Dissociation of the tubulin dimer is extremely slow,thermodynamically very unfavorable,and reversible in the absence of an energy source

The finding that exchange of tubulin subunits between tubulin dimers (alpha-beta + alpha'beta' <--> alpha'beta + alphabeta') does not occur in the absence of protein cofactors and GTP hydrolysis conflicts with the assumption that pure tubulin dimer and monomer are in rapid equilibrium. This assumption underlies the many physical chemical measurements of the K(d) for dimer dissociation. To resolve this discrepancy we used surface plasmon resonance to determine the rate constant for dimer dissociation. The half-time for dissociation was approximately 9.6 h with tubulin-GTP, 2.4 h with tubulin-GDP, and 1.3 h in the absence of nucleotide. A Kd equal to 10(-11) M was calculated from the measured rate for dissociation and an estimated rate for association. Dimer dissociation was found to be reversible, and dimer formation does not require GTP hydrolysis or folding information from protein cofactors, because 0.2 microM tubulin-GDP incubated for 20 h was eluted as dimer when analyzed by size exclusion chromatography. Because 20 h corresponds to eight half-times for dissociation, only monomer would be present if dissociation were an irreversible reaction and if dimer formation required GTP or protein cofactors. Additional evidence for a 10(-11) M K(d) was obtained from gel exclusion chromatography studies of 0.02-2 nM tubulin-GDP. The slow dissociation of the tubulin dimer suggests that protein tubulin cofactors function to catalyze dimer dissociation, rather than dimer assembly. Assuming N-site-GTP dissociation is from monomer, our results agree with the 16-h half-time for N-site GTP in vitro and 33 h half-life for tubulin N-site-GTP in CHO cells.     

Microtubular protein reaction with nucleotides.

The dissociation constants for GTP and GDP with tubulin were determined to be equal to 1.1 ± 0.4 × 10^?7 M and 1.5 ± .6 × 10^?7 (4°), respectively. A lower limit for the dissociation constant for ATP was established as equal to 6 × 10^?4 M. The equivalent binding of GTP and GDP is not readily consistent with a mechanism in which the role of GTP in microtubule assembly is to bind to the protein to induce a conformation which is able to polymerize. An ATP-induced polymerization of tubulin apparently involves a transphosphorylation reaction in which GTP is formed and mediates the assembly. For this reaction to occur with desalted tubulin trace amounts of GDP are required; in the reaction of 0.1 mM ATP with 22.0 μM tubulin, 0.1 μM GDP induces about 80% as much tubule formation as is seen with 0.1 mM GTP alone.     

Polymerization and calcium binding of the tubulin-colchicine complex in the GDP state

The tubulin-colchicine complex instead of tubulin was used in an imidazole buffer throughout experiments. The interaction with calcium was examined, especially in the GDP state. The high affinity sites of calcium took part in the polymerization of the complex in the GTP state, while the low ones participated in the depolymerization. The complex had 2 high affinity sites with the dissociation constant of 11.5 x 10(-6) M, and 16 low affinity sites with the dissociation constant of 2.27 x 10(-4) M in the GTP state. In the case of GDP state, the dissociation constant of the high affinity site was 7.2 x 10(-6) M, and the low affinity site was not observed. The ultracentrifugal experiment indicated a little compact structure in the GTP state compared with the GDP state. This agreed with the results of calcium binding.     

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.     

Euglena gracilis chloroplast elongation factor Tu. Interaction with guanine nucleotides and aminoacyl-tRNA

