects of added Mg2+ on tubulin polymerization, GDP inhibition of polymerization, binding of GDP and GTP to tubulin, and GTP hydrolysis. Exogenously added Mg2+ had markedly different effects on these reactions. The order of their sensitivity for a requirement for added Mg2+ was as follows: GTP binding greater than GTP hydrolysis greater than polymerization greater than GDP binding. Inhibition of polymerization by GDP varied inversely with the Mg2+ concentration and was greatest in the absence of the cation. These results indicate that GDP and GDP-Mg2+ interact with similar affinity at the exchangeable site, while GTP-Mg2+ has a higher affinity for tubulin than does free GTP. Nevertheless, under appropriate conditions, free GTP can interact sufficiently well with tubulin to permit both nucleation and elongation reactions.     

Neurotubule assembly at substoichiometric nucleotide levels using a GTP regenerating system

Tubulin derived from cold depolymerized bovine microtubules has been gel filtered to obtain a tubulin preparation with only 3% of the tubulin dimers containing exchangeable [³H]-guanine nucleotide. In the presence of acetyl-P and bacterial acetate kinase, this preparation polymerizes to form microtubules which are morphologically indistinguishable from microtubules formed in the presence of excess GTP. The extent of microtubule formation at substoichiometric nucleotide levels using the GTP regenerating system exceeds the extent of assembly obtained with excess GTP. It is concluded that the exchangeable guanine nucleotide site can be virtually unoccupied in intact neurotubules and this finding indicates that GDP can “catalyze” tubule assembly in the presence of a GTP regenerating system.     

Reexamination of the role of nonhydrolyzable guanosine 5'-triphosphate analogues in tubulin polymerization: reaction conditions are a critical factor for effective interactions at the exchangeable nucleotide site

Recently it was proposed [O'Brien, E. T., & Erickson, H. P. (1989) Biochemistry 28, 1413-1422] that tubulin polymerization supported by guanosine 5'-(beta,gamma-imidotriphosphate) [p(NH)ppG], guanosine 5'-(beta,gamma-methylenetriphosphate) [p(CH2)ppG], and ATP might be due to residual GTP in reaction mixtures and that these nucleotides would probably support only one cycle of assembly. Since we had observed polymerization with these three compounds, we decided to study these reactions in greater detail in two systems. The first contained purified tubulin and a high concentration of glycerol, the second tubulin and microtubule-associated proteins (MAPs). In both systems, reactions supported by nucleotides other than GTP were most vigorous at lower pH values. In the glycerol system, repeated cycles of polymerization were observed with ATP and p(CH2)ppG, but not with p(NH)ppG. With p(NH)ppG, a single cycle of polymerization was observed, and this was caused by contaminating GTP. In the MAPs system, repeated cycles of polymerization were observed with both nonhydrolyzable GTP analogues, even without contaminating GTP, but ATP was not active at all in this system. Binding to tubulin of p(NH)ppG, p(CH2)ppG, and, to a lesser extent, ATP was demonstrated indirectly, since high concentrations of the three nucleotides displaced radiolabeled GDP originally bound in the exchangeable site, with p(NH)ppG the most active of the three compounds in this displacement assay. The failure of GTP-free p(NH)ppG to support tubulin polymerization in our glycerol system even though it displaced GDP from the exchangeable site was further investigated by examining the effects of p(NH)ppG on polymerization and polymer-bound nucleotide with low concentrations of GTP. The two nucleotides appeared to act synergistically in supporting polymerization, so that a reaction occurred with a subthreshold GTP concentration if p(NH)ppG was also in the reaction mixture. Analysis of radiolabeled exchangeable-site nucleotide in polymers formed in reaction mixtures containing both GTP and p(NH)ppG demonstrated that p(NH)ppG which entered polymer did so primarily at the expense of GDP originally bound in the exchangeable site rather than at the expense of GTP. It appears that in the glycerol reaction condition, tubulin-p(NH)ppG cannot initiate tubulin polymerization but that it can participate in polymer elongation. ATP and p(CH2)ppG also entered the exchangeable site during polymerization without GTP in glycerol, as demonstrated by displacement of radiolabeled GDP from polymer when these alternate nucleotides were used.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tubulin exchanges divalent cations at both guanine nucleotide-binding sites

