The effects of elevated pH and high salt concentrations on tubulin

The effects of incubating phosphocellulose-purified bovine tubulin at 4 degrees C in nucleotide-free buffers at alkaline pH or at high concentrations of NaCl, KCl, (NH4)2SO4, or NH4Cl have been studied. At pH greater than or equal to 7.5 or at NaCl concentrations greater than or equal to 0.7 M, tubulin releases bound nucleotides irreversibly and loses, with apparent first-order kinetics, the ability to assemble into microtubules. In 0.1 M 1,4-piperazinediethanesulfonic acid buffer, pH 6.9, in the presence of 1.3 M NH4Cl, tubulin undergoes more rapid loss of capacity to assemble than it does in NaCl and KCl, but 1.3 M (NH4)2SO4 causes no detectable change in tubulin after 1-h incubation. Incubation at high pH or at high neutral salt concentrations also causes an apparently irreversible change in the ultraviolet difference spectrum and in the sedimentation velocity profile of tubulin. At elevated salt concentrations a decrease of approximately 10% in the molar ellipticity within the wavelength range 220-260 nm is observed. The changes that occur during 1-h exposure to pH 8.0 can be completely prevented by including 1 mM guanosine 5'-triphosphate (GTP) or 4 M glycerol in the buffer, but those which occur at pH 9.0 cannot be prevented by these additions. In 1 M NaCl when the ratio of bound guanine nucleotide to tubulin reaches approximately 1.0, tubulin loses the abilities to assemble into microtubules and to bind colchicine. The rate of loss of nucleotide in 2 M NaCl is decreased in the presence of 1 mM GTP, and tubulin is protected almost completely from 1 M NaCl-induced loss of GTP (and retains the ability to exchange [3H]GTP as well) in the presence of bound colchicine. Investigators who anticipate exposing tubulin to buffers of elevated pH or high concentrations of chaotropic salts should be extremely cautious in interpreting the resulting data unless they can demonstrate that irreversible alteration of the protein has not occurred.     

Effect of guanine nucleotides on the hydrophobic interaction of tubulin

The influence of guanine nucleotides on the binding of tubulin to hydrophobic components is investigated. Tubulin binds to a hydrophobic phenyl-Sepharose gel in a reversible, nucleotide-dependent way. Assembly-competent tubulin is released with ion-free water as eluent. It contains one guanosine triphosphate per dimer. More denatured tubulin needs a mixture of ethanol-water to elute. Consequently, hydrophobic interaction chromatography over phenyl-Sepharose represents an easy method for preparing polymerizable tubulin free of nucleotides at the exchangeable sites. While, in the absence of guanine nucleotide, the binding of tubulin to phenyl-Sepharose is rapid and immediately reversible on nucleotide addition, the binding of the nucleotide-dependent hydrophobic sites of tubulin to 1,8-ANS is slow, and its dissociation on nucleotide addition is poor. No differences are observed between the shielding of hydrophobic sites in the presence of GTP or GDP. Neither inorganic phosphate nor A1F4- is found to directly influence guanine nucleotides in their ability to shield hydrophobic sites.     

Relationship between tubulin SH groups and bound guanine nucleotides.

Guanine nucleotides bound to both the non-exchangeable sites (N sites) and exchangeable sites (E sites) of tubulin were completely released after 7 moles of SH groups per tubulin subunit (55,000 molecular weight) had reacted with PCMPS. The blockage of 2 moles of SH groups in the glycerol-reassembly buffer or 1 mole of SH groups in glycerol-free reassembly buffer resulted in complete loss of tubulin polymerizability. However, under both sets of experimental conditions, the amount of guanine nucleotides released from the E sites was less than 8% and the loss of total guanine nucleotides was only 5%. Addition of GSH did not induce reassociation of released guanine nucleotides, although it restored tubulin polymerizability. These results indicate that the loss of tubulin polymerizability on blockage of the SH groups was not due to dissociation of bound guanine nucleotides and that the binding sites of the nucleotides were independent of the SH groups in tubulin required for polymerization. Furthermore, blockage of SH groups did not change the ratio of GTP to GDP bound to tubulin.     