The interaction of the chloroplast elongation factor Tu (EF-Tuchl) from Euglena gracilis with guanine nucleotides and aminoacyl-tRNA has been investigated. The apparent dissociation constant at 37 degrees C for the EF-Tuchl X GDP complex is about 3 X 10(-7) M and for the EF-Tuchl X GTP complex, it is about 1 order of magnitude higher. The sulfhydryl modifying reagent N-ethylmaleimide severely inhibits the polymerization activity of Euglena EF-Tuchl. In the presence of N-ethylmaleimide, the dissociation constant for the modified EF-Tuchl X GDP complex is increased by an order of magnitude. Conversely, both GDP and GTP protect EF-Tuchl from the modification. The polymerization activity of EF-Tuchl is also sensitive to the antibiotic kirromycin. In the presence of kirromycin, the apparent dissociation constant for the EF-Tuchl X GTP complex is lowered 10-fold. The interaction of aminoacyl-tRNA with EF-Tuchl was investigated by examining the ability of EF-Tuchl to prevent the spontaneous hydrolysis of Phe-tRNA and by gel filtration chromatography. The binding of aminoacyl-tRNA to EF-Tuchl occurs only in the presence of GTP indicating the formation of the ternary complex EF-Tuchl X GTP X Phe-tRNA. The effect of kirromycin on the interaction was also investigated. In the presence of kirromycin, no interaction between EF-Tuchl and Phe-tRNA is observed, even in the presence of GTP.     

Equilibrium studies of a fluorescent paclitaxel derivative binding to microtubules

A fluorescent derivative of paclitaxel, 3'-N-m-aminobenzamido-3'-N-debenzamidopaclitaxel (N-AB-PT), has been prepared in order to probe paclitaxel-microtubule interactions. Fluorescence spectroscopy was used to quantitatively assess the association of N-AB-PT with microtubules. N-AB-PT was found equipotent with paclitaxel in promoting microtubule polymerization. Paclitaxel and N-AB-PT underwent rapid exchange with each other on microtubules assembled from GTP-, GDP-, and GMPCPP-tubulin. The equilibrium binding parameters for N-AB-PT to microtubules assembled from GTP-tubulin were derived through fluorescence titration. N-AB-PT bound to two types of sites on microtubules (K(d1) = 61 +/- 7.0 nM and K(d2) = 3.3 +/- 0.54 microM). The stoichiometry of each site was less than one ligand per tubulin dimer in the microtubule (n(1) = 0.81 +/- 0.03 and n(2) = 0.44 +/- 0.02). The binding experiments were repeated after exchanging the GTP for GDP or for GMPCPP. It was found that N-AB-PT bound to a single site on microtubules assembled from GDP-tubulin with a dissociation constant of 2.5 +/- 0.29 microM, and that N-AB-PT bound to a single site on microtubules assembled from GMPCPP-tubulin with a dissociation constant of 15 +/- 4.0 nM. It therefore appears that microtubules contain two types of binding sites for paclitaxel and that the binding site affinity for paclitaxel depends on the nucleotide content of tubulin. It has been established that paclitaxel binding does not inhibit GTP hydrolysis and microtubules assembled from GTP-tubulin in the presence of paclitaxel contain almost exclusively GDP at the E-site. We propose that although all the subunits of the microtubule at steady state are the same "GDP-tubulin-paclitaxel", they are formed through two paths: paclitaxel binding to a tubulin subunit before its E-site GTP hydrolysis is of high affinity, and paclitaxel binding to a tubulin subunit containing hydrolyzed GDP at its E-site is of low affinity.     

Kinetics and thermodynamics of the interaction of elongation factor Tu with elongation factor Ts, guanine nucleotides, and aminoacyl-tRNA

The exchange of elongation factor Tu (EF-Tu)-bound GTP in the presence and absence of elongation factor Ts (EF-Ts) was monitored by equilibrium exchange kinetic procedures. The kinetics of the exchange reaction were found to be consistent with the formation of a ternary complex EF-Tu X GTP X EF-Ts. The equilibrium association constants of EF-Ts to the EF-Tu X GTP complex and of GTP to EF-Tu X EF-Ts were calculated to be 7 X 10(7) and 2 X 10(6) M-1, respectively. The dissociation rate constant of GTP from the ternary complex was found to be 13 s-1. This is 500 times larger than the GTP dissociation rate constant from the EF-Tu X GTP complex (2.5 X 10(-2) s-1). A procedure based on the observation that EF-Tu X GTP protects the aminoacyl-tRNA molecule from phosphodiesterase I-catalyzed hydrolysis was used to study the interactions of EF-Tu X GTP with Val-tRNAVal and Phe-tRNAPhe. Binding constants of Phe-tRNAPhe and Val-tRNAVal to EF-Tu X GTP of 4.8 X 10(7) and 1.2 X 10(7)M-1, respectively, were obtained. The exchange of bound GDP with GTP in solution in the presence of EF-Ts was also examined. The kinetics of the reaction were found to be consistent with a rapid equilibrium mechanism. It was observed that the exchange of bound GDP with free GTP in the presence of a large excess of the latter was accelerated by the addition of aminoacyl-tRNA. On the basis of these observations, a complete mechanism to explain the interactions among EF-Tu, EF-Ts, guanine nucleotides, and aminoacyl-tRNA has been developed.     

