Interaction of tubulin with guanosine 5'-O-(1-thiotriphosphate) diastereoisomers: specificity of the alpha-phosphate binding region

The exchangeable nucleotide-binding site of tubulin has been studied using diastereoisomers A (Sp) and B (Rp) of guanosine 5'-O-(1-thiotriphosphate) (GTP alpha S) in which the phosphorus atom to which sulfur is attached is chiral. GTP alpha S(A) (10 microM) nucleated assembly of purified tubulin (20 microM) into microtubules in buffer containing 0.1 M 2-(N-morpholino)ethanesulfonic acid with 3 mM Mg2+ and 1 mM EGTA, pH 6.6 at 37 degrees C. With 0.2 mM GTP alpha S(A), the critical concentration (Cc; minimum protein concentration required for assembly) was 8 microM tubulin. Neither 0.2 mM GTP nor GTP alpha S(B) promoted microtubule assembly in buffer with 0.5-6.75 mM Mg2+ and 20-70 microM tubulin. The Cc values for GTP alpha S-(A)-induced assembly of tubulin in buffer with 30% glycerol and of microtubule protein (tubulin and microtubule-associated proteins) in buffer were lower than for GTP. GTP alpha S(A)-induced microtubules were more stable to the cold and to Ca2+. GTP alpha S(A) and GTP but not GTP alpha S(B) bound tightly to tubulin at 4 degrees C. Although GTP alpha S(B) did not nucleate assembly, it did bind to tubulin since it was incorporated into the growing microtubule. Both isomers were hydrolyzed in the microtubules. These studies show that GTP alpha S(A) promotes tubulin assembly better than GTP and GTP alpha S(B) and that there is stereoselectivity at the alpha-phosphate binding region of tubulin. The stereoselectivity may be due to different MgGTP alpha S(A) and -(B) interactions with tubulin.     

Tubulin-nucleotide interactions during the polymerization and depolymerization of microtubules

The interactions of nucleotides and their role in the polymerization of tubulin have been studied in detail. GTP promotes polymerization by binding to the exchangeable site (E site) of tubulin. The microtubules formed contain only GDP at the E site, indicating that hydrolysis of E site GTP occurs during or shortly after polymerization. Tubulin prepared by several cycles of polymerization and depolymerization will polymerize in the presence of ATP as well as GTP. Polymerization in ATP is preceded by a distinct lag period which is shorter at higher concentrations of ATP. As reported by others ATP will transphosphorylate bound GDP to GTP. Under polymerizing conditions the maximum level of GTP formation occurs at about the same time as the onset of polymerization, and the lag probably reflects the time necessary to transphosphorylate a critical concentration of tubulin. The transphosphorylated protein can be isolated and will polymerize without further addition of nucleotide. The transphosphorylated GTP is hydrolyzed and the phosphate released during polymerization. About 25% of the phosphate transferred from ATP is noncovalently bound to the subunit as inorganic phosphate and this fraction is also released during polymerization. The nonhydrolyzable analogue of GTP, GMPPNP, will promote microtubule assembly at high concentration. GMPPNP assembled microtubules do not depolymerize in Ca concentrations several fold greater than that which will completely depolymerize GTP assembled tubules; however, addition of Ca prior to inducing polymerization in GMPPNP prevents the formation of microtubules. Thus GTP hydrolysis appears to promote depolymerization rather than polymerization. GDP does not promote microtubule assembly but can inhibit GTP binding and GTP induced polymerization. GDP does not, however, induce the depolymerization of formed microtubules. These experiments demonstrate that tubulin polymerization can not be treated as a thermodynamically reversible process, but must involve one or more irreversible steps. Exchange experiments with [3H]GTP indicate that the "E" site on both microtubules and ring aggregates of tubulin is blocked and does not exchange rapidly. However, during polymerization and depolymerization induced by raising or lowering the temperature, respectively, all the E sites become transiently available and will exchange their nucleotide. This observation does not suggest a direct morphological transition between rings and microtubules. The presence of a blocked E site on the rings explains the apparent transphosphorylation and hydrolysis of "N" site nucleotide reported by others.     