The tubulin heterodimer binds a molecule of GTP at the nonexchangeable nucleotide-binding site (N-site) and either GDP or GTP at the exchangeable nucleotide-binding site (E-site). Mg2+ is known to be tightly linked to the binding of GTP at the E-site (Correia, J. J., Baty, L. T., and Williams, R. C., Jr. (1987) J. Biol. Chem. 262, 17278-17284). Measurements of the exchange of Mn2+ for bound Mg2+ (as monitored by atomic absorption and EPR) demonstrate that tubulin which has GDP at the E-site possesses one high affinity metal-binding site and that tubulin which has GTP at the E-site possesses two such sites. The apparent association constants are 0.7-1.1 x 10(6) M-1 for Mg2+ and approximately 4.1-4.9 x 10(7) M-1 for Mn2+. Divalent cations do bind to GDP at the E-site, but with much lower affinity (2.0-2.3 x 10(3) M-1 for Mg2+ and 3.9-6.6 x 10(3) M-1 for Mn2+). These data suggest that divalent cations are involved in GTP binding to both the N- and E-sites of tubulin. The N-site metal exchanges slowly (kapp = 0.020 min-1), suggesting a mechanism involving protein "breathing" or heterodimer dissociation. The N-site metal exchange rate is independent of the concentration of protein and metal, an observation consistent with the possibility that a dynamic breathing process is the rate-limiting step. The exchange of Mn2+ for Mg2+ has no effect on the secondary structure of tubulin at 4 degrees C or on the ability of tubulin to form microtubules. These results have important consequences for the interpretation of distance measurements within the tubulin dimer using paramagnetic ions. They are also relevant to the detailed mechanism of divalent cation release from microtubules after GTP hydrolysis.     

Localization of the ATP binding site on alpha-tubulin

The binding site for ATP to tubulin was established by use of the photoaffinity label [gamma-32P]N3ATP. Photolysis of the analog in the presence of tubulin resulted in covalent modification of the protein as revealed by autoradiography of electropherograms. Scanning the autoradiograms showed that the ATP analog was bound mainly to the alpha subunit of the tubulin dimer; the alpha subunit was two to three times more radioactive than was the beta subunit. The location of a particular site on the alpha subunit was further defined by peptide maps. The alpha and beta subunits from affinity-labeled tubulin were separated and digested with Staphylococcus protease. Radioactivity was found predominantly in one peptide band from the alpha subunit. The location of the [gamma-32P]N3ATP binding site on the alpha subunit distinguishes it from the previously known exchangeable GTP binding site which is on the beta subunit. Moreover, excess GTP did not compete with [gamma-32P]N3ATP binding. The ATP binding site is distinct from the nonexchangeable GTP binding site. The GTP content of tubulin was the same after dialysis in 0.5 mM ATP as it was following dialysis against ATP-free buffer. Proof that the binding site for [gamma-32P]N3ATP is the same as that for ATP was obtained by competition experiments. In the presence of ATP, photolysis of the affinity analog did not label the alpha subunit preferentially.     

Direct photoaffinity labeling of tubulin with guanosine 5'-triphosphate

Irradiation of tubulin in the presence of [3H]GTP or [3H]GDP at 254 nm led to the covalent incorporation of nucleotide into the protein. The specific nature of the labeling was shown in the following manner: with tubulin depleted of exchangeable nucleotide, the amount of labeling increased to a plateau value as the [3H]GTP concentration was increased, with saturation being reached at a ratio of approximately 1.5; the same amount of labeling was obtained with GTP/tubulin ratios of 1 and 100; [3H]GMP was not incorporated into the dimer, nor did GMP inhibit the incorporation of [3H]GTP; [3H]ATP was not incorporated; [3H]GTP incorporation did not occur into denatured tubulin or into serum albumin. When [alpha-32P]GTP was used in the irradiation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the carboxymethylated protein demonstrated that the incorporated label was associated with the beta subunit. The radiation treatment did cause changes in the tubulin molecule resulting in a decrease in assembly competence and in sulfhydryl groups, but these effects were minimized when a large excess of GTP was present during irradiation. Labeling of tubulin in the assembled state was much less than that observed in the free state.     

Magnesium ion exerts a central role in the regulation of inhibitory adenosine receptors.

