Biomedical subjects

D Pantaloni

Publications and source records attributed to D Pantaloni.

At least 73 records · Page 4Linked to original sources

PubMed1985

Polymerization of ADP-actin and ATP-actin under sonication and characteristics of the ATP-actin equilibrium polymer.

Polymerization under sonication has been developed as a new method to study the rapid polymerization of actin with a large number of elongating sites. The theory proposed assumes that filaments under sonication are maintained at a constant length by the constant input of energy. The data obtained for the reversible polymerization of ADP-actin under sonication have been successfully analyzed according to the proposed model and, therefore, validate the model. The results obtained for the polymerization of ATP-actin under sonication demonstrate the involvement of ATP hydrolysis in the polymerization process. At high actin concentration, polymerization was fast enough, as compared to ATP hydrolysis on the F-actin, to obtain completion of the reversible polymerization of ATP-actin before significant hydrolysis of ATP occurred. A critical concentration of 3 microM was determined as the ratio of the dissociation and association rate constants for the interaction of ATP-actin with the ATP filament ends in 1 mM MgCl2, 0.2 mM ATP. The plot of the rate of elongation of filaments versus actin monomer concentration exhibited an upward deviation at high actin concentration that is consistent with this result. The fact that F-actin at steady state is more stable than the ATP-F-actin polymer at equilibrium suggests that the interaction between ADP-actin and ATP-actin subunits at the end of the ATP-capped filament is much stronger than the interaction between two ATP-actin subunits.

Actins↗

PubMed1985

The interaction between ATP-actin and ADP-actin. A tentative model for actin polymerization.

The involvement of interactions between ATP-actin and ADP-actin in actin polymerization has been studied. It has been found that ATP-actin and ADP-actin can copolymerize and that the rate of nucleation is enhanced when both ATP-actin and ADP-actin are present in solution. The fact that the heterologous interaction between ATP-actin (T) and ADP-actin (D) is stronger than either of the homologous reactions, T-T and D-D, agrees with the kinetic data in the accompanying paper (Carlier, M.-F., Pantaloni, D., and Korn, E.D. (1985) J. Biol. Chem. 260, 6565-6571) which show that filament ends having the DT conformation are more stable than those having the TT conformation. These data are incorporated into a model for actin polymerization in ATP in which the kinetic parameters for polymerization depend on the nature of the nucleotide (ADP or ATP) bound to the three terminal subunits of the actin filament.

4-Chloro-7-nitrobenzofurazan↗

PubMed1985

4',6-Diamidino-2-phenylindole, a fluorescent probe for tubulin and microtubules.

A new fluorophor for tubulin which has permitted the monitoring of microtubule assembly in vitro is reported. DAPI (4',6-diamidino-2-phenylindole), a fluorophor already known as a DNA intercalator, was shown to bind specifically to a unique tubulin site as a dimer (KD(app) = 43 +/- 5 microM at 37 degrees C) or to tubulin associated in microtubules (KD(app) = 6 +/- 2 microM at 37 degrees C) with the same maximum enhancement in fluorescence. When tubulin polymerization was induced with GTP, the change in DAPI affinity for tubulin resulted in an enhancement of DAPI binding and, consequently, of fluorescence intensity. DAPI, whose binding site is different from that of colchicine, vinblastine, or taxol, did not interfere greatly with microtubule polymerization. It induced a slight diminution of the critical concentration for tubulin assembly due to a decrease in the depolymerizing rate constant. Moreover, DAPI did not interfere with GTP hydrolysis correlated with tubulin polymerization, but it decreased the GTPase activity at the steady state of tubulin assembly. Even at substoichiometric levels DAPI can be used to follow the kinetics of microtubule assembly.

Animals↗

PubMed1985

A model for actin polymerization and the kinetic effects of ATP hydrolysis.

A model for actin polymerization is proposed in which the rate of elongation of actin filaments depends on whether adenosine 5'-triphosphate or adenosine 5'-diphosphate is bound to the two terminal subunits of the filament. This model accounts quantitatively for the experimental data on the kinetics of filament elongation and explains the effect of ATP hydrolysis on actin polymerization.

Actin Cytoskeleton↗

PubMed1985

High molecular weight microtubule-associated proteins from pig brain are immunologically related to human erythrocyte membrane proteins spectrin, ankyrin, proteins 4.1 and 4.2.