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)     

Assembly of pure tubulin in the absence of free GTP: effect of magnesium, glycerol, ATP, and the nonhydrolyzable GTP analogues

We describe in vitro microtubule assembly that exhibits, in bulk solution, behavior consistent with the GTP cap model of dynamic instability. Microtubules assembled from pure tubulin in the absence of free nucleotides could undergo one cycle of assembly, but could not sustain an assembly plateau. After the initial peak of assembly was reached and bound E-site GTP hydrolyzed to GDP, the microtubules gradually disassembled. We studied buffer conditions that maximized this disassembly while still allowing robust assembly to take place. While both glycerol and glutamate increased the rate of initial assembly and then slowed disassembly, magnesium promoted initial assembly and, surprisingly, enhanced disassembly. After cooling, a second cycle of assembly was unsuccessful unless GTP or the hydrolyzable GTP analogue GMPCPOP was readded. The nonhydrolyzable GTP analogues GMPPNP and GMPPCP could not support the second assembly cycle in the absence of E-site GTP. Analysis using HPLC found no evidence that GMPPNP, GMPPCP, or ATP could bind to free tubulin, and these nucleotides did not compete with GTP for the E-site. We have, however, demonstrated that the nonhydrolyzable GTP analogues and ATP do have an important effect on microtubule assembly. GMPPNP, GMPPCP, and ATP could each enhance the rate of assembly and stabilize the plateau of assembled microtubules against disassembly, while not binding appreciably to free tubulin. We conclude that these nucleotides, as well as GTP itself, enhance assembly by binding to a site on microtubules that is not present on free, unpolymerized tubulin. We estimate the affinity (KD) of the polymeric site for nucleotide triphosphates to be approximately 10(-4)M.     

Characterization of [3H]Guanine Nucleotide Binding Sites in Brain Membranes

[3H]GTP [guanosine triphosphate] and [3H]GMP-PNP [guanosine 5'-(beta, 8-imino)triphosphate, a nonmetabolized analog of GTP] have been utilized as ligands to characterize binding sites of guanine nucleotides to rat brain membranes. Binding of both [3H]GTP and [3H]GMP-PNP is saturable, with respective KD values of 0.76 and 0.42 microM. The number of binding sites for GMP-PNP (4 nmol/g) is three times greater than for GTP (1.5 nmol/g). This discrepancy is caused by rapid degradation of GTP to guanosine by brain membranes, which can be partially prevented by addition of 100 microM-ATP. The binding of [3H]guanine nucleotides is selective, with approximately equipotent inhibition by GTP, GDP, and GMP-PNP (at 0.2--1.0 microM), but no inhibition by other nucleotides at 100 microM concentrations. The bindings sites for guanine nucleotides in brain membranes appear not to be associated with microtubules, since treatments that reduce [3H]colchicine binding by 65% have no effect on [3H]GTP binding. [3H]Guanine nucleotide binding is widely distributed in various organs, with highest levels in liver and brain and lowest levels in skeletal muscle. The characteristics of these binding sites in brain show specificity properties of sites that regulate neurotransmitter receptors and adenylate cyclase.     

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.     

A second guanyl nucleotide-binding site associated with adenylate cyclase. Distinct nucleotides activate adenylate cyclase and permit ADP-ribosylation by cholera toxin

There are two functionally and physically distinct types of guanyl nucleotide site associated with the adenylate cyclase system of pigeon erythrocytes. One is on the well known regulatory protein, N, that mediates the adenylate cyclase response to hormones, guanyl nucleotides and fluoride, and is the substrate for ADP-ribosylation by cholera toxin. We now describe a second site that must be occupied by GTP or an analog of GTP before N can be ADP-ribosylated. We call this second site S. It differs from the site on N in many respects. GTP appears to be rapidly hydrolyzed when it is bound to N but not when bound at S. GTP analogs such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) bind stably to both sites but the binding of GTP gamma S to N is more sensitive to EDTA and is more easily prevented by guanosine 5'-O-(2-thiodiphosphate). The nucleotide binding only to S is promoted by the cytosolic protein required by cholera toxin. Isoproterenol decreases GTP gamma S binding to S while indirectly increasing GTP gamma S binding to N. By adjusting the binding conditions, the nucleotides bound functionally to N and S can be varied independently and then the effect of ADP-ribosylation upon the adenylate cyclase activity can be seen to depend on the type of nucleotide bound to N. This activity rises, falls slightly, or remains at zero, if N is occupied by GTP, GTP gamma S, or guanosine 5'-O-(2-thiodiphosphate, respectively.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