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.     

Preparation and characterization of nucleotide-free and metal ion-free p21 "apoprotein"

p21 isolated under nondenaturing conditions is obtained as a complex with guanosine nucleotides and magnesium ions. We have developed a high performance liquid chromatography method which removes greater than 95% of bound nucleotide and the metal ion very rapidly under mild conditions. At the same time, p21 is purified from minor protein impurities. The protein thus prepared is thermally much less stable than the complexed p21, but can be used for studying its interaction with nucleotides and metal ions at low temperatures. The association rate constant for p21 and GDP is 1.47 X 10(6) M-1 s-1 and for GTP is 2.9 X 10(6) M-1 s-1 at 0 degree C. By using appropriately determined dissociation rate constants we have determined the binding constant for p21.GDP and p21.GTP in the presence of excess Mg2+ to be 5.7 X 10(10) M-1 and 6.0 X 10(10) M-1, respectively, at 0 degree C.     

Interaction of 6-mercapto-GTP with bovine brain tubulin. Equilibrium aspects

In the presence of glycerol, the thionucleotide 2-amino-6-mercapto-9-ribofuranosyl purine 5'-triphosphate (S6-GTP) promotes the assembly of 6 S tubulin to form microtubules. Microtubules assembled with this analog show normal stability properties. In the absence of glycerol, few microtubules are formed with S6-GTP; however, many twisted ribbons are evident. Binding of S6-GTP to tubulin from which the associated proteins and exchangeable nucleotide have been removed (Tu(-] produces about a 16% quenching of intrinsic tubulin fluorescence. Fluorescence titrations indicate an apparent Kd for the tubulin S6-GTP complex of about 3 X 10(-8)M. Binding of S6-GTP to Tu(-) also produces a change in its absorption spectrum. The observed difference spectrum has a maximum at 350 nm and negative extrema at 323 and 338 nm. This suggests that the environment of the thioguanine ring is relatively hydrophobic. Competitive displacement studies yield apparent Kd values of about 1.7 X 10(-8)M for GTP and 8.3 X 10(-8) M for GDP. The changes in absorbance and fluorescence which accompany binding provide an excellent approach to the study of the kinetics and mechanisms of nucleotide binding as well as studies of the kinetics of displacement of GTP, GDP, and their analogs.     

Microtubule elongation and guanosine 5'-triphosphate hydrolysis. Role of guanine nucleotides in microtubule dynamics

The tubulin concentration dependence of the rates of microtubule elongation and accompanying GTP hydrolysis has been studied over a large range of tubulin concentration. GTP hydrolysis followed the elongation process closely at low tubulin concentration and became gradually uncoupled at higher concentrations, reaching a limiting rate of 35-40 s-1. The kinetic parameters for microtubule growth were different at low and high tubulin concentrations. Elongation of microtubules has also been studied in solutions containing GDP and GTP in variable proportions. Only traces of GTP present in GDP were necessary to confer a high stability (low critical concentration) to microtubules. Pure GDP-tubulin was found unable to elongate microtubules in the absence of GTP but blocked microtubule ends with an equilibrium dissociation constant of 5-6 microM. These data were accounted for by a model within which, in the presence of GTP-tubulin at high concentration, microtubules grow at a fast rate with a large GTP cap; the GTP cap may be quite short in the region of the critical concentration; microtubule stability is linked to the strong interaction between GTP and GDP subunits at the elongating site; dimeric GDP-tubulin does not have the appropriate conformation to undergo reversible polymerization. These results are discussed with regard to possible role of GDP and GTP and of GTP hydrolysis in microtubule dynamics.     

