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D Pantaloni

Publications and source records attributed to D Pantaloni.

At least 55 records · Page 3Linked to original sources
PubMed1989

Mechanism of GTP hydrolysis in tubulin polymerization: characterization of the kinetic intermediate microtubule-GDP-Pi using phosphate analogues.

Beryllium fluoride (BeF3-) has previously been shown to bind tightly to microtubules as a structural analogue of Pi and to mimic the GDP-Pi transient state in tubulin polymerization [Carlier, M.-F., Didry, D., Melki, R., Chabre, M., & Pantaloni, D. (1988) Biochemistry 27, 3555-3559]. The interaction of BeF3- with tubulin is analyzed here in greater detail. BeF3- binds to and dissociates from microtubule GDP subunits at very slow rates (k+ congruent to 100 M-1 s-1; k- congruent to 6 x 10(-4) s-1), suggesting that a slow conformation change of tubulin, linked to the stabilization of the microtubule structure, follows BeF3- binding. The possibility is evoked that BeF3- acts as a transition-state analogue in the GTPase reaction of tubulin. BeF3- does not bind to dimeric nor to oligomeric GDP-tubulin with high affinity. Substoichiometric binding of BeF3- to microtubules provides extensive stabilization of the structure. An original mechanistic model that accounts for the data is proposed. The kinetic parameters for microtubule elongation in the presence of GTP- and GDP-tubulin with and without BeF3- have been determined. Data support the following views: (i) Microtubules at steady state and in a regime of slow growth in the presence of GTP are stabilized by a cap of GDP-Pi subunits functionally similar to GDP-BeF3 subunits. (ii) In the presence of BeF3-, microtubules elongate from GDP-tubulin within the following sequence of reactions: initial nonproductive binding of GDP-tubulin to microtubule ends is followed by the binding of BeF3- and the associated conformation change allowing sustained elongation.

Animals↗
PubMed1988

Stabilization of microtubules by inorganic phosphate and its structural analogues, the fluoride complexes of aluminum and beryllium.

In order to elucidate how the elementary reactions of GTP cleavage and subsequent inorganic phosphate (Pi) release, which accompany microtubule assembly, regulate microtubule dynamics, the effect of Pi and of its structural analogues AlF4- and BeF3- on the stability of GDP-microtubules has been investigated. Inorganic phosphate binds to microtubules with a low affinity (KD = 25 mM) and slows down the rate of GDP-subunit dissociation by about 2 orders of magnitude. AlF4- and BeF3- exhibit phosphate-like effects with 1000-fold higher affinity. Evidence has been obtained for direct binding of BeF3- to microtubules with a stoichiometry of 1 mol of BeF3- per mole of GDP-subunit and an equilibrium dissociation constant of 12-15 microM. AlF4- and Pi compete for this site. Phosphate analogues abolish oscillatory polymerization kinetics and slow down microtubule turnover at steady state. In view of these results, we propose that Pi and its structural analogues bind to the site of the gamma-phosphate of GTP in the E site and reconstitute a GDP-Pi-microtubule, from which tubulin subunits dissociate very slowly. We therefore understand that, following GTP cleavage on microtubules, Pi release in the medium is accompanied by a structural change resulting in a large destabilization of the polymer. A cap of slowly dissociating GDP-Pi-subunits prevents depolymerization of the microtubule GDP-core at steady state. The similarity with the actin system [Carlier, M.-F., & Pantaloni, D. (1988) J. Biol. Chem. 263, 817-825] is underlined.

Aluminum↗
PubMed1988

Binding of phosphate to F-ADP-actin and role of F-ADP-Pi-actin in ATP-actin polymerization.

Our previous work (Carlier, M.-F., and Pantaloni, D. (1986) Biochemistry 25, 7789-7792) had shown that F-ADP-Pi-actin is a major intermediate in ATP-actin polymerization, due to the slow rate of Pi release following ATP cleavage on filaments. To understand the mechanism of ATP-actin polymerization, we have prepared F-ADP-Pi-actin and characterized its kinetic parameters. 32Pi binds to F-ADP-actin with a stoichiometry of 1 mol/mol of F-actin subunit and an equilibrium dissociation constant Kpi of 1.5 mM at pH 7.0 Kpi increases with pH, indicating that the H2PO-4 species binds to F-actin. ADP-Pi-actin subunits dissociate much more slowly from filament ends than ADP-actin subunits; therefore, the stability of filaments in ATP is due to terminal ADP-Pi subunits. The slow rate of dissociation of ADP-Pi-actin also explains the decrease in critical concentration of ADP-actin in the presence of Pi reported by Rickard and Sheterline (Richard, J. E., and Sheterline, P. (1986) J. Mol. Biol. 191, 273-280). The effect of Pi on the rate of actin dissociation from filaments is much more pronounced at the barbed end than at the pointed end. Using gelsolin to block the barbed end, we have shown that the two ends are energetically different in the presence of ATP and saturating Pi, but less different than in the absence of Pi. The results are interpreted within a new model for actin polymerization. It is possible that phosphate binding to F-actin can regulate motile events in muscle and nonmuscle cells.