The microtubule-associated proteins MAPs 1 and 2 from pig brain have been found to react with antibodies directed against human ankyrin and spectrin, respectively (Bennett and Davis, 1981; Davis and Bennett, 1982). In a complementary approach we have prepared antibodies against MAP1 alpha. MAP1 gamma and MAP2 purified from pig brain and tested their reactivity with human erythrocyte membrane proteins. Anti-MAP1 alpha was shown to react with alpha and beta-spectrin and with protein 4.1; anti-MAP1 gamma reacted with alpha-spectrin and ankyrin and with a 60 K peptide which copurified with human spectrin. Finally anti-MAP2 was specific for beta-spectrin and protein 4.2. The biological function of protein 4.2 is still unknown but details on the interactions between ankyrin, spectrin and protein 4.1 and their role in mediating the linkage of oligomeric actin on the erythrocyte membrane are well documented. The present results, which demonstrate extended immunological analogies between pig brain high molecular weight MAPs and human erythrocyte membrane proteins, may reflect the presence, in the two families of proteins, of similar functionally important epitopes.

Animals↗

PubMed1984

Nucleoside diphosphate kinase from brain. Purification and effect on microtubule assembly in vitro.

Tubulin strictly requires GTP for its polymerization. Nevertheless, microtubule assembly can be observed in the presence of ATP as the only nucleotide triphosphate, due to the nucleoside diphosphate kinase (NDP kinase) present in microtubule preparations, and which phosphorylates the GDP into GTP. We have purified this enzyme from pig brain to homogeneity, and shown that its relative mass is close to 100 000 in its native state, and 17 000 under denaturing conditions. Therefore it is probably a hexamer, as previously shown for the enzyme from other sources, and also presents a microheterogeneity, with the major isoforms between pI 5.0 and 6.0. The enzyme is transiently phosphorylated during catalysis, as expected within a ping-pong bi-bi mechanism. The effect of the NDP kinase on pure tubulin polymerization was studied: in the presence of NDP kinase, the lag time observed in the kinetics of microtubule assembly was shorter and the final extent of assembly was unchanged. The effect of the enzyme was observed at enzyme concentrations 900-fold lower than tubulin concentration, which shows that the NDP kinase acts catalytically. Kinetic data show that the catalytic effect of the NDP kinase is faster than the rate of nucleotide exchange on tubulin under the same conditions. This result demonstrates that the tubulin-GDP complex itself is a substrate for the enzyme, which may indicate that the GDP bound to tubulin at the E site is exposed on the surface of dimeric tubulin.

Animals↗

PubMed1984

Evidence for an ATP cap at the ends of actin filaments and its regulation of the F-actin steady state.

The correlation between the time courses of actin polymerization under continuous sonication and the associated ATP hydrolysis has been studied. ATP hydrolysis was not mechanistically coupled to polymerization, i.e. not necessary for polymerization, but occurred on F-actin in a subsequent monomolecular reaction. Under sonication, polymerization was complete in 10 s while hydrolysis of ATP on the polymer required 200 s. A value of 0.023 s-1 was found for the first order rate constant of ATP hydrolysis on the polymer at 25 degrees C, pH 7.8, in the presence of 0.2 mM ATP, 0.1 mM CaCl2, and 1 mM MgCl2, independent of the F-actin concentration. The conversion of ATP X F-actin to ADP X F-actin was accompanied by an increase in fluorescence of a pyrenyl probe covalently attached to actin, consistent with a 2-fold greater fluorescence for ADP X F-actin than for ATP X F-actin, with a rate constant of 0.022 s-1. In contrast, the fluorescence of F-actin labeled with 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole did not change significantly when ATP or ADP was bound. The direct consequence of the uncoupling between polymerization and ATP hydrolysis is the formation of an ATP cap at the ends of the filaments, which maintains the stability of the polymer, while most of the filament contains bound ADP. The heterogeneity of the filament with respect to ATP and ADP results in a nonlinear relationship between the rate of elongation and the concentration of G-actin with a discontinuity at the critical concentration, where the rate of growth is zero. In this respect, F-actin in ATP behaves similarly to microtubules in GTP.

Actins↗

PubMed1984

Steady state length distribution of F-actin under controlled fragmentation and mechanism of length redistribution following fragmentation.