Studies on the mechanism of oxidative phosphorylation. ADP promotion of GDP phosphorylation

The process of ATP or GTP synthesis by bovine heart submitochondrial particles involves the binding of ADP or GDP to 3 exchangeable sites I, II, and III, and only upon substrate occupation of site III does rapid ATP or GTP synthesis take place. The dissociation constants determined for ADP were KADPI less than or equal to 10(-8) M, KADPII approximately 10(-7) M, and KADPIII (equivalent to apparent KADPm), approximately 3 x 10(-6) M in the low Km mode and KADPIII approximately 150 x 10(-6) M in the high Km mode. For GDP, these constants were KGDPI approximately 10(-6)-10(-5) M, KGDPII approximately 10(-4) M, and KGDPIII approximately 10(-3) M when NADH was the respiratory substrate (Matsuno-Yagi, A., and Hatefi, Y. (1990) J. Biol. Chem. 265, 82-88). Because of its low affinity for the above binding sites, GDP at micromolar concentrations does not lead to GTP synthesis. However, as shown in this paper, micromolar [GDP] undergoes phosphorylation in the presence of micromolar concentrations of ADP. Under these conditions, both ATP and GTP are synthesized. GDP inhibits ATP synthesis with KGDPi congruent to 7 microM, while ADP promotes GTP synthesis in a reaction that requires inorganic phosphate (apparent KPim = 2-3 mM) and is inhibited by uncouplers and inhibitors of the ATP synthase complex. The ADP-promoted GTP synthesis exhibited an "apparent" KGDPm = 4 microM and an "apparent" Vmax = 11 nmol of GTP (min.mg of protein)-1. These results were interpreted to mean that (a) micromolar [ADP] occupies sites I and II, allowing site III to bind and phosphorylate GDP, and (b) the KGDPm and Vmax calculated under these conditions represent values for the low Km-low Vmax mode of GTP synthesis, which in the absence of ADP is not detectable because of the positive cooperativity phase of GTP synthesis with the high KGDPII approximately 10(-4) M.     

Interaction of vinblastine with calf brain tubulin: multiple equilibria

The binding of the anticancer drug vinblastine to calf brain tubulin was measured by a batch gel filtration method in PG buffer (0.01 M NaPi, 10(-4) M GTP, pH 7.0) at three different protein concentrations. The Scatchard binding isotherms obtained were curvilinear. The binding of the first vinblastine molecule to each tubulin alpha-beta dimer (Mr 110,000) was enhanced by an increase in the protein concentration. Additional binding of vinblastine to the protein was independent of the protein concentration. Theoretical ligand binding isotherms were calculated for a ligand-induced macromolecule self-association involving various ligand stoichiometries and association schemes. Fitting of the experimental data to these isotherms showed that the system can be described best by a one-ligand-induced isodesmic indefinite self-association. The pathway giving the best fit consists of a ligand-mediated plus -facilitated self-association mechanism. The self-association-linked bound vinblastine binds specifically at a site with an intrinsic binding constant K1 = 4 X 10(4) M-1. Additional vinblastine molecules can bind less strongly to tubulin in probably nonspecific fashion, and the previous reports of two specific sites on alpha-beta tubulin for binding vinblastine are incorrect. The self-association constant K2 for liganded tubulin is 1.8 X 10(5) M-1. This analysis is fully consistent with the conclusions derived earlier from the linked function analysis of the vinblastine-induced tubulin self-association [Na, G. C., & Timasheff, S. N. (1980) Biochemistry 19, 1347-1354; Na, G. C., & Timasheff, S. N. (1980) Biochemistry 19, 1355-1365].     

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.     