New tumor markers of testis cancer

Microtubules are filamentous polar polymers with plus and minus ends. This polarity plays a crucial role in a variety of cellular functions such as chromosome movement and organelle transport. To examine the relationship between the growth polarity of microtubules and guanine nucleotide dependence, we polymerized microtubules from axonemes of sea urchin sperm flagella either with GTP or with GTP and GDP, and observed individual microtubules by dark-field microscopy. Tubulin concentrations were adjusted in each case to grow microtubules from only one end of each axoneme. The growth polarity of microtubules was determined using N-ethylmaleimide-modified tubulin (NEM-tubulin). In the presence of GTP only and at low tubulin concentrations, microtubules grew from the plus ends of axonemes. Surprisingly, in the presence of GTP and GDP, microtubules grew from the minus ends, even at high tubulin concentrations. To confirm these results, we used a perfusion chamber to monitor the growth polarity of microtubules from the same axoneme under different conditions. Exchanging a solution containing only GTP for one containing GTP and GDP elicited a switch in the growth polarity of microtubules from the plus ends to the minus ends. These results suggest that GDP directly affects microtubule polymerization and inverts microtubule growth polarity, probably by inhibiting microtubule growth at the plus ends.     

Effects of nocodazole and taxol on glycine evoked currents on rat spinal cord neurones in culture

We examined the effect of altering the cytoskeleton polymerization state by treatment with nocodazole and taxol on glycine-evoked currents in patch-clamp recordings from cultured spinal cord neurones. Adding ATP and GTP to the pipette solution did not prevent the rundown of the peak current. In the absence or in the presence of ATP, the proportion of the non-desensitizing part of the glycine evoked-current declined with time. Adding intracellular GTP and ATP stabilized glycine-evoked responses although the proportion of non-inactivating current was reduced. Nocodazole reduced by itself the proportion of the non-inactivating current whereas taxol (with ATP and GTP) had an opposite effect. These results suggest that the polymerization state of microtubules has functional consequences on glycine receptors.     

Assembly of purified GDP-tubulin into microtubules induced by taxol and taxotere: reversibility, ligand stoichiometry, and competition

Purified tubulin fully liganded to GDP at the exchangeable nucleotide binding site has been prepared by a new direct nucleotide exchange procedure. This normally inactive GDP-tubulin is driven to assemble into microtubules by the binding of the antitumor drug taxol or its more soluble side-chain analogue Taxotere in Mg(2+)-containing buffer, and it disassembles by cooling the solution. Therefore this ligand-induced equilibrium microtubule assembly system dispenses with the requirement of a gamma-phosphate-metal cation ligand bound at the nucleotide site for tubulin to be active. GDP-tubulin can also form characteristic pseudo-ordered aggregates of double rings. These aggregates dissociate upon warming or by addition of GTP. Back-substitution of the nucleotide gamma-phosphate permits glycerol-induced assembly without taxol and reduces the critical protein concentration required for drug-induced microtubule assembly by a factor of 2.6 +/- 0.1. The ligand-induced assembly is maximal at taxol or Taxotere concentrations equimolar with tubulin, and both drugs bind to assembled tubulin with a stoichiometry of 0.99 +/- 0.04 ligand per alpha beta dimer. Taxotere apparently competes with taxol for the same binding site, with 1.9 +/- 0.1 times larger effective affinity. Similarly, the Taxotere-induced assembly of GDP-tubulin or GTP-tubulin proceeds with a critical protein concentration 2.1 +/- 0.1 times smaller than with taxol.     