Actins↗
PubMed1988

Oscillations in microtubule polymerization: the rate of GTP regeneration on tubulin controls the period.

The essential reactions involved in the oscillatory kinetics of microtubule polymerization have been investigated. The rate of GDP dissociation from tubulin decreased cooperatively upon increasing tubulin concentration above 20 microM, consistent with the formation of GDP oligomers whose dissociation is rate limiting in nucleotide exchange. The apparent rate constant for nucleotide exchange at high tubulin concentration was 0.02 s-1 at 37 degrees C, which is the exact value needed in previous theoretical simulations to obtain oscillations with the real period of 70-80 s. A glass filter assay separating microtubules from oligomers and tubulin allowed nucleotide bound to non-microtubular tubulin during the oscillations to be monitored. In agreement with nucleotide exchange data, tubulin-bound GDP was found to oscillate in antiphase with microtubules. By varying the concentration of an enzymatic GTP-regenerating system, we could demonstrate that the period of the oscillations is directly controlled by the rate at which GTP is regenerated on tubulin, and oscillations can be observed under conditions where the dissociation of oligomers is no longer rate limiting. The possible physiological significance of GTP-regenerating systems in establishing synchrony in a microtubule population is evoked. The present data confirm and extend the model that we previously proposed to account for the oscillations.

Animals↗
PubMed1987

Actin polymerization and ATP hydrolysis.

F-actin is the major component of muscle thin filaments and, more generally, of the microfilaments of the dynamic, multifunctional cytoskeletal systems of nonmuscle eukaryotic cells. Polymeric F-actin is formed by reversible noncovalent self-association of monomeric G-actin. To understand the dynamics of microfilament systems in cells, the dynamics of polymerization of pure actin must be understood. The following model has emerged from recent work. During the polymerization process, adenosine 5'-triphosphate (ATP) that is bound to G-actin is hydrolyzed to adenosine 5'-diphosphate (ADP) that is bound to F-actin. The hydrolysis reaction occurs on the F-actin subsequent to the polymerization reaction in two steps: cleavage of ATP followed by the slower release of inorganic phosphate (Pi). As a result, at high rates of filament growth a transient cap of ATP-actin subunits exists at the ends of elongating filaments, and at steady state a stabilizing cap of ADP.Pi-actin subunits exists at the barbed ends of filaments. Cleavage of ATP results in a highly stable filament with bound ADP.Pi, and release of Pi destabilizes the filament. Thus these two steps of the hydrolytic reaction provide potential mechanisms for regulating the monomer-polymer transition.

Actin Cytoskeleton↗
PubMed1987

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.

Animals↗
PubMed1987

The mechanisms of ATP hydrolysis accompanying the polymerization of Mg-actin and Ca-actin.

The hydrolysis of ATP that accompanies actin polymerization occurs on the F-actin subsequent to the elongation step. For Mg-actin, the rate of ATP hydrolysis is similar to the rate of elongation at low concentrations of G-actin but increases more slowly as the G-actin concentration is increased. This behavior can be quantitatively modeled by assuming that ATP hydrolysis occurs predominantly, but not exclusively, on a single subunit of Mg-F-actin at the interface between an ATP-subunit cap and an ADP-subunit core. The rates of elongation of Ca-actin and Mg-actin are similar but the rate of ATP hydrolysis on Ca-F-actin is appreciably slower than the rate of elongation at all concentrations of Ca-G-actin. The data for Ca-actin can be modeled by assuming that ATP hydrolysis occurs essentially randomly on Ca-F-actin within a large ATP cap which can be as long as 2,000 subunits in a 10,000-subunit long filament.

4-Chloro-7-nitrobenzofurazan↗
PubMed1987

Synchronous oscillations in microtubule polymerization.