Purification of angiotensin-converting enzyme from rabbit lung and human plasma by affinity chromatography

Lisinopril (N alpha-[(S)-1-carboxy-3-phenylpropyl]L-lysyl-L-proline), a potent angiotensin-converting enzyme inhibitor, is an exceptionally selective affinity chromatography ligand for this enzyme. Affinity chromatography furnishes electrophoretically homogeneous enzyme directly from crude homogenates of rabbit lung tissue, a 1,000-fold purification; also, it affords a 100,000-fold enrichment of the more rare human plasma enzyme in a single step. The affinity of angiotensin-converting enzyme for the Sepharose-spacer-lisinopril matrix (Ki matrix = 1 X 10(-5) M) is weak compared to its affinity for free lisinopril (Ki = 1 X 10(-10) M). The capacity of the affinity column is described quantitatively as a function of Ki matrix, lisinopril, and enzyme concentrations. The recovery of bound enzyme is low in chromatography of crude tissue samples (10-40%), although it approaches a reversible process (70-100%) with pure enzyme. The holoenzyme is converted to Zn2+-free apoenzyme to effect removal of lisinopril. In this process, the rate constant for spontaneous dissociation of Zn2+ from free enzyme is 1 X 10(-2) s-1 (t 1/2 = 1 min), which places a lower limit of 3 X 10(-10) M on the dissociation constant of Zn2+ at neutral pH from angiotensin-converting enzyme. The exceptional selectivity of lisinopril as an affinity chromatography ligand for angiotensin-converting enzyme suggests it is among the most specific inhibitors designed for any enzyme.     

Binding of colchicine to purified microtubule protein.

The binding of colchicine to tubulin, purified by two cycles of assembly-disassembly, has been studied. Equilibrium studies indicated a dissociation constant which declined during incubation approaching a minimum value of approximately 0.30 times 10- minus 6 M after 13 hours of incubation. Because tubulin is unstable during prolonged incubation (t1/2 of 5.2 hours for free tubulin, t1/2 of 12.5 hours for tubulin bound to colchicine), the equilibrium Kd was felt to be an overestimation of the true Kd. The rate constant of dissociation (k-1 equal to 0.009 hour- minus 1 hour- minus 1) and the rate constant of association (k1 equal to 0.37 times 10-6 M-minus 1) were measured under conditions designed to circumvent or correct for tubulin instability. The dissociation constant determined by the ratio k-1/k1 was 0.024 times -minus 6 M. To determine whether the discrepancy between the "equilibrium" and "kinetic" determined dissociation constants could be accounted for on the basis of tubulin instability, the binding reaction was computer-simulated using the measured association and dissociation rate constants and the rate constants for decay of bound and free tubulin. Computer simulation was in close agreement with the experimentally determined behavior of the reaction during a 13-hour incubation. It is concluded that the Kd determined by equilibrium methodology results in a considerable overestimation due to the instability of tubulin, and that the best estimate for the Kd of the colchicine-tubulin equilibrium is the value determined by the ratio of the rate constants.     

Effects of pH on tubulin-nucleotide interactions

Significant GTP-independent, temperature-dependent turbidity development occurs with purified tubulin stored in the absence of unbound nucleotide, and this can be minimized with a higher reaction pH. Since microtubule assembly is optimal at lower pH values, we examined pH effects on tubulin-nucleotide interactions. While the lowest concentration of GTP required for assembly changed little, GDP was more inhibitory at higher pH values. The amounts of exogenous GTP bound to tubulin at all pH values were similar, but the amounts of exogenous GDP bound and endogenous GDP (i.e., GDP originally bound in the exchangeable site) retained by tubulin rose as reaction pH increased. Endogenous GDP was more efficiently displaced by exogenous GTP than GDP at all pH values, but displacement by GTP was 10-15% greater at pH 6 than at pH 7. Dissociation constants for GDP and GTP were about 1.0 microM at pH 6 and 0.02 microM at pH 7. A small increase in the affinity of GDP relative to that of GTP occurs at pH 7 as compared to pH 6, together with a 50-fold absolute increase in the affinity of both nucleotides for tubulin at pH 7. The time courses of microtubule assembly and GTP hydrolysis were compared at pH 6 and pH 7. At pH 6, the two reactions were simultaneous in onset and initially stoichiometric. At pH 7, although the reactions began simultaneously, hydrolysis seemed to lag substantially behind assembly. Unhydrolyzed radiolabeled GTP was not incorporated into microtubules, however, indicating that GTP hydrolysis is actually closely coupled to assembly. The apparent lag in hydrolysis probably results from a methodological artifact rather than incorporation of GTP into the microtubule with delayed hydrolysis.     

