Kinetic analysis of the binding of guanine nucleotides to bovine brain rhoB p20, a ras p21-like GTP-binding protein

We have investigated the kinetics of the binding of guanine nucleotides to bovine brain rhoB p20, a ras p21-like GTP-binding protein with GTPase activity. The initial velocities of the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to GDP-bound rhoB p20 and the dissociation of GDP from this protein were markedly increased by decreasing Mg2+ concentrations. The initial velocity of the binding of GTP gamma S to GDP-free rhoB p20 was not affected by changing Mg2+ concentrations. These results indicate that the dissociation of GDP from rhoB p20 limits the binding of GTP to this protein, and suggest that there is a factor stimulating the dissociation of GDP from rhoB p20 and thereby stimulating the binding of GTP to this protein in mammalian tissues. Consistently, the factor stimulating the dissociation of GDP, but not of GTP gamma S, from rhoB p20 was detected in bovine brain cytosol.     

Molecular cloning of smg p21B and identification of smg p21 purified from bovine brain and human platelets as smg p21B

We have previously purified smg p21 from bovine brain membranes and isolated its cDNA from a bovine brain cDNA library. In the present studies, we have performed extensive screening of the bovine brain cDNA library with the cloned smg p21 cDNA as a probe and isolated another cDNA encoding a protein highly homologous to smg p21. The proteins encoded by the previously and newly isolated cDNAs are designated as smg p21A and -B, respectively. Since the partial amino acid sequences determined previously from the smg p21 purified from bovine brain were identical with the common amino acid sequences between smg p21A and -B, we have further sequenced smg p21 and identified it as smg p21B. We have also further sequenced the smg p21 purified from human platelet membranes and identified it as smg p21B. Amino acid sequence analysis indicates that smg p21A is identical with the rap1A and Krev-1 proteins and smg p21B is identical with the rap1B protein.     

Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein

A novel regulatory protein for smg p25A, a ras p21-like GTP-binding protein, was purified to near homogeneity from bovine brain cytosol. This regulatory protein, designated here as smg p25A GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP, but not of guanosine 5'-(3-O-thio)triphosphate (GTPgamma S), from smg p25A. smg p25A GDI also inhibited the binding of GTPgamma S to the GDP-bound form of smg p25A but not of that to the guanine nucleotide-free form. GDI did not stimulate the GTPase activity of smg p25A and by itself showed neither GTPgammaS-binding nor GTPase activity. GDI was inactive for other ras p21/ras p21-like GTP-binding proteins including c-Ha-ras p21, rhoB p20, and smg p21. The Mr value of GDI was estimated to be about 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, about 65,000 from the S value (4.5 S), and about 82,000 by gel filtration. The isoelectric point of GDI was about pH 5.6. The activities of GDI were killed by tryptic digestion or heat boiling. These results indicate that bovine brain cytosol contains a regulatory protein for smg p25A that inhibits the dissociation of GDP from and thereby the subsequent binding of GTP to this protein.     

Localization and subcellular distribution of smg p25A, a ras p21-like GTP-binding protein, in rat brain

We have made a monoclonal antibody which specifically recognizes smg p25A among many ras p21/ras p21-like GTP-binding proteins thus far purified from bovine brain membranes. By use of this antibody, we have investigated the localization and subcellular distribution of smg p25A in rat brain by light and electron microscopic immunocytochemistry and by immunoblotting. By light microscopic immunocytochemistry, specific immunoreactivity is widely distributed, most abundant in neuropil, weak in neuronal somata, and absent from white matter. By electron microscopic immunocytochemistry, intense labeling is demonstrated on most of the synapses and concentrated in the presynaptic area where synaptic vesicles are observed. Presynaptic plasma membranes are weakly labeled but mitochondria, postsynaptic plasma membranes, and postsynaptic densities are unlabeled. In subcellular fractionation analysis of cerebrum, about one-fifth of smg p25A is found in the soluble cytosol fraction and the rest is found in the particulate fraction. About half of the particulate-bound smg p25A is recovered in the P2 fraction containing synaptosomes, mitochondria, and myelin, among which a major portion of smg p25A is recovered in the synaptosomal fraction. In the synaptosomal fraction, smg p25A is concentrated about 8-fold in the fraction containing synaptic vesicles and about 3-fold in the fraction containing synaptic plasma membranes compared with the original homogenate. smg p25A is present at a low level in the fraction containing synaptosomal soluble substances but almost absent from the fractions containing intrasynaptosomal mitochondria or post-synaptic densities. These results suggest that smg p25A plays important roles in the regulation of synaptic functions such as exo-endocytotic recycling of synaptic vesicles during neurotransmitter release.     