Guanine nucleotides and Mg2+ differentially regulate agonist binding to adenosine (Ri) receptors in fat-cell plasma membranes. GTP alone decreases binding of the agonist ligand [3H]N6-cyclohexyladenosine (CHA) by increasing the dissociation constant (Kd). Mg2+ alone also decreases [3H]CHA binding, which is associated with a decrease in the number of receptors and in the dissociation constant. In the presence of Mg2+, the effect of GTP is to increase [3H]CHA binding by increasing the total number of receptors. It thus appears that Mg2+ acts specifically at a bivalent-cation site which, with GTP, regulates agonist binding. This putative Mg site is highly sensitive to alkylating agents. Mild treatment with N-ethylmaleimide (NEM) abolishes the characteristic GTP effect on agonist binding in the presence of Mg2+. In addition, the effect of Mg2+ alone is also eliminated. The effect of GTP alone is largely unaltered. Studies of the adenylate cyclase activity indicate that this NEM treatment also abolishes the inhibition of basal activity by adenosine analogues, whereas guanylyl imidodiphosphate inhibition of forskolin-stimulated activity is only slightly impaired at this NEM concentration. These observations indicate that a Mg2+ 'site' or 'component' is required for the integration of receptor (Ri) occupancy with regulation of catalytic activity (C). The regulatory role of Mg2+ is more demonstrable in receptor-GTP-regulatory-protein (Ri-Ni) interactions than in GTP-regulatory-protein-catalytic-unit (Ni-C) interactions.     

Mg2+ dependence of guanine nucleotide binding to tubulin

The relationship between the concentration of Mg2+ and the binding of GDP and GTP to tubulin dimers was investigated by measuring the displacement of the nucleotide bound at the exchangeable site (E-site) by radiolabeled GDP and GTP. A wide range of concentrations of GTP, GDP, and Mg2+ was explored. In the near absence of Mg2+, the affinity of tubulin for GDP was found to be much greater than its affinity for GTP. In the presence of 1.0 mM Mg2+, however, its affinity for GDP was slightly less than for GTP. The results could be quantitatively described in terms of a small number of reversible equilibria. Equilibrium constants, pertaining to measurements at 0 degrees C, in 0.1 M piperazine-N,N'-bis(2-ethanesulfonic acid), 0.2 mM dithioerythritol, 2 mM EGTA, pH 6.9, were obtained by nonlinear least squares fitting of the data. When the association constant of tubulin for GDP uncomplexed with Mg2+ was taken to be 1.6 X 10(7) M-1, that for uncomplexed GTP was found to be no larger than 1.4 x 10(4) M-1, at least 1100-fold smaller. The association constant of tubulin for the GDP.Mg2+ complex was found to be 2.5-2.7 x 10(7) M-1, while that for the GTP.Mg2+ complex is 6.4-9.0 x 10(7) M-1.     

Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites

Dolastatin 10, a potent antimitotic peptide from a marine animal, strongly inhibits microtubule assembly, tubulin-dependent GTP hydrolysis, and the binding of vinca alkaloids to tubulin. In studies of the binding of [3H]vincristine to the protein, with vinblastine as a control for competitive inhibition (Ki, 6.6 microM), we found that the macrolide antimitotic agents maytansine and rhizoxin were also competitive inhibitors (Ki values, 3.1 and 12 microM). Dolastatin 10 and an unrelated peptide antimitotic, phomopsin A, were more potent but noncompetitive inhibitors (Ki values, 1.4 and 2.8 microM). Since maytansine and, to a much lesser extent, vinblastine interfere with nucleotide exchange on tubulin, all drugs were examined for effects on nucleotide interactions at the exchangeable GTP site. Rhizoxin had effects intermediate between those of vinblastine and maytansine. Both peptides inhibited binding of radiolabeled GTP to tubulin even more strongly than did maytansine, but no drug displaced nucleotide from tubulin. The drugs were evaluated for stabilizing effects on the colchicine binding activity of tubulin. The peptides prevented loss of this activity, and vinblastine provided partial protection, while rhizoxin and maytansine did not stabilize tubulin. A tripeptide segment of dolastatin 10 also effectively inhibits tubulin polymerization and GTP hydrolysis. The tripeptide did not significantly inhibit either vincristine binding or nucleotide exchange, nor did it stabilize colchicine binding. These findings are rationalized in terms of a model with two distinct drug binding sites in close physical proximity to each other and to the exchangeable GTP site on beta-tubulin.     

Affinity labeling of the exchangeable nucleotide binding site in platelet tubulin

The exchangeable nucleotide binding site of platelet tubulin was labeled with [14C]p-fluorosulfonyl benzoylguanosine (FSBG). FSBG promoted polymerization of tubulin but depolymerization did not occur in the presence of this nucleoside analogue. GTP was able to block FSBG binding to tubulin. [14C]Iodoacetamide-treated tubulin which was first reacted with FSBG was digested with trypsin. The resultant peptides were analyzed by reverse phase high pressure liquid chromatography. One FSBG-labeled peptide could be identified both by its radioactivity and the characteristic UV absorbance spectrum associated with it. This may represent the exchangeable nucleotide site. A second peptide with a distinct nucleotide absorbance peak was found both in FSBG-treated and untreated tubulin preparations. This evidence is suggestive of the non-exchangeable nucleotide binding site.     