Head-to-tail polymerization of microtubules in vitro

The kinetics of microtubule polymerization to steady-state and the ability of tubulin subunits to exchange with polymer at steady-state were examined to determine the applicability of the head-to-tail polymerization mechanism (Wegner, 1976) to microtubule assembly in vitro. Under conditions where self-nucleation was a rare event, tubulin was induced to polymerize by the addition of short microtubule fragments, and the kinetics of elongation were analyzed as a pseudofirst-order reaction. At steady-state, a trace amount of [³H]tubulin, prepared by labeling in vivo of chick brain protein, was added to polymerized microtubules and the kinetics of label uptake into polymer were monitored by a rapid centrifugal assay. The isotope exchange kinetics were analyzed according to a theoretical model previously applied to actin polymerization (Wegner, 1976) and extended for the case of microtubule polymerization. The rate of head-to-tail polymerization, expressed as the steady-state subunit flux, was 27·6 ± 7·6 per second at 37 °C. The head-to-tail parameter s, a measure of the efficiency of subunit flux, was 0·26 ± 0·07, indicating that four association and four dissociation events resulted in the flux of one subunit through the polymer at steady-state.The role of GTP in this mechanism of microtubule polymerization was examined by replacement of the nucleotide occupying the exchangeable binding site of tubulin with the non-hydrolyzable GTP analog guanosine 5′-(β,γ-methylene)triphosphate. It was found that the rate of steady-state flux was reduced by two orders of magnitude compared to tubulin polymerized with GTP. The head-to-tail parameter approached its limiting value of zero, indicating greatly reduced efficiency of subunit flux through the polymer in the presence of this analog.In summary, this study demonstrates that microtubules exhibit significant headto-tail polymerization in the presence of GTP and, in keeping with theoretical considerations, provides evidence that nucleotide hydrolysis is required for subunit flux through the polymer.     

Purification of tubulin from bovine brain and its interaction with guanine nucleotides.

A rapid and sensitive assay for [3H]GTP binding activity of tubulin has been developed. This assay method is based on the quantitative retention of [3H]GTP. Tubulin complex on a nitrocellulose membrane filter. It was also found that bovine brain tubulin is markedly stablized by glycerol and GTP against denaturation. A large-scale purification of bovine brain tubulin was achieved using the new assay procedure and by the inclusion of glycerol and GTP in a buffer solution used for column chromatograph. The purified tubulin could be stored at -80degrees in the presence of glycerol and GTP for at least a year without any apprecialbe loss of [3H]GTP- and [3H]colchicine binding activities. The interaction of tubulin with guanine nucleotides was also studied using the nitorcellulose membrane filter procedure. It was found that the binding of [3H]GTP to tubulin with an empty exchangeable site proceeded promptly within k sec while the exchange of [3H]GTP- with a GTP-tubulin complex in which the exchangeable site had been occupied with unlabeled GTP occured more slowly. The dissociation constants for GTP and GDP at the exchangeable site of tubulin were determined as 0.5 times 10-6M and 1.9 times 10-6M, respectively. 5'-Guanylylimidodiphosphate could interact, although less strongly, with tubulin at this site, whereas the interaction of other nucleoside triphosphates includint ATP, CTP, UTP, and 5'-guanylyl methylenediphosphonate was very weak, if it occured at all. The presence of Mg2+ and a free sulfhydryl group was found to be essential for binding of [3H]GTP to tubulin. Ca2+ was found to replace Mg2+ in this binding reaction.     

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)     

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.     

Divalent cation-nucleotide complex at the exchangeable nucleotide binding site of tubulin

Tubulin was first treated with alkaline phosphatase-agarose to vacate the exchangeable nucleotide binding site and then tested for manganese binding sites by Mn(II) EPR. Buttlaire et al. ((1980) J. Biol. Chem. 255, 2164-2168) have shown that high affinity manganese binding occurs at a single site normally occupied by magnesium. We report that the number of high affinity manganese binding sites per mol of tubulin depends on the number of occupied exchangeable nucleotide binding sites. Thus, removal of nucleotides results in a loss of high affinity manganese binding sites. The EPR spectra of manganese bound to tubulin and to GTP are found to be qualitatively similar. These data indicate that high affinity manganese binding is the result of the formation of a metal-nucleotide complex at the exchangeable nucleotide binding site. In addition it was found that zinc, cobalt, and magnesium bind with approximately equal affinity to this site whereas calcium binds only weakly.     