Under conditions where microtubule nucleation and growth are fast (i.e., high magnesium ion and tubulin concentrations and absence of glycerol), microtubule assembly in vitro exhibits an oscillatory regime preceding the establishment of steady state. The amplitude of the oscillations can represent greater than 50% of the maximum turbidity change and oscillations persist for up to 20 periods of 80 s each. Oscillations are accompanied by extensive length redistribution of microtubules. Preliminary work suggests that the oscillatory kinetics can be simulated using a model in which many microtubules undergo synchronous transitions between growing and rapidly depolymerizing phases, complicated by the kinetically limiting rate of nucleotide exchange on free tubulin.

Guanosine Diphosphate↗
PubMed1987

Cyclic AMP specifically blocks proliferation of rat 3T3 cells transformed by polyomavirus.

Elevated exogenous and intracellular levels of cyclic AMP could totally block proliferation of polyomavirus (PyV) transformants derived from rat 3T3 cells without affecting proliferation of normal cells or simian virus 40 (SV40)-induced transformants. Concanavalin A (ConA) had the opposite effect; it could totally block proliferation of both normal cells and SV40 transformants but reduced proliferation of PyV transformants only twofold. Adenylate cyclase was threefold less active in membranes of PyV transformants, and the number of ConA receptors was similar to that of normal cells. Proliferating PyV transformants contained threefold less cyclic AMP than did proliferating SV40 transformants. The sensitivity to cyclic AMP did not correlate with the degree of transformation: cells transformed by Rous sarcoma virus and tumor cells derived from SV40 transformants were not sensitive to cyclic AMP. The differential effect of cyclic AMP and ConA on proliferation was probably due to the activity of an intact middle t protein. The presence of both large T and small t together with middle t was also required for cyclic AMP sensitivity.

Adenylyl Cyclases↗
PubMed1986

Direct evidence for ADP-Pi-F-actin as the major intermediate in ATP-actin polymerization. Rate of dissociation of Pi from actin filaments.

The sequence of reactions involved in the polymerization of ATP-actin and accompanying hydrolysis of ATP has been investigated by using a new glass-fiber filter assay. The assay allows the rapid separation of filaments from monomeric actin, and therefore the straightforward identification of the nucleotide bound to F-actin in the time course of polymerization, using double-labeled [gamma-32P,3H]ATP. The data bring a direct confirmation of the existence of the previously proposed ATP-F-actin intermediate in the time course of polymerization. Moreover, comparison of the hydrolyzed ATP (i.e., acid-labile [32P]Pi) and of 32P bound to F-actin provides direct evidence for the second intermediate ADP-Pi-F-actin in the polymerization process. This latter species is the major transient in the polymerization of ATP-actin, its lifetime being of the order of minutes.

Actins↗
PubMed1986

The effects of Mg2+ at the high-affinity and low-affinity sites on the polymerization of actin and associated ATP hydrolysis.

Actin contains a single high-affinity cation-binding site, for which Ca2+ and Mg2+ can compete, and multiple low-affinity cation-binding sites, which can bind Ca2+, Mg2+, or K+. Binding of cations to the low-affinity sites causes polymerization of monomeric actin with either Ca2+ or Mg2+ at the high-affinity site. A rapid conformational change occurs upon binding of cations to the low-affinity sites (G----G) which is apparently associated with the initiation of polymerization. A much slower conformational change (G----G', or G----G' if the low-affinity sites are also occupied) follows the replacement of Ca2+ by Mg2+ at the high-affinity site. This slow conformational change is reflected in a 13% increase in the fluorescence of G-actin labeled with the fluorophore 7-chloro-4-nitrobenzene-2-oxadiazole (NBD-labeled actin). The rate of the ATP hydrolysis that accompanies elongation is slower with Ca-G-actin than with Mg-G'-actin (i.e. with Ca2+ rather than Mg2+ at the high-affinity site) although their rates of elongation are similar. The slow ATP hydrolysis on Ca-F-actin causes a lag in the increase in fluorescence associated with the elongation of actin labeled with the fluorophore N-pyrene iodoacetamide (pyrenyl-labeled actin), even though there is no lag in the elongation rate, because pyrenyl-labeled ATP-F-actin subunits have a lower fluorescence intensity than pyrenyl-labeled ADP-F-actin subunits. The effects of the cation bound to the high-affinity binding site must, therefore, be considered in quantitatively analyzing the kinetics of polymerization of NBD-labeled actin and pyrenyl-labeled actin. Although their elongation rates are not very different, the rate of nucleation is much slower for Ca-G-actin than for Mg-G'-actin, probably because of the slower rate of ATP hydrolysis when Ca2+ is bound to the high-affinity site.