Stabilization of microtubule dynamics by estramustine by binding to a novel site in tubulin: a possible mechanistic basis for its antitumor action

The cellular targets for estramustine, an antitumor drug used in the treatment of hormone-refractory prostate cancer, are believed to be the spindle microtubules responsible for chromosome separation at mitosis. Estramustine only weakly inhibits polymerization of purified tubulin into microtubules by binding to tubulin (Kd, approximately 30 microM) at a site distinct from the colchicine or the vinblastine binding sites. However, by video microscopy, we find that estramustine strongly stabilizes growing and shortening dynamics at plus ends of bovine brain microtubules devoid of microtubule-associated proteins at concentrations substantially below those required to inhibit polymerization of the microtubules. Estramustine strongly reduced the rate and extent both of shortening and growing, increased the percentage of time the microtubules spent in an attenuated state, neither growing nor shortening detectably, and reduced the overall dynamicity of the microtubules. Significantly, the combined suppressive effects of vinblastine and estramustine on the rate and extent of shortening and dynamicity were additive. Thus, like the antimitotic mechanisms of action of the antitumor drugs vinblastine and taxol, the antimitotic mechanism of action of estramustine may be due to kinetic stabilization of spindle microtubule dynamics. The results may explain the mechanistic basis for the benefit derived from combined use of estramustine with vinblastine or taxol, two other drugs that target microtubules, in the treatment of hormone-refractory prostate cancer.     

Female genital mutilation: an overview

A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4 degrees C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60-68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.     

The development of a new method to detect the adulteration of commercial aloe gel powders

Recent high-resolution analysis of tubulin's structure has led to the prediction that the taxol binding site and a tubulin acetylation site are on the interior of microtubules, suggesting that diffusion inside microtubules is potentially a biologically and clinically important process. To assess the rates of transport inside microtubules, predictions of diffusion time scales and concentration profiles were made using a model for diffusion with parameters estimated from experiments reported in the literature. Three specific cases were considered: 1) diffusion of alpha beta-tubulin dimer, 2) diffusion/binding of taxol, and 3) diffusion/binding of an antibody specific for an epitope on the microtubule's interior surface. In the first case tubulin is predicted to require only approximately 1 min to reach half the equilibrium concentration in the center of a 40 microns microtubule open at both ends. This relatively rapid transport occurs because of a lack of appreciable affinity between tubulin and the microtubule inner surface and occurs in spite of a three-fold reduction in diffusivity due to hindrance. By contrast the transport of taxol is much slower, requiring days (at nM concentrations) to reach half the equilibrium concentration in the center of a 40 microns microtubule having both ends open. This slow transport is the result of fast, reversible taxol binding to the microtubule's interior surface and the large capacity for taxol (approximately 12 mM based on interior volume of the microtubule). An antibody directed toward an epitope in the microtubule's interior is predicted to require years to approach equilibrium. These results are difficult to reconcile with previous experimental results where substantial taxol and antibody binding is achieved in minutes, suggesting that these binding sites are on the microtubule exterior. The slow transport rates also suggest that microtubules might be able to serve as vehicles for controlled-release of drugs.     

Mechanism of action cryptophycin. Interaction with the Vinca alkaloid domain of tubulin

Cryptophycin is a potent antitumor agent that depletes microtubules in intact cells, including cells with the multidrug resistance phenotype. To determine the mechanism of action of cryptophycin, its effects on tubulin function in vitro were analyzed. Cryptophycin reduced the in vitro polymerization of bovine brain microtubules by 50% at a drug:tubulin ratio of 0.1. Cryptophycin did not alter the critical concentration of tubulin required for polymerization, but instead caused substoichiometric reductions in the amount of tubulin that was competent for assembly. Consistent with its persistent effects on intact cells, cryptophycin-treated microtubule protein remained polymerization-defective even after cryptophycin was reduced to sub-inhibitory concentrations. The effects of cryptophycin were not due to denaturation of tubulin and were associated with the accumulation of rings of microtubule protein. The site of cryptophycin interaction with tubulin was examined using functional and competitive binding assays. Cryptophycin blocked the formation of vinblastine-tubulin paracrystals in intact cells and suppressed vinblastine-induced tubulin aggregation in vitro. Cryptophycin inhibited the binding of [3H]vinblastine and the hydrolysis of [gamma32P]GTP by isolated tubulin, but did not block the binding of colchicine. These results indicate that cryptophycin disrupts the Vinca alkaloid site of tubulin; however, the molecular details of this interaction are distinct from those of other antimitotic drugs.     