Direct photoaffinity labeling by dolastatin 10 of the amino-terminal peptide of beta-tubulin containing cysteine 12

Tubulin with bound [5-3H]dolastatin 10 was exposed to ultraviolet light, and 8-10% of the bound drug cross-linked to the protein, most of it specifically. The primary cross-link was to the peptide spanning amino acid residues 2-31 of beta-tubulin, but the specific amino acid could not be identified. Indirect studies indicated that cross-link formation occurred between cysteine 12 and the thiazole moiety of dolastatin 10. An equipotent analog of dolastatin 10, lacking the thiazole ring, did not form an ultraviolet light-induced cross-link to beta-tubulin. Preillumination of tubulin with ultraviolet light, known to induce cross-link formation between cysteine 12 and exchangeable site nucleotide, inhibited the binding of [5-3H]dolastatin 10 and cross-link formation more potently than it inhibited the binding of colchicine or vinblastine to tubulin. Conversely, binding of dolastatin 10 to tubulin inhibited formation of the cross-link between cysteine 12 and the exchangeable site nucleotide. Dithiothreitol inhibited formation of the beta-tubulin/dolastatin 10 cross-link but not the beta-tubulin/exchangeable site nucleotide cross-link. Modeling studies revealed a highly favored binding site for dolastatin 10 at the + end of beta-tubulin in proximity to the exchangeable site GDP. Computational docking of an energy-minimized dolastatin 10 conformation at this site placed the thiazole ring of dolastatin 10 8-9 A from the sulfur atom of cysteine 12. Dolastatin 15 and cryptophycin 1 could also be docked into positions that overlapped more extensively with the docked dolastatin 10 than with each other. This result was consistent with the observed binding properties of these peptides.     

Direct incorporation of guanosine 5'-diphosphate into microtubules without guanosine 5'-triphosphate hydrolysis

Using highly purified calf brain tubulin bearing [8-14C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins (both components containing negligible amounts of nucleoside diphosphate kinase and nonspecific phosphatase activities), we have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly.     

Magnesium requirements for guanosine 5'-O-(3-thiotriphosphate) induced assembly of microtubule protein and tubulin

Two conflicting interpretations on the role of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in microtubule protein and tubulin assembly have been previously reported. One study finds that GTP gamma S promotes assembly while another study reports that GTP gamma S is a potent inhibitor of microtubule assembly. We have examined the potential role of Mg2+ to learn if the conflicting interpretations are due to a metal effect. Turbidity, electron microscopy, and nucleotide binding and hydrolysis were used to analyze the effect of the Mg2+ concentration on GTP gamma S-induced assembly of microtubule protein (tubulin + microtubule-associated proteins) in the presence of buffer +/- 30% glycerol and in buffer with GTP added before or after GTP gamma S. GTP gamma S substantially lowers the Mg2+ concentration required to induce cross-linked or clustered rings of tubulin. These cross-linked rings do not assemble well into microtubules, and GTP only partially restores microtubule assembly. However, taxol will promote GTP gamma S-induced cross-linked rings of microtubule protein to assemble into microtubules. The effect of GTP gamma S on microtubule protein assembly in the presence of Zn2+ with and without added Mg2+ suggests that GTP gamma S also effects the formation of Zn2+-induced sheet aggregates. Purified tubulin was used in assembly experiments with Mg2+, Zn2+, and taxol to better understand GTP gamma S interactions with tubulin. The optimal Mg2+ concentration for assembly of tubulin is lower with GTP gamma S than with GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of a GTP photoaffinity probe to resolve aspects of the mechanism of tubulin polymerization.

8-Azidoguanosine 5'-triphosphate (8-N3GTP) was used in a photoactivatable probe to examine the role of GTP in microtubule assembly. 8-N3GTP was able to substitute for GTP in the promotion of tubulin polymerization and was hydrolyzed at 37 degrees C in the presence or absence of colchicine or calcium. Photolysis of the analog in the presence of microtubular protein resulted in its covalent incorporation onto a GTP-specific site of the beta monomer. The efficiency of this incorporation was different when 8-N3GDP (which does not affect polymerization) was used in place of 8-N3GTP, implying a different orientation of the nucleoside diphosphate within the receptor site. During microtubule assembly, 8-N3GTP was hydrolyzed in situ at the tubulin-GTP exchangeable site in a process that was dependent upon polymerization. The use of [beta, gamma-32P]8-N3GTP and [gamma-32P]8-N3GTP indicated that this hydrolysis occurred concurrently with polymerization and that only nucleoside diphosphate remained bound to the polymerized tubulin.     