Chemical modification of bovine brain tubulin with the guanine nucleotide affinity analogue 5'-p-fluorosulfonylbenzoylguanosine

Bovine brain tubulin purified in the absence of GTP and MgCl2, reacts with 5'-p-fluorosulfonylbenzoylguanosine (5'-FSBG)2, an affinity analog of GTP and two moles of the reagent are incorporated per mole of tubulin at 0 degree C. 5'-FSBG is unable to promote the polymerization of tubulin into microtubules. 2 mM GTP, podophyllotoxin and vinblastine provide almost 50% protection against the modification, when added individually. Combination of these ligands gives maximal protection. Tubulin modified with 5'-FSBG lost two sulfhydryl groups per mole of tubulin and reduction with beta-mercaptoethanol led to the loss of the 2 moles of FSBG that had been incorporated. These data are interpreted on the basis that the modification of tubulin by 5'-FSBG proceeds via a thiosulfonate intermediate between the analogue and a reactive thiol group at or near that portion of the GTP binding site of tubulin where the phosphate moiety of GTP binds.     

Regulation of follicle-stimulating hormone binding to receptors on bovine calf testis membranes by cholera toxin-sensitive guanine nucleotide binding protein

In a previous study we reported that FSH receptors in bovine testes membranes are physically and functionally associated with a guanine nucleotide-binding protein (N protein). In this study we examined the mechanism whereby GTP binding to N protein regulates FSH binding to its receptors. Binding of FSH to receptors decreased in the presence of GTP in a dose-dependent and noncompetitive manner. This effect did not require the presence of Mg+2 and is in contrast to the reported requirement for Mg+2 for GTP effects on human CG binding to ovarian receptors. Equilibrium binding experiments indicated that decreased hormone binding in the presence of GTP was not due to a decrease in the number of FSH receptors per se; rather, the altered binding isotherm was the result of a decrease in affinity of receptors for FSH. Moreover, the dissociation of [125I]human FSH from preformed FSH-receptor complex was rapid in onset and significantly accelerated in the presence of GTP. In a series of nucleotides, GTP was most effective in causing this effect. Evidently, occupancy of GTP binding sites on the N protein, including low affinity and high capacity sites, is necessary for GTP regulation of FSH binding to receptors. The fact that pretreatment of bovine testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminates the GTP effect on FSH binding to its receptors suggests that the GTP regulatory binding protein mediating the GTP regulation of FSH binding is probably Ns and not Ni. Further characterization of FSH receptor sensitivity to GTP, however, indicated that the N protein involved does not exhibit all of the characteristics reported for Ns. For example, the affinity of GTP for N protein is relatively low even under conditions where GTP hydrolysis has a minimal effect in reducing the total concentration of GTP. Also, the absence of a requirement for Mg+2 in high affinity FSH receptor-N protein coupling is different from the requirement for Mg+2 seen with the beta-adrenergic receptor and Ns. Moreover, the N protein which mediates GTP regulation of FSH-receptor binding appears to be relatively insensitive to N-ethylmaleimide, unlike the N-ethylmaleimide sensitivity of the turkey erythrocyte Ns. These results suggest that differences may exist in the structure-function features of GTP regulatory binding protein associated with different types of hormone ligands and receptors.     