Roles of nucleoside triphosphates in microtubule assembly

Depolymerization of microtubules in the ATP-reassembly buffer permitted the preparation of GDPETNGTP. Incubation of this tubulin fraction at 35 degrees with ATP induced the phosphorylation of E-site GDP into GTP, which was then dephosphorylated during microtubule assembly. Incubation of GDPETNGTP with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40] also induced polymerization. Depolymerization of microtubules in the GTP-reassembly buffer yielded GTPETNGTP, which was capable of polymerizing into microtubules even in the absence of free GTP. In the presence of 4 M glycerol, GDPETNGTP assembled into microtubules with no change in the bound nucleotides.     

The microtubule-stabilizing agent discodermolide competitively inhibits the binding of paclitaxel (Taxol) to tubulin polymers, enhances tubulin nucleation reactions more potently than paclitaxel, and inhibits the growth of paclitaxel-resistant cells

The lactone-bearing polyhydroxylated alkatetraene (+)-discodermolide, which was isolated from the sponge Discodermia dissoluta, induces the polymerization of purified tubulin with and without microtubule-associated proteins or GTP, and the polymers formed are stable to cold and calcium. These effects are similar to those of paclitaxel (Taxol), but discodermolide is more potent. We confirmed that these properties represent hypernucleation phenomena; we obtained lower tubulin critical concentrations and shorter polymers with discodermolide than paclitaxel under a variety of reaction conditions. Furthermore, we demonstrated that discodermolide is a competitive inhibitor with [3H]paclitaxel in binding to tubulin polymer, with an apparent Ki value of 0.4 microM. Multidrug-resistant human colon and ovarian carcinoma cells overexpressing P-glycoprotein, which are 900- and 2800-fold resistant to paclitaxel, respectively, relative to the parental lines, retained significant sensitivity to discodermolide (25- and 89-fold more resistant relative to the parental lines). Ovarian carcinoma cells that are 20-30-fold more resistant to paclitaxel than the parental line on the basis of expression of altered beta-tubulin polypeptides retained nearly complete sensitivity to discodermolide. The effects of discodermolide on the reorganization of the microtubules of Potorous tridactylis kidney epithelial cells were examined at different times. Intracellular microtubules were reorganized into bundles in interphase cells much more rapidly after discodermolide treatment compared with paclitaxel treatment. A variety of spindle aberrations were observed after treatment with both drugs. The proportions of the different types of aberration were different for the two drugs and changed with the length of drug treatment.     

Differential regulation of mitogen-activated protein kinases by microtubule-binding agents in human breast cancer cells

Drug design targeted at microtubules has led to the advent of some potent anti-cancer drugs. In the present study, we demonstrated that microtubule-binding agents (MBAs) taxol and colchicine induced immediate early gene (c-jun and ATF3) expression, cell cycle arrest, and apoptosis in the human breast cancer cell line MCF-7. To elucidate the signal transduction pathways that mediate such biological activities of MBAs, we studied the involvement of mitogen-activated protein (MAP) kinases. Treatment with taxol, colchicine, or other MBAs (vincristine, podophyllotoxin, nocodazole) stimulated the activity of c-jun N-terminal kinase 1 (JNK1) in MCF-7 cells. In contrast, p38 was activated only by taxol and none of the MBAs changed the activity of extracellular signal-regulated protein kinase 2 (ERK2). Activation of JNK1 or p38 by MBAs occurred subsequent to the morphological changes in the microtubule cytoskeleton induced by these compounds. Furthermore, baccatine III and beta-lumicolchicine, inactive analogs of taxol and colchicine, respectively, did not activate JNKI or p38. These results suggest that interactions between microtubules and MBAs are essential for the activation of these kinases. Pretreatment with the antioxidants N-acetyl-L-cysteine (NAC), ascorbic acid or vitamin E, blocked H2O2- or doxorubicin-induced JNKI activity, but had no effect on JNKI activation by MBAs, excluding a role for oxidative stress. However, BAPTA/AM, a specific intracellular Ca2+ chelator, attenuated JNK1 activation by taxol but not by colchicine, and had no effect on microtubule changes induced by taxol. Thus, stabilization or depolymerization of microtubules may regulate JNK1 activity via distinct downstream signaling pathways. The differential activation of MAP kinases opens up a new avenue for addressing the mechanism of action of antimicrotubule drugs.     

