guanosine-triphosphate and beryllium-fluoride

The size and chemical nature of the stabilizing cap at microtubule (MT) ends has remained enigmatic, in large part because it has been difficult to detect and measure it directly. By pulsing steady-state suspensions of bovine brain microtubules (MTs) with trace quantities of [gamma(32)P]GTP and sedimenting the MTs through 50% sucrose cushions to reduce background contaminating (32)P to negligible levels, we were able to detect a small number of (32)P molecules that remain stably bound to the MTs (a mean of 25.5 molecules of (32)P per MT). Analysis of the chemical form of the stably bound (32)P by thin-layer chromatography revealed that it was all (32)P-orthophosphate ((32)P(i)). The (32)P(i) was determined to be located at the MT ends because colchicine and vinblastine, drugs that suppress tubulin incorporation into the MT by binding specifically at MT ends, reduced the quantity of the stably bound (32)P(i). Taxol, a drug that stabilizes MT dynamics by binding along the MT surface rather than at the ends, did not affect the stoichiometry of the bound (32)P(i). If the bound (32)P is equally distributed between the two ends, each end would contain 12-13 molecules of (32)P(i). Beryllium fluoride (BeF(3-)) and aluminum fluoride (AlF(4-)), inorganic phosphate analogues, suppressed the dynamic instability behavior of individual MTs and, thus, stabilized them. For example, BeF(3-) (70 microM) reduced the MT shortening rate by 2.5-fold and decreased the transition frequency from the growing or the attenuated state to rapid shortening by 2-fold. The data support the hypothesis that the stabilizing cap at MT ends consists of a single layer of tubulin GDP-P(i) subunits. The data also support the hypothesis that the mechanism giving rise to the destabilized GDP-tubulin core involves release of P(i) rather than hydrolysis of the GTP.

Topics: Aluminum Compounds; Animals; Beryllium; Binding Sites; Brain Chemistry; Cattle; Fluorides; Guanosine Diphosphate; Guanosine Triphosphate; Male; Microscopy, Video; Microtubules; Phosphates; Phosphorus Radioisotopes; Polymers; Sea Urchins; Spermatozoa; Thermodynamics; Tubulin

Hitherto ras-related GTP-binding proteins have been considered not to bind phosphate analogs (Kahn, R. A. (1991) J. Biol. Chem. 266, 15595-15597), at least in the absence of activating proteins (Mittal, R., Reza, M., Goody, R., and Wittinghofer, A. (1996) Science 273, 115-117). In this work, we have used a fluorescent active mutant (Y32W) of p21(Ha-)ras to demonstrate that BeF3- binds to the GDP. p21(Ha-ras) complex in the absence of activating proteins. It induces a conformational change leading to a state with fluorescence properties similar to those of the active state. The binding has a low affinity (Kd at 25 degrees C = 8.1 +/- 0.3 mM) and is endothermic (DeltaH = 22.3 +/- 1.6 kJ mol-1). The similarity between the GTP-bound form and the GDP.BeF3--bound form has been confirmed using lifetime analysis of the tryptophan fluorescence. The kinetic analysis of the process indicates that the binding can be divided into a first bimolecular step, which accounts for the association of the anion with its binding site, and a second step, which corresponds to an internal conformational transition of the GDP. BeF3-.p21(Ha-)ras complex to its final state. Both steps are endothermic (DeltaH1 = 15 +/- 2 kJ mol-1 and DeltaH2 = 8 +/- 2 kJ mol-1). The kinetically determined enthalpy change of 23 +/- 4 kJ mol-1 is in excellent agreement with the equilibrium analysis.

Topics: Anions; Beryllium; Flow Injection Analysis; Fluorides; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Models, Chemical; Mutation; Phosphates; Protein Conformation; Proto-Oncogene Proteins p21(ras); Recombinant Proteins; Spectrometry, Fluorescence

A study was made of the role of G proteins in the K+ conductance increases elicited by cholinergic and glutamatergic agonists in identified Aplysia neurons. The cholinergic response, previously shown to be G protein mediated, was occluded by dialysis with either nonhydrolyzable GTP analogs (GTP gamma S or Gpp(NH)p) or beryllium fluoride and was blocked by pertussis toxin as well as by dialysis with a nonhydrolyzable GDP analog (GDP beta S). In contrast, the glutamatergic response, studied simultaneously in the same cell, persisted throughout all of the above manipulations and hence does not appear to depend upon G protein activation. This characteristic differentiates the glutamatergic response from most other transmitter- or hormone-induced increases in K+ conductance elicited in either neurons or other cell types, whether vertebrate or invertebrate.