cGMP influences guanine nucleotide binding to frog photoreceptor G-protein

A rapid light-induced decrease in cGMP is thought to play a role in regulating the permeability or light sensitivity of photoreceptor membranes. Photo-excited rhodopsin activates a guanine nucleotide-binding protein (G-protein) by catalyzing the exchange of bound GDP for GTP. This G-protein X GTP complex activates the phosphodiesterase resulting in a decrease in cGMP concentration. We have observed two processes in vitro which may be relevant for the regulation of G-protein activation. First, we have found that free GDP binds to G-protein with an affinity similar to that of GTP. These two nucleotides appear to compete for a common site. Since G-protein X GDP does not activate phosphodiesterase, light-induced changes in the GTP/GDP ratio known to occur on illumination may serve to reduce G-protein activation and hence reduce phosphodiesterase activation. Second, addition of cGMP in the presence of equimolar GTP and GDP causes GTP binding to G-protein to be enhanced compared to GDP binding. This effect increases as the cGMP concentration is increased from 0.05 to 2 mM. Thus, light-induced decreases in cGMP concentration may also act as a feedback control in reducing G-protein activation. One or both of these processes may be involved in the desensitization (light adaptation) of rod photoreceptors.     

Effect of Mg2+ on guanine nucleotide sensitivity of ligand binding to serotonin1A receptors from bovine hippocampus

The serotonin1A (5-HT1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein coupled receptors (GPCRs). We report here that guanine nucleotide sensitivity of agonist binding to hippocampal 5-HT1A receptors is dependent on the concentration of Mg2+. Our results show that agonist binding to 5-HT1A receptors is relatively insensitive to guanine nucleotides in the absence of Mg2+. In contrast to this, the specific antagonist binding is insensitive to guanine nucleotides, even in the presence of Mg2+. These results point out the requirement of an optimal concentration of Mg2+ which could be used in assays toward determining guanine nucleotide sensitivity of ligand binding to GPCRs such as the 5-HT1A receptor. Our results provide novel insight into the requirement and concentration dependence of Mg2+ in relation to guanine nucleotide sensitivity for the 5-HT1A receptor in particular, and GPCRs in general.     

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.     

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin.

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.     

Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

Prostaglandin E2 (PGE2) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet (Negishi, M., Ito, S., Tanaka, T., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1987) J. Biol. Chem. 262, 12077-12084). In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, we purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its alpha subunit from two known pertussis toxin substrate G-proteins (Gi and Go) purified from bovine brain. The molecular weight of the alpha subunit was 40,000, which is between those of Gi and Go. The purified protein was also distinguished immunologically from Gi and Go and was referred to as Gam. PGE receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and freed from G-proteins by wheat germ agglutinin column chromatography. Reconstitution of the PGE receptor with pure Gam, Gi, or Go in phospholipid vesicles resulted in a remarkable restoration of [3H]PGE2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. The displacement of [3H]PGE2 binding was specific for PGE1 and PGE2. Furthermore, addition of PGE2 stimulated the GTPase activity of the G-proteins in reconstituted vesicles. These results indicate that the PGE receptor can couple functionally with Gam, Gi, or Go in phospholipid vesicles and suggest that Gam may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.     

Two forms of the bovine brain Go that stimulate the inositol trisphosphate-mediated Cl- currents in Xenopus oocytes. Distinct guanine nucleotide binding properties.

Heterotrimeric GTP-binding proteins from bovine brain were resolved by fast protein liquid chromatography chromatography using Mono Q columns. Two distinct forms of the protein Go were identified. Both forms had stochiometric amounts of alpha- and beta gamma-subunits. The a-subunits of both forms were recognized by an alpha o-specific antiserum, but not by any of the alpha i-specific antisera. The two forms showed distinct migration patterns on 9% sodium dodecyl sulfate-polyacrylamide gels containing 4-8 M urea gradients. Neither form comigrated with the recombinant alpha o1. Both the recombinant alpha o1 and the most abundant form of Go were recognized by an antiserum, H-660, against a peptide encoding amino acids 3-17 of alpha i2. H-660 has been shown previously to recognize alpha o and alpha i (Mumby, S. M., Pang, I. K., Gilman, A. G., and Sternweis, P. C. (1988) J. Biol. Chem. 263, 2020-2026). This more abundant form is called Go A most likely corresponds to the cloned alpha o1. The less abundant form, Go B, was not recognized by H-660. However, both forms of bovine brain Go were recognized by GC/2, an antiserum against the N-terminal region of alpha o1. Hence alpha oA and alpha oB may be different in their N terminus regions. Neither form of bovine brain Go was recognized by an antisera made to a peptide encoding the unique regions of the cloned alpha o2 from HIT cells (Hsu W. H., Rudolph, U., Sanford, J., Bertrand, P., Olate, J., Nelson, C., Moss, L.E., Boyd, A. E., III, Codina, J., and Birnbaumer, L. (1990) J. Biol. Chem. 265, 11220-11226). Go A and Go B have similar guanine nucleotide binding and release properties. Both release GDP within 1 min in the absence of added Mg2+. Both bind guanosine (GTP gamma S) rapidly as well. However Go A binds GTP gamma S about 2.5-fold faster than Go B, in the absence of added Mg2+ ion. Both forms of Go as well as the recombinant alpha o (alpha o1) can increase muscarinic stimulation of inositol trisphosphate-mediated Cl- current in Xenopus oocytes. These data indicate that we have identified two structurally distinct forms of Go that have different guanine nucleotide binding properties and are capable of functioning in the receptor-regulated phospholipase C pathway in Xenopus oocytes.     