Inability to detect Chlamyodomonas microtubule assembly in vitro: possible implications to the in vivo regulation of microtubule assembly

It has been demonstrated that the in vitro assembly of microtubules from Chlamydomonas preparations does not occur under a wide range of conditions, including those efficacious for mammalian brain tubulin. This incompetence of Chlamydomonas extracts to form microtubules is independent of the tubulin concentration, the presence of added nucleotides or an added seed, temperature, or the concentration of divalent cation. However, an amorphous aggregate was observed under certain conditions, who composition was mainly tubulin. The in vitro reassembly of microtubules in gerbil brain extracts is inhibited by Chlamydomonas preparations. Fractionation of the Chlamydomonas extracts by column chromatography suggests that the inhibitory component is Chlamydomonas tubulin itself. The mechanism of this inhibition is unknown, but reassembly experiments indicate that the 2 types of tubulins cannot copolymerize. We suggest that the Chlamydomonas tubulin, derived from a cytoplasmic pool, requires to be activated prior to its in vivo polymerization into microtubules.     

Detection of GTP and Pi in wild-type and mutated yeast microtubules: implications for the role of the GTP/GDP-Pi cap in microtubule dynamics

Microtubule dynamics are believed to be controlled by a stabilizing cap of tubulin dimers at microtubule ends that contain either GTP or GDP and Pi in the exchangeable nucleotide site (E-site) of the beta-subunit. However, it has been difficult to obtain convincing evidence to support this hypothesis because the quantity of GTP and Pi in the E-site of assembled brain tubulin (the tubulin used in most studies thus far) is extremely low. In this study, we have measured the amount of GTP and Pi in the E-site of wild-type and mutated yeast assembled tubulins. In contrast to brain microtubules, 6% of the tubulin in a wild-type yeast microtubule contains a combination of E-site GTP and Pi. This result indicates that GTP hydrolysis and Pi release are not coupled to dimer addition to the end of the microtubule and supports the hypothesis that microtubules contain a cap of tubulin dimers with GTP or Pi in their E-sites. In addition, we have measured the E-site content of GTP and Pi in microtubules assembled from two yeast tubulins that had been mutated at residues T107 and T143 in beta-tubulin, sites thought to interact with the nucleotide bound in the E-site. Previous studies have shown that microtubules containing these mutated tubulins have modified dynamic behavior in vitro. The results from these experiments indicate that the GTP or GDP-Pi cap model does not adequately explain yeast microtubule dynamic behavior.     

Influence of catheter materials and tissue composition on low dose rate iridium-192 seed implant dosimetry

Drug sensitivity was studied for the tubulin inhibitors taxol, taxotere, rhizoxin and for doxorubucin and cisplatin, in human lung and breast cancer cell lines, including drug-selected cell lines, overexpressing the membrane transporter P-glycoprotein (Pgp) or the multidrug resistance protein (MRP). All tubulin-inhibiting agents were more potent than doxorubicin and cisplatin in all cell lines. In the drug resistance-selected cell lines (doxorubicin or mitoxantrone resistant) there was cross-resistance between the tubulin inhibitors and the selecting agent; however, MRP overexpressing cells were relatively less resistant to taxanes than the Pgp overexpressing cells. Polymerization of microtubules after exposure to taxol was observed in drug sensitive cell lines, but not in resistant cell lines, even at high taxol concentrations and after long exposure times. In the Pgp overexpressing cell lines, steady accumulation of 14C-taxol was defective and could be reverted by verapamil. MRP overexpressing cells did not have a significant accumulation defect of taxol, compared to the parental cell lines, and verapamil did not have any effect. These data confirm that the Pgp overexpression is an important mechanism of resistance to taxanes and rhizoxin in human lung and breast tumor cells. However, the presence of mechanisms other than transport defects may play an important role in non-Pgp expressing cells, and these may include an altered function of tubulins.     