Actins↗
PubMed1986

Fluorescence measurements of the binding of cations to high-affinity and low-affinity sites on ATP-G-actin.

The binding of cations to ATP-G-actin has been assessed by measuring the kinetics of the increase in fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Ca2+ and Mg2+ compete for a single high-affinity site on ATP-G-actin with KD values of 1.5-15 nM for Ca2+ and 0.1-1 microM for Mg2+, i.e. with affinities 3-4 orders of magnitude higher than previously reported (Frieden, C., Lieberman, D., and Gilbert, H. R. (1980) J. Biol. Chem. 255, 8991-8993). As proposed by Frieden (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886), the Mg-actin complex undergoes a slow isomerization (Kis = 0.03-0.1) to a higher affinity state (K'D = 4-40 nM). The replacement of Ca2+ by Mg2+ at this high-affinity site causes a slow 10% increase in fluorescence that is 90% complete in about 200 s at saturating concentrations of Mg2+. Independently, Ca2+, Mg2+, and K+ bind to low-affinity sites (KD values of 0.15 mM for Ca2+ and Mg2+ and 10 mM for K+) which causes a rapid 6-8% increase in fluorescence (complete in less than 5 s). We propose that the activation step that converts Ca-G-actin to a polymerizable species upon addition of Mg2+ is the binding of Mg2+ to the low-affinity sites and not the replacement of Ca2+ by Mg2+ at the high-affinity site.

Actins↗
PubMed1986

Interaction of cytochalasin D with actin filaments in the presence of ADP and ATP.

Cytochalasin D strongly inhibits the faster components in the reactions of actin filament depolymerization and elongation in the presence of 10 mM Tris-Cl-, pH 7.8, 0.2 mM dithiothreitol, 1 mM MgCl2, 0.1 mM CaCl2, and 0.2 mM ATP or ADP. Assuming an exclusive and total capping of the barbed end by the drug, the kinetic parameters derived at saturation by cytochalasin D refer to the pointed end and are 10-15-fold lower than at the barbed end. In ATP, the critical concentration increases with cytochalasin D up to 12-fold its value when both ends are free; as a result of the lowering of the free energy of nucleation by cytochalasin D, short oligomers of F-actin exist just above and below the critical concentration. Cytochalasin D interacts strongly with the barbed ends independently of the ADP-G-actin concentration (K = 0.5 nM-1). In contrast, the affinity of cytochalasin D decreases cooperatively with increasing ATP-G-actin concentration. These data are equally well accounted for by two different models: either cytochalasin D binds very poorly to ATP-capped filament ends whose proportion increases with actin concentration, or cytochalasin D binds equally well to ATP-ends and ADP-ends and also binds to actin dimers in ATP but not in ADP. A linear actin concentration dependence of the rate of growth was found at the pointed end, consistent with the virtual absence of an ATP cap at that end.

Actin Cytoskeleton↗
PubMed1986

Butyrate converts rat 3T3 fibroblasts into giant cells.

Butyrate inhibits proliferation of rat 3T3 cells by blocking the cell cycle in late G1. In these cells, DNA synthesis is completely arrested 24 h after butyrate addition, whereas RNA and protein synthesis proceed unaffected. This partial inhibition of proliferation progressively converts normal cells into giant ones. Transcription and protein synthesis are both more intense in the giant cells than in normal cells. Cell enlargement is inhibited by cell-to-cell contact and the conversion of a normal into a giant cell is reversible. Giant cells may be of use when designing new approaches to the study of cell structure and motility as well as differentiation and proliferation.

Animals↗
PubMed1985

Bundling of microtubules by glyceraldehyde-3-phosphate dehydrogenase and its modulation by ATP.

Glyceraldehyde-3-phosphate dehydrogenase from different origins (brain, muscle, erythrocytes) binds to microtubules polymerized from pure brain tubulin and causes bundle formation in vitro. ATP is shown to dissociate these bundles into individual microtubules, while the dehydrogenase is not displaced from the polymers by this nucleotide. ATP can be replaced by adenosine 5'-(beta, gamma-imido]triphosphate, a nonhydrolyzable analog of ATP. These data are interpreted in terms of dissociation of the glyceraldehyde-3-phosphate dehydrogenase tetramer into dimers by ATP. The enzyme is also efficiently purified by a tubulin-Sepharose affinity chromatography.

Adenosine Triphosphate↗