High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor. G protein interactions

The binding of the non-selective muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions, [3H]QNB bound to a homogenous population of muscarinic receptors (Kd, 118 +/- 19 pM; Bmax, 572 +/- 42 fmol/mg membrane protein, n = 12). The addition of G protein activators AlF4- or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) + Mg2+ increased the Kd by 77 +/- 7% (n = 4, P less than 0.05) and 83 +/- 27% (n = 7, P less than 0.05), respectively, without a change in the Bmax or homogeneity of the binding site. GTP gamma S added without exogenous Mg2+ did not affect [3H]QNB binding. Thus, optimal QNB binding requires a muscarinic receptor/G protein interaction.     

Direct evidence for GTP and GDP-Pi intermediates in microtubule assembly

Identification of the kinetic intermediates in GTP hydrolysis on microtubules and characterization of their assembly properties is essential in understanding microtubule dynamics. By using an improved glass filter assay that selectively traps microtubules with a dead time of 2 s and monitoring taxol-induced rapid assembly of microtubules from [gamma-32P,3H]GTP-tubulin 1:1 complex, direct evidence has been obtained for GTP- and GDP-Pi-microtubule transient states in the early stages of the polymerization process. A simple kinetic analysis of GTP hydrolysis on microtubules within two sequential pseudo-first-order processes led to apparent first-order rate constants of 0.065 s-1 for the cleavage of the gamma-phosphate and 0.02 s-1 for the liberation of Pi, assuming a simple random model. Apparent rate constants for GTP hydrolysis and Pi release were independent of the composition of the buffer used to polymerize tubulin. The significance of these values with respect to those derived from previous studies from this and other laboratories and the possibility of a vectorial model for GTP hydrolysis are discussed.     

Ionic and GTP regulation of binding of platelet-activating factor to receptors and platelet-activating factor-induced activation of GTPase in rabbit platelet membranes

Specific binding of 3H-labeled platelet-activating factor (PAF) to rabbit platelet membranes was found to be regulated by monovalent and divalent cations and GTP. At 0 degrees C, inhibition of [3H]PAF binding by sodium is specific, with an ED50 of 6 mM, while Li+ is 25-fold less effective. On the contrary, K+, Cs+, and Rb+ enhance the binding. The divalent cations, Mg2+, Ca2+, and Mn2+ enhance the specific binding 8-10-fold. From both Scatchard and Klotz analyses, the inhibitory effect of Na+ is apparently due to an increase in the equilibrium dissociation constant (KD) of PAF binding to its receptors. However, the Mg2+-induced enhancement of the PAF specific binding may be attributed to an increased affinity of the receptor and an increased availability of the receptor sites. In the presence of Na+, PAF receptor affinity decreased with increasing temperature with a 100-fold sharp discontinuous decrease in receptor affinity at 24 degrees C. In contrast, the Mg2+-induced increase is independent of temperature suggesting that the Mg2+ regulatory site is different from Na+ regulatory site. [3H]PAF binding is also specifically inhibited by GTP; other nucleotides have little effect. PAF also stimulates hydrolysis of [gamma-32P]GTP with an ED50 of 0.7 nM, whereas 3-O-hexadecyl-2-O-acetyl-sn-glyceryl-1-phosphorylcholine showed no activity even at 10 microM. Moreover, such stimulatory effect of PAF is dependent on Na+ and can be abolished by the PAF-specific receptor antagonist, kadsurenone, but not by an inactive analog, kadsurin B. These results suggest that the PAF receptor may be coupled with the adenylate cyclase system via an inhibitory guanine nucleotide regulatory protein.     

Stoichiometry for guanine nucleotide binding to tubulin under polymerizing and nonpolymerizing conditions

The maximal stoichiometry for [³H]GTP binding to depolymerized tubulin with saturating amounts of added [³H]GTP is 0.4 mol/110,000 g protein. In contrast, 1 mol of radioactive nucleotide is incorporated into microtubules as a result of polymerization with [³H]GTP. The different stoichiometries result from a difference in the nucleotide binding properties of ring protein under polymerizing and nonpolymerizing conditions: ring protein at 0 °C is devoid of binding activity but binds added radioactive guanine nucleotide during microtubule assembly. The radioactive nucleotide which is incorporated into rings during microtubule assembly is not displaced by excess GDP, although it is at a site which is distinct from the N site.