Zinc ion-induced assembly of tubulin

Zinc ion-induced assembly of tubulin was followed using electron microscopy and turbidimetric measurements. A scheme utilizing repeated cycles of assembly and disassembly was used to prepare tubulin and microtubule-associated proteins (MAPs) (Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 765-768). Tubulin was further purified by phosphocellulose chromatography to remove the MAPs (Weingarten, M., Lockwood, A. H., Hwo, S-Y, and Kirschner, M. W. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1858-1862). In tubulin preparations containing MAPs and added GTP, Zn2+-induced sheets of 15 to 60 protofilaments oriented in parallel. In the absence of MAPs and/or added GTP, Zn2+ induced the formation of sheets which wrapped quite specifically and serial sections were often consistent with a tubular structure of approximately 220 nm. The assembly of recycled tubulin + GTP and 0 to 1 mM Zn2+ was analyzed by A350 as a function of time at 30 degrees. The greater the concentration of Zn2+, the shorter the lag time, the faster the rate after the lag, and the greater the plateau value of A350. Although turbidimetric measurements can be used to quantitate microtubules, they are not quantitative for Zn2+-induced sheets.     

The C terminus of beta-tubulin regulates vinblastine-induced tubulin polymerization

Oligoanions such as sodium triphosphate or GTP prevent and/or reverse vinblastine-induced polymerization of tubulin. We now show that the anions of glutamate-rich extreme C termini of tubulin are similarly involved in the regulation of the vinblastine effect. Cleavage of the C termini by limited proteolysis with subtilisin enhances vinblastine-induced tubulin polymerization and abolishes the anion effect. Only the beta-tubulin C terminus needs to be removed to achieve these changes and the later cleavage of the alpha-tubulin C terminus has little additional effect. In fact, vinblastine concentrations >20 microM block cleavage of the alpha-tubulin C terminus in the polymer, whereas cleavage of the beta-tubulin C terminus proceeds unimpeded over the time used. The vinblastine effect on tubulin polymerization is also highly pH-dependent between pH 6.5 and 7.5; this is less marked, but not absent, after subtilisin treatment. A working model is proposed wherein an anionic domain proximal to the extreme C terminus must interact with a cationic domain to permit vinblastine to promote polymerization. Both exogenous and extreme C-terminal anions compete for the cationic domain with the proximal anionic domain to prevent vinblastine-induced polymerization. We conclude that the electrostatic regulation of tubulin polymerization induced by vinblastine resides primarily in the beta-tubulin C terminus but that additional regulation proximal in the tubulin molecule also plays a role.     

Alteration of tubulin-Gi protein interaction in rat cerebral cortex with aging

The ability of the tubulin dimer to interact with and to modulate the Gi function inhibiting adenylyl cyclase was examined in cerebral cortex membranes from 2-month-old and 24-month-old rats. The hydrolysis-resistant GTP analogue 5'-guanylylimidodiphosphate (GppNHp)-dependent inhibition of adenylyl cyclase was significantly decreased in cerebral cortex membranes from 24-month-old rats. Tubulin, prepared from rat brains by polymerization with GppNHp, caused inhibition of adenylyl cyclase (approximately 28%) in 2-month-old rats. Tubulin-GppNHp-dependent inhibition of adenylyl cyclase in 24-month-old rats was significantly attenuated (approximately 15%). In 2-month-old rats, when tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analogue [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to Gi alpha. Transfer of AAGTP from tubulin to Gi alpha was reduced in 24-month-old rats. Furthermore, photoaffinity labeling of [32P]AAGTP to Gi alpha in cortex membranes was significantly decreased in 24-month-old rats. No differences were observed in the amounts of Gs alpha, Gi alpha, or G beta subunits and tubulin, estimated by immunoblotting, in cortex membranes from 2-month-old and 24-month-old rats. These results suggest that the ability of tubulin to interact with Gi and thereby modulate the inhibitory regulation of adenylyl cyclase is reduced in the cerebral cortex of 24-month-old rats.