Nucleoside triphosphate specificity of tubulin

We have determined the binding affinity for binding of the four purine nucleoside triphosphates GTP, ITP, XTP, and ATP to E-site nucleotide- and nucleoside diphosphate kinase-depleted tubulin. The relative binding affinities are 3000 for GTP, 10 for ITP, 2 for XTP, and 1 for ATP. Thus, the 2-exocyclic amino group in GTP is important in determining the nucleotide specificity of tubulin and may interact with a hydrogen bond acceptor group in the protein. The 6-oxo group also makes a contribution to the high affinity for GTP. NMR ROESY experiments indicate that the four nucleotides have different average conformations in solution. ATP and XTP are characterized by a high anti conformation, ITP by a medium anti conformation, and GTP by a low anti conformation. Possibly, the preferred solution conformation contributes to the differences in affinities. When the tubulin E-site is saturated with nucleotide, there appears to be little difference in the ability of the four nucleotides to stimulate assembly. The critical protein concentration is essentially identical in reactions using the four nucleotides. All four of the nucleotides were hydrolyzed during the assembly reaction, and the NDPs were incorporated into the microtubule. We also examined the binding of two gamma-phosphoryl-modified GTP photoaffinity analogues, p(3)-1, 4-azidoanilido-GTP and p(3)-1,3-acetylanilido-GTP. These analogues are inhibitors of the assembly reaction and bind to tubulin with affinities that are 15- and 50-fold lower, respectively, than the affinty for GTP. The affinity of GTP is less sensitive to substitutions at the gamma-phosphoryl position that to changes in the purine ring.     

Escherichia coli FtsZ polymers contain mostly GTP and have a high nucleotide turnover

The cell division protein FtsZ is a GTPase structurally related to tubulin and, like tubulin, it assembles in vitro into filaments, sheets and other structures. To study the roles that GTP binding and hydrolysis play in the dynamics of FtsZ polymerization, the nucleotide contents of FtsZ were measured under different polymerizing conditions using a nitrocellulose filter-binding assay, whereas polymerization of the protein was followed in parallel by light scattering. Unpolymerized FtsZ bound 1 mol of GTP mol(-1) protein monomer. At pH 7.5 and in the presence of Mg(2+) and K(+), there was a strong GTPase activity; most of the bound nucleotide was GTP during the first few minutes but, later, the amount of GTP decreased in parallel with depolymerization, whereas the total nucleotide contents remained invariant. These results show that the long FtsZ polymers formed in solution contain mostly GTP. Incorporation of nucleotides into the protein was very fast either when the label was introduced at the onset of the reaction or subsequently during polymerization. Molecular modelling of an FtsZ dimer showed the presence of a cleft between the two subunits maintaining the nucleotide binding site open to the medium. These results show that the FtsZ polymers are highly dynamic structures that quickly exchange the bound nucleotide, and this exchange can occur in all the subunits.     

Guanine nucleotide regulation of VIP binding to rat peritoneal macrophage membranes.

In the present study, we have examined the effect of guanine nucleotides on VIP binding to rat peritoneal macrophage membranes. Both guanosine 5'-triphosphate (GTP) and its nonhydrolizable analog guanosine 5'-beta, Y-imidotriphosphate [Gpp(NH)p] inhibited, in a dose-dependent manner, the VIP binding to its specific binding sites. Half-maximal inhibition (IC50) was observed at 5.4 +/- 0.5 microM GTP. The inhibitory effect of GTP was due to an increase of the dissociation rate of peptide bound to membranes. The specificity of the binding inhibition was assessed from the lack of action of the other nucleotides tested. These results directly suggest the coupling of VIP binding sites with guanine nucleotide binding proteins in rat peritoneal macrophage membranes.     