A radioimmunoassay for human epidermal growth factor receptor

The development of a radioimmunoassay (RIA) for the human epidermal growth factor receptor solubilized with nonionic detergents which employs iodinated epidermal growth factor (125I-EGF) as the specific ligand is described. A monoclonal antibody (R1) that binds specifically to human EGF receptors [Waterfield, M. D., et al. (1982) J. Cell Biochem. 20, 149-161] was used to separate solubilized receptors saturated with 125I-EGF from free ligand by absorption to protein A-Sepharose, and the bound radioactivity was determined. The RIA was linear when increasing amounts of solubilized membrane protein were added and, when compared to the standard polyethylene glycol assay, was more reproducible. In addition, the background nonspecific binding obtained in the presence of a hundred-fold excess of unlabeled EGF was less in the RIA. Substitution of normal mouse serum for the monoclonal antibody gave very low nonspecific background ligand binding and avoided the use of large amounts of unlabeled EGF in the assay. Two major classes of binding sites for EGF were observed in membrane preparations from the cervical carcinoma cell line A431 or from normal human placental tissue. These were present in approximately equal amounts, with apparent dissociation constants of 4 X 10(-10) and 4 X 10(-9) M. Upon solubilization with the nonionic detergent Triton X-100, only one class of EGF binding sites was detected in both cases, with a dissociation constant of 3 X 10(-8) M. The RIA can be used to monitor receptor purification and for quantitation of receptor number and affinity in various cell types.     

The Caulobacter crescentus CgtA Protein Displays Unusual Guanine Nucleotide Binding and Exchange Properties

The Caulobacter crescentus CgtA protein is a member of the Obg-GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA specifically bound GTP and GDP but not GMP or ATP. CgtA bound GTP and GDP with moderate affinity at 30 degrees C and displayed equilibrium binding constants of 1.2 and 0.5 microM, respectively, in the presence of Mg(2+). In the absence of Mg(2+), the affinity of CgtA for GTP and GDP was reduced 59- and 6-fold, respectively. N-Methyl-3'-O-anthranoyl (mant)-guanine nucleotide analogs were used to quantify GDP and GTP exchange. Spontaneous dissociation of both GDP and GTP in the presence of 5 to 12 mM Mg(2+) was extremely rapid (k(d) = 1.4 and 1.5 s(-1), respectively), 10(3)- to 10(5)-fold faster than that of the well-characterized eukaryotic Ras-like GTP-binding proteins. The dissociation rate constant of GDP increased sevenfold in the absence of Mg(2+). Finally, there was a low inherent GTPase activity with a single-turnover rate constant of 5.0 x 10(-4) s(-1) corresponding to a half-life of hydrolysis of 23 min. These data clearly demonstrate that the guanine nucleotide binding and exchange properties of CgtA are different from those of the well-characterized Ras-like GTP-binding proteins. Furthermore, these data are consistent with a model whereby the nucleotide occupancy of CgtA is controlled by the intracellular levels of guanine nucleotides.     

Evidence for a distinct ligand binding site on tubulin discovered through inhibition by GDP of paclitaxel-induced tubulin assembly in the absence of exogenous GTP

GDP inhibits paclitaxel-induced tubulin assembly without GTP when the tubulin bears GDP in the exchangeable site (E-site). Initially, we thought inhibition was mediated through the E-site, since small amounts of GTP or Mg²⁺, which favors GTP binding to the E-site, reduced inhibition by GDP. We thought trace GTP released from the nonexchangeable site (N-site) by tubulin denaturation was required for polymer nucleation, but microtubule length was unaffected by GDP. Further, enhancing polymer nucleation reduced inhibition by GDP. Other mechanisms involving the E-site were eliminated experimentally. Upon finding that ATP weakly inhibited paclitaxel-induced assembly, we concluded that another ligand binding site was responsible for these inhibitory effects, and we found that GDP was not binding at the taxoid, colchicine, or vinca sites. There may therefore be a lower affinity site on tubulin to which GDP can bind distinct from the E- and N-sites, possibly on α-tubulin, based on molecular modeling studies.