Under controlled levels of fragmentation by sonication in the presence of ATP, actin filaments reached different length distributions corresponding to different steady states of polymerization. When the sonication was interrupted, a spontaneous decrease in the number of filaments and corresponding increase in length was observed. The kinetics of the process of length redistribution subsequent to fragmentation was studied for the simpler case of F-actin filaments at equilibrium in the presence of ADP. Analysis of the data shows that a diffusion-like random walk mechanism of length redistribution quantitatively accounts for the observations much better than the previously proposed end-to-end reannealing of filaments. The involvement of this process in the kinetics of actin polymerization and in establishing the length distribution at steady state is discussed.

Actins↗

PubMed1984

The critical concentration of actin in the presence of ATP increases with the number concentration of filaments and approaches the critical concentration of actin.ADP.

F-actin at steady state in the presence of ATP partially depolymerized to a new steady state upon mechanical fragmentation. The increase in critical concentration with the number concentration of filaments has been quantitatively studied. The data can be explained by a model in which the preferred pathway for actin association-dissociation reactions at steady state in the presence of ATP involves binding of G-actin . ATP to filaments, ATP hydrolysis, and dissociation of G-actin . ADP which is then slowly converted to G-actin . ATP. As a consequence of the slow exchange of nucleotide on G-actin, the respective amounts of G-actin . ATP and G-actin . ADP coexisting with F-actin at steady state depend on the filament number concentration. G-actin coexisting with F-actin at zero number concentration of filaments would then consist of G-actin . ATP only, while the critical concentration obtained at infinite number of filaments would be that for G-actin . ADP. Values of 0.35 and 8 microM, respectively, were found for these two extreme critical concentrations for skeletal muscle actin at 20 degrees C, pH 7.8, 0.1 mM CaCl2, 1 mM MgCl2, and 0.2 mM ATP. The same value of 8 microM was directly measured for the critical concentration of G-actin . ADP polymerized in the presence of ADP and absence of ATP, and it was unaffected by fragmentation. These results have important implications for experiments in which critical concentrations are compared under conditions that change the filament number concentrations.

Actins↗

PubMed1984

Polymorphism of tubulin oligomers in the presence of microtubule-associated proteins. Implications in microtubule assembly.

The interaction between tubulin and microtubule-associated proteins (MAPs) in solutions of cycled microtubule protein has been studied by using radioactively labeled MAPs. Kinetic data of MAP association to microtubules in the polymerization process indicate that an oligomer P of tubulin and MAPs is the polymerizing species. Analysis of MAP binding to microtubules formed from solutions in which the ratio MAPs/tubulin was varied shows evidence for a polymorphism of tubulin-MAP oligomers. When the ratio MAPs/tubulin is decreased by addition of dimeric tubulin to 3 times cycled microtubule protein, an oligomer P' less rich in MAPs than P and unable to incorporate in microtubules is formed. The data further show that while tau, MAP1, and MAP2 can bind to oligomer P, only MAP1 and MAP2 can bind to oligomer P'. Therefore, the interactions of tau factor and of MAP1 and MAP2 with tubulin follow different patterns.

Animals↗

PubMed1983

Taxol effect on tubulin polymerization and associated guanosine 5'-triphosphate hydrolysis.

Taxol has been used as a tool to investigate the relationship between microtubule assembly and guanosine 5'-triphosphate (GTP) hydrolysis. The data support the model previously proposed [Carlier, M.-F., & Pantaloni, D. (1981) Biochemistry 20, 1918] that GTP hydrolysis is not tightly coupled to the polymerization process but takes place as a monomolecular process following polymerization. The results further indicate that the energy liberated by GTP hydrolysis is not responsible for the subsequent blockage of GDP on polymerized tubulin. When tubulin is polymerized in the presence of 10-100 microM taxol, the rapid formation of a large number of very short microtubules (l less than 1 micron) is accompanied by the development of turbidity to a lesser extent than what is observed when the same weight amount of longer microtubules (l = 5 microns) is formed. A slower subsequent turbidity increase corresponds to the length redistribution of these short microtubules into 3-5-fold longer ones without any change in the weight amount of polymer. The evolution of the rate of length redistribution with the concentration of taxol suggests a model within which taxol would bind to dimeric tubulin and to tubulin present at the ends of microtubules with a somewhat 10-fold lower affinity than to polymerized tubulin embedded in the bulk of microtubules. In agreement with this model, binding of taxol to the tubulin-colchicine complex in the dimeric form could be measured from the increase in the GTPase activity of the tubulin-colchicine complex accompanying taxol binding.