Guanosine 5'-O-(3-thiotriphosphate), a potent nucleotide inhibitor of microtubule assembly

Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and the two diastereoisomers of guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) were prepared enzymatically, and their interactions with tubulin and microtubule-associated proteins (MAPs) in 0.1 M 2-(N-morpholino)ethanesulfonate, 0.5 mM MgCl2 were examined. GTP gamma S did not support microtubule assembly but instead inhibited the reaction. This analog was 1.5-2 times more potent than GDP in inhibiting both tubulin polymerization and GTP hydrolysis under conditions in which these reactions were dependent on MAPs. In contrast to the analog's inhibitory effects on polymerization and hydrolysis, however, radiolabeled GTP gamma S was only feebly bound by purified tubulin at 0 degrees C relative to the binding of GDP and GTP. There was a marked increase in the amount of GTP gamma S bound when the reaction temperature was raised to 37 degrees C or when MAPs were included in the reaction mixture. Only when both MAPs were present and the higher reaction temperature was used did the binding of GTP gamma S exceed that of GDP. Since substitution of sulfur for oxygen in a molecule should decrease its hydrophilic properties, these findings suggest that the exchangeable nucleotide binding site of tubulin becomes more hydrophobic at higher temperatures and in the presence of MAPs. The two isomers of GTP beta S were able to support MAP-dependent polymerization, although a 50-100-fold higher concentration of the analogs as compared to GTP was required. Neither isomer of GTP beta S had a significant inhibitory effect on GTP hydrolysis dependent on tubulin + MAPs.     

Annexin proteins PP4 and PP4-X. Comparative characterization of biological activities of placental and recombinant proteins.

Several G-proteins (GTP-binding proteins) were identified by SDS/PAGE in the cytosol (105,000 g supernatant) and membrane fractions of the oestrogen-dependent human mammary-tumour cell line ZR-75-1. These proteins, with molecular masses in the range 18-29 kDa, specifically bind [alpha-32P]GTP, which can be displaced by unlabelled GTP, GDP and their non-hydrolysable analogues guanosine 5'-[delta-thio]triphosphate (GTP[S]) and guanosine 5'-[beta-thio]diphosphate (GDP[S]), but not by GMP, ATP, ADP, AMP and other unrelated nucleotides. The apparent dissociation constant for GTP was approx. 2 x 10(-8)M. Homogenization of ZR-75-1 cells in high-salt buffer (1 M-KCl), and successive washing of the membrane fraction, suggested that, among the major G-proteins found, the 18 kDa protein is predominantly soluble, whereas the 27-29 kDa complex is primarily bound to the membrane fraction under the experimental conditions employed. Possible translocation of these G-proteins between membrane and cytosol was analysed. No redistribution of the 27-29 kDa complex was observed, whereas GTP[S] in the presence of Mg2+ caused apparent translocation of the 18 kDa protein to the membrane fraction. This effect was specific for GTP and stable GTP analogues, whereas GDP, GMP, ATP, ADP, AMP and other unrelated nucleotides were ineffective. GTP[S] and guanosine 5'-[beta gamma-imido]-triphosphate (p[NH]ppG) were equally potent (apparent Kd approximately 5 x 10(-6)M), whereas GTP was rather weak. The nucleotide effect is temperature-, time- and concentration-dependent. The translocation process was reversible, slow, and reached its maximum between 30 and 60 min at 37 degrees C. The apparent translocation of this small G-protein from the cytosol to the membrane fraction, and the specific effect of GTP analogues, suggest that this process may have functional significance in mammary-tumour cells.     

Porphyrins affect the self-assembly of tubulin in solution

Self-assembly of tubulin heterodimers in solution has been studied in the past to predict the effects that ligands and/or conformational changes have on the formation of tubulin filaments. Self-assembly of tubulin in solution has produced formations similar to cellular microtubules (MTs). The present study reports on the effects that two porphyrins (protoporphyrin IX, PPIX and tetrakis(4-sulfonatophenyl)porphyrin, TPPS) produce on the self-assembly of tubulin α,β-heterodimers in buffer solution. The study shows that, when incubated simultaneously with MT-stabilizing ligands (i.e., paclitaxel and guanosine triphosphate, GTP), porphyrins do not affect the ability of tubulin to form MT. However, if paclitaxel and GTP are added after tubulin has been allowed to self-assemble in the presence of either porphyrin, the ability to form MT-like structures is reduced or suppressed. We suggest that this effect is due to the formation of porphyrin-mediated aggregates that cannot be broken or elongated by the addition of GTP or paclitaxel.