Topics: Animals; Aplysia; Beryllium; Cholinergic Agonists; Dialysis; Electric Conductivity; Excitatory Amino Acid Agonists; Fluorides; Glutamic Acid; GTP-Binding Proteins; Guanosine Triphosphate; Neurons; Pertussis Toxin; Potassium; Virulence Factors, Bordetella

Carlier et al. (1988, Biochemistry 27, 3555-3559; 1989, Biochemistry 28, 1783-1791) described enhancement of tubulin polymerization and stabilization of glycerol-induced microtubules by BeF3- (by addition of both BeSO4 and NaF to reaction mixtures). We were able to confirm the stabilization of glycerol-induced polymer reported by these workers, provided Mg2+ was also present in the reaction. When we examined polymerization dependent on microtubule-associated proteins (MAPs), however, we obtained very different results. BeF3- had no significant effect on this reaction, or the polymer formed, under any condition examined. Lower concentrations of BeSO4 alone, in contrast to a negligible effect in glycerol, enhanced polymerization with MAPs provided the concentrations of both Mg2+ and GTP were low; and Be2+ stabilized the polymer, if the GTP concentration was low, at both low and high Mg2+ concentrations. Higher concentrations of BeSO4 precipitated tubulin, an effect which was not affected by Mg2+, partially prevented but not reversed by MAPs, and prevented or reversed by either NaF or nucleotides at adequate concentrations. These results suggest that Be2+ binds at site(s) distinct from Mg2+ site(s), and that partial occupancy of these site(s) at lower Be2+ concentrations enhances tubulin polymerization and polymer stability, while extensive occupancy at higher Be2+ concentrations results in tubulin precipitation. Effects of Be2+ and BeF3- on polymerization dependent on dimethyl sulfoxide or glutamate were also evaluated. The dimethyl sulfoxide system displayed properties similar to those of the glycerol system, while the glutamate system was similar to the MAPs system.

Topics: Animals; Beryllium; Brain; Cattle; Dimethyl Sulfoxide; Fluorides; Glutamates; Glutamic Acid; Glycerol; Guanosine Triphosphate; Kinetics; Macromolecular Substances; Magnesium Sulfate; Microtubule-Associated Proteins; Sodium Fluoride; Tubulin

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.

Topics: Animals; Beryllium; Brain; Fluorides; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Kinetics; Macromolecular Substances; Mathematics; Microtubules; Models, Theoretical; Phosphates; Structure-Activity Relationship; Swine; Tubulin

Fluoride activation of G proteins requires the presence of aluminium or beryllium and it has been suggested that AIF4- acts as an analogue of the gamma-phosphate of GTP in the nucleotide site. We have investigated the action of AIF4- or of BeF3- on transducin (T), the G protein of the retinal rods, either indirectly through the activation of cGMP phosphodiesterase, or more directly through their effects on the conformation of transducin itself. In the presence of AIF4- or BeF3-, purified T alpha subunit of transducin activates purified cyclic GMP phosphodiesterase (PDE) in the absence of photoactivated rhodopsin. Activation is totally reversed by elution of fluoride or partially reversed by addition of excess T beta gamma. Activation requires that GDP or a suitable analogue be bound to T alpha: T alpha-GDP and T alpha-GDP alpha S are activable by fluorides, but not T alpha-GDP beta S, nor T alpha that has released its nucleotide upon binding to photoexcited rhodopsin. Analysis of previous works on other G proteins and with other nucleotide analogues confirm that in all cases fluoride activation requires that a GDP unsubstituted at its beta phosphate be bound in T alpha. By contrast with alumino-fluoride complexes, which can adopt various coordination geometries, all beryllium fluoride complexes are tetracoordinated, with a Be-F bond length of 1.55 A, and strictly isomorphous to a phosphate group. Our study confirms that fluoride activation of transducin results from a reversible binding of the metal-fluoride complex in the nucleotide site of T alpha, next to the beta phosphate of GDP, as an analogue of the gamma phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Aluminum; Aluminum Compounds; Animals; Beryllium; Cattle; Fluorides; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Photoreceptor Cells; Protein Binding; Rod Cell Outer Segment