Alkaloids↗

PubMed1983

Copolymerization of tubulin-colchicine complex and unliganded tubulin in a nonmicrotubular polymer.

We have shown previously [Saltarelli, D., & Pantaloni, D. (1982) Biochemistry 21, 2996-3006] that the tubulin-colchicine complex is able to polymerize in vitro into peculiar "curly" polymers, under the solution conditions permitting polymerization of unliganded tubulin into microtubules. Here it is further demonstrated that unliganded tubulin can be incorporated into these "curly" polymers. The partial critical concentration of tubulin-colchicine is decreased upon incorporation of unliganded tubulin into the copolymer. GTP hydrolysis occurs on unliganded tubulin upon incorporation in the copolymer. Tubulin-podophyllotoxin does not copolymerize with tubulin-colchicine to form a large polymer but interacts with it, preventing tubulin-colchicine polymerization. The data have been analyzed within a model of random copolymerization of unliganded tubulin and tubulin-colchicine into "curly" polymers. A corollary is that unliganded tubulin is virtually able to self-assemble into curly polymers with a critical concentration 10-fold higher than the critical concentration found for microtubule assembly. Consequently, these peculiar tubulin homopolymers cannot be observed except as transients at high concentrations, or when microtubule assembly is inhibited. Kinetic measurements of the T-TC copolymerization process and associated GTP hydrolysis at different T/TC ratios provide supplementary information about some privileged interactions between tubulin and tubulin-colchicine molecules. A comprehensive phase diagram of the various possible polymers formed in the presence of tubulin and tubulin-colchicine is presented.

Animals↗

PubMed1982

Polymerization of the tubulin-colchicine complex and guanosine 5'-triphosphate hydrolysis.

The tubulin-colchicine (1:1) complex was shown to be able to polymerize in vitro under the buffer conditions of microtubule assembly from pure native tubulin. The physical characteristics of this peculiar polymer have been investigated under a variety of conditions and compared with those of microtubules. Polymerization consisted of the nucleation followed by the growth process, was characterized by a critical concentration, and exhibited divalent ion, temperature, and pH dependences very similar to those of microtubules, Guanosine 5'-triphosphate (GTP) or 5' -guanylyl methylene-diphosphate (GMPPCP) was required for polymerization, and guanosine 5'-diphosphate (GDP) was a potent inhibitor. GTP hydrolysis was totally disconnected from the polymerization process and occurred as well under nonpolymerizing conditions. The results are discussed in view of the different types of protein-protein interactions exhibited by tubulin and of the possible relationship between the conformation of the GTP site and the interaction areas.

Animals↗

PubMed1982

Assembly of microtubule protein: role of guanosine di- and triphosphate nucleotides.

A quantitative analysis of the interplay between guanosine 5'-triphosphate (GTP) and guanosine 5'-diphosphate (GDP) in microtubule assembly and accompanying GTP hydrolysis has been performed when tubulin was polymerized in the presence of microtubule-associated proteins (MAPs) which display an interfering GTPase activity. The use of adenylyl beta-imidodiphosphate, which specifically inhibits the MAPs GTPase activity, and of vinblastine (or podophyllotoxin), which specifically inhibits GTP hydrolysis due to tubulin, made possible a study of the extensive GTP hydrolysis associated to microtubule assembly. The results indicate that GDP binds to microtubule ends with an affinity comparable to GTP, thus strongly inhibiting both the elongation process and the steady-state GTP hydrolysis at microtubule ends. GDP shifts the equilibrium between tubulin and microtubules toward disassembly. The MAPs which are released from the microtubules during the GDP-driven depolymerization cluster on the remaining microtubules. The resulting increased stability of microtubules is quantitatively consistent with the decrease in the critical concentration of the polymerizing species GTP-tubulin.

Animals↗

PubMed1982

Mechanism of tubulin assembly: guanosine 5'-triphosphate hydrolysis decreases the rate of microtubule depolymerization.