Photo-cross-linking of guanine nucleotides to the nucleotide binding site of elongation factor G

Elongation factor G (EF-G) is rapidly inactivated when irradiated at 253.7 nm. The inactivation follows first-order single-hit kinetics with a quantum efficiency of 3.15 × 10^?5 μmol/μE. Inclusion of either GTP or GDP in the irradiation mixture does not alter the kinetics of inactivation, but does result in the covalent attachment of nucleotide to between 10 and 20% of the EF-G. This relatively low percentage of cross-linking is due to the rapid rate of photoinactivation as compared to the slower rate of covalent attachment. If EF-G is reacted before irradiation with N-ethylmaleimide, a modification known to block the nucleotide binding site [Rohrbach and Bodley (1976) J. Biol. Chem.251, 930], essentially no nucleotide can be photo-cross-linked to EF-G. Treatment of the photo-cross-linked GTP-EF-G with Raney nickel led to the liberation of the nucleotide moiety, indicating that the photo-cross-link to EF-G occurred through a sulfur atom. Although the formation of the EF-G nucleotide complex has been shown to be an obligatory first step in the formation of the EF-G nucleotide ribosome complex [Rohrbach and Bodley (1976) Biochemistry15, 4565], the covalent EF-G-nucleotide adduct cannot form a ternary complex with the ribosome. The presence of both nucleotide and ribosomes during irradiation drastically alters the kinetics of inactivation. The inactivation under these conditions follows multiple-hit kinetics with an initial period during which no EF-G activity is lost. Following this lag period, EF-G is inactivated at the same rate at which ribosomes lose their ability to bind EF-G. No nucleotide is cross-linked to EF-G or the ribosome under these conditions.     

Kinetic analysis of calcium binding to concanavalin A

The kinetics of calcium binding to concanavalin A was studied utilizing ultraviolet difference spectral measurements. The results show that calcium binds to the lectin in a biphasic process: a rapid and reversible phase, followed by a relaxation phase with a k_obs of 0.012 sec^?1. Kinetic measurements were used to calculate the association constant, K_a, for calcium binding to concanavalin A of 2.7 x 10⁴ M^?1, in reasonable agreement with values obtained by equilibrium methods.     

Localization of smg p25A/rab3A p25, a small GTP-binding protein, at the active zone of the rat neuromuscular junction.

smg p25A is a small G protein which has been suggested to regulate neurotransmitter release from the synapses. We investigated here the ultrastructural localization of this small G protein in the rat neuromuscular junction by an immunoperoxidase method. The results showed that smg p25A was distributed non-uniformly on the presynaptic plasma membrane and among the synaptic vesicles with the focal accumulation on the discrete presynaptic sites which corresponded to the active zones, the regions of the presynaptic plasma membrane specialized for the exocytosis of the synaptic vesicles. This unique distribution of smg p25A suggests that it plays an important role in the attachment and fusion of the synaptic vesicles with the active zones.     

A Drosophila homolog of bovine smg p25a GDP dissociation inhibitor undergoes a shift in isoelectric point in the developmental mutant quartet.