The rate of depolymerization of microtubules upon lowering the temperature was found to depend on the amount of time elapsed since the beginning of the assembly process. In the first minutes following self-assembly at 37 degrees C, microtubules are more cold sensitive and depolymerize faster than later at the steady state. In the meanwhile, no change occurred in the average length nor in the shape of the distribution of microtubules. On the other hand, the evolution with time of the apparent dissociation rate constant of tubulin from microtubules was in good correlation with the GTP content of microtubules following assembly, showing that GTP hydrolysis modifies the tubulin-tubulin interactions. Microtubule-bound GTP was not exchangeable for GDP, but steady-state GTP hydrolysis was inhibited by GDP. This result indicates that GDP and GTP exhibit different affinities for tubulin in the body and at the ends of microtubules. It is proposed that GTP-tubulin dissociates faster from microtubules than GDP-tubulin. In other words GTP hydrolysis contributes to the stabilization of microtubules.

Animals↗

PubMed1981

Stimulation by tubulin of an adenylate cyclase from murine plasmacytoma.

Different brain tubulin preparations were shown to stimulate membrane-bound adenylate cyclase from the murine plasmacytoma MOPC 173. Purified tubulin devoid of microtubule-associated proteins and of nucleoside-diphosphate kinase activity was responsible for this stimulation. Activation of the basal adenylate cyclase activity occurred in less than 2 min at 32 degrees C and was amplified by a 4 degree C preincubation of tubulin with plasma membranes. Tubulin affected the Km and the V of the enzyme and was shown to be associated with the membrane during the activation phenomenon. Tubulin was more active on the basal adenylate cyclase activity than that stimulated by fluoride or guanosine 5'-[beta, gamma-imido]triphosphate. GTP has no effect on the tubulin-stimulated enzyme.

Adenylyl Cyclases↗

PubMed1981

Mechanism of tubulin assembly: role of rings in the nucleation process and of associated proteins in the stabilization of microtubules.

Several types of experiments were designed to elucidate the mechanism of initiation of tubulin assembly in the presence of microtubule-associated proteins (MAPs): (1) The evolution of the proportion of the double ring species with temperature was examined in the ultracentrifuge, under nonpolymerizing conditions (guanosine diphosphate = 0.5 mM). A net dissociation of rings into dimers occurred when temperature increased. The transition took place above 20 degrees C. (2) The kinetic parameters of the exchange at equilibrium between free tubulin dimers and tubulin in rings were studied at different temperatures, using the technique of isotopic exchange at equilibrium with radioactively labeled tubulin. The values found for the half-time of the exchange reaction varied between or approximately 100 min at 0 degrees C and 10 min at 2 degrees C. (3) The relative participation of unlabeled rings and labeled dimers to microtubules in the time course of assembly was studied at 22 degrees C. The time dependence of microtubule specific radioactivity showed that tubulin incorporated in the initial stages of assembly came predominantly from rings (85-90%). This result indicates that oligomers directly issued from rings are the first intermediates in the assembly process and suggests that at the beginning of polymerization incorporation of isomers or fragments of rings proceeds at a faster rate than their dissociation into dimers. (4) Polymerization experiments in the presence of MAPs with increasing concentrations of tubulin dimers indicated that MAPs are in rapid equilibrium with the microtubules and are distributed along the wall in a more or less loose lattice depending on the relative concentrations of tubulin and MAPs in the solution.

Animals↗

PubMed1981

Kinetic analysis of guanosine 5'-triphosphate hydrolysis associated with tubulin polymerization.

The correlation between the time courses of pure tubulin assembly and accompanying guanosine 5'-triphosphate (GTP) hydrolysis has been studied at different tubulin concentrations in the range where the rate of assembly varies with a strong cooperativity. One GTP molecule was found hydrolyzed per molecule of tubulin dimer incorporated in the microtubule. This hydrolysis was not strictly coupled to polymerization and occurred in a subsequent step. Consequently, in the first stages of assembly, tubulin-GtP complex is the transient major constituent of microtubules. Kinetic data of GTP hydrolysis have been treated within a model of two consecutive first-order reactions: [tubulin-GTP]free k1 leads to [tubulin-GTP]MT k2 leads to [tubulin-GDP]MT + [Pi] GTP hydrolysis proceeded at an intrinsic rate k2 = 0.25 min-1 independent of tubulin concentration. Simultaneous measurements of polymerization, GTPase activity, and incorporation of [3H]GTP followed by unlabeled GTP chase indicated that before its hydrolysis GTP bound to microtubules was exchangeable while after hydrolysis GDP remained locked in the E site. The possibility is discussed that after assembly tubulin undergoes a conformation change which could trigger GTP hydrolysis and sequestration of GDP.

Animals↗