The Drosophila developmental mutation quartet causes late larval lethality and small imaginal discs and, when expressed in the adult female, has a lethal effect on early embryogenesis. These developmental defects are associated with mitotic defects, which include a low mitotic index in larval brains and incomplete separation of chromosomes in mitosis in the early embryo. quartet mutations also have a biochemical effect, i.e., a basic shift in isoelectric point in three proteins. We have purified one of these proteins, raised an antibody to it, and isolated and sequenced its cDNA. At the amino acid level, the sequence shows 68% identity and 81% similarity to bovine smg p25a GDP dissociation inhibitor (GDI), a regulator of ras-like small GTPases of the rab/SEC4/YPT1 subfamily. The correlation between a basic shift in isoelectric point in Drosophila GDI in quartet mutant tissue and the quartet developmental phenotype raises the possibility that a posttranslational modification of GDI is necessary for its function and that GDI function is essential for development.     

Microtubule end binding: EBs sense the guanine nucleotide state

EB proteins accumulate at the tips of growing microtubules and recruit to them a multitude of factors to regulate microtubule functions. A new study suggests that EBs recognize microtubule ends by distinguishing between different states of the tubulin-bound guanine nucleotide.     

Agonist binding promotes a guanine nucleotide reversible increase in the apparent size of the bovine anterior pituitary dopamine receptors

Dopamine receptors, solubilized from bovine anterior pituitary membranes with the detergent digitonin, retained a typical dopaminergic specificity for the binding of both agonists and antagonists. The affinities of antagonists for binding to the soluble receptors are virtually identical with those observed with the membrane-bound receptors. The affinities of agonists however, correspond to those for the form of the receptors in the membranes having low affinity for those agonists (De Lean, A., Kilpatrick, B. F., and Caron, M. G. (1982) Mol. Pharmacol. 22, 290-297). Thus, after solubilization, agonist high affinity interactions with the receptor and their sensitivity to modulation by guanine nucleotides are lost. However, high affinity agonist binding and its sensitivity to guanine nucleotides can be preserved if the membrane-bound receptors are prelabeled with the agonist [3H]n-propylapomorphine prior to solubilization. In order to investigate the molecular basis for these changes in the properties of agonist binding, the solubilized receptors were characterized by chromatographic procedures. Using molecular exclusion high pressure liquid chromatography, [3H]n-propylapomorphine-prelabeled receptors elute as an apparent larger molecular species than either unlabeled or antagonist [( 3H]spiroperidol)-pre-labeled receptors. Moreover, incubation of the pooled agonist-prelabeled receptor peak with guanine nucleotides effects a decrease in the apparent size of the receptors such that upon rechromatography they elute in a position coincidental with the 3H-antagonist-pre-labeled receptor peak. Thus, occupancy of the receptors by agonists promotes the formation of a guanine nucleotide-sensitive agonist high affinity form of the receptor which is of larger apparent size presumably due to the association of the receptor with a guanine nucleotide regulatory protein.     

Membrane-dependent guanine nucleotide binding and GTPase activities of soluble protein from bovine rod cell outer segments.

Soluble proteins can be extracted by osmotic shock of purified rod (photoreceptor cell) outer segments that have intact plasma membranes. The soluble proteins include a component that contains tightly bound GDP-Exchange of this GDP with exogenous nucleotide is catalyzed by (and requires) the membranes from the outer segments. ATP does not participate in these reactions. Approximately one-half of the binding sites in the soluble component require GTP as the source of exogenous nucleotide; the remainder accept GTP or GDP with equal facility. When exogenous GTP is the source of bound nucleotide, it is found in the complex in the form of GDP. Exchange of bound nucleotide with GTP is stoichiometrically related to GTPase activity; this activity is highly dependent upon the presence of both membranes and soluble protein. The soluble nucleotide binding protein was purified by making use of the fact that it binds tightly to the membranes (under conditions of moderate ionic strength) in the absence of GTP and can be eluted by solutions containing low concentrations of GTP (but not GDP or ATP, nor can it be eluted by GTP-free solutions of low ionic strength). The purified protein contains two polypeptide chains of molecular weights 41,000 and 37,000; these are the major species that can be extracted from the outer segments by osmotic shock, and they constitute approximately 7% of the total protein of the isolated organelle.     

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.