Aluminum fluoride inhibition of glucocorticoid receptor inactivation and transformation

Fluoride, in the presence of aluminum ions, reversibly inhibits the temperature-mediated inactivation of unoccupied glucocorticoid receptors in cytosol preparations from mouse L cells. The effect is concentration-dependent, with virtually complete stabilization of specific glucocorticoid-binding capacity at 2 mM fluoride and 100 microM aluminum. These concentrations of aluminum and fluoride are ineffective when used separately. Aluminum fluoride also stabilizes receptors toward inactivation by gel filtration and ammonium sulfate precipitation. Aluminum fluoride prevents temperature-dependent transformation of steroid-receptor complexes to the DNA-binding state. Aluminum fluoride does not inhibit calf intestine alkaline phosphatase, and unoccupied receptors inactivated by this enzyme in the presence of aluminum fluoride can be completely reactivated by dithiothreitol. The effects of aluminum fluoride are due to stabilization of the complex between the glucocorticoid receptor and the 90-kDa mammalian heat-shock protein hsp90, which suggests that aluminum fluoride interacts directly with the receptor. Endogenous thermal inactivation of receptors in cytosol is not accompanied by receptor dephosphorylation. However, inactivation is correlated with dissociation of hsp90 from the unoccupied receptor. These results support the proposal that hsp90 is required for the receptor to bind steroid and dissociation of hsp90 is sufficient to inactivate the unoccupied receptor.     

RecA protein-promoted cleavage of LexA repressor in the presence of ADP and structural analogues of inorganic phosphate, the fluoride complexes of aluminum and beryllium

Complexes formed from A13+ or Be2+ and fluoride inhibit the single-stranded DNA-dependent ATPase activity of RecA protein. In contrast, poly(dT)-RecA-ADP complexes, which are inactive for cleavage of LexA protein, become fully active in the presence of AlF4- or BeF3- ions. These data suggest that fluoride complexes of aluminum and beryllium (called herein X) convert RecA-ADP complexes, which bind weakly to single-stranded DNA, into RecA-ADP-X complexes, which bind tightly to single-stranded DNA, the ADP-X moiety behaving as a nonhydrolyzable analogue of ATP. We propose that AlF4- and BeF3- ions act as analogues of inorganic phosphate by binding to the site of the gamma-phosphate of ATP on RecA-ADP complexes, hence mimicking the single-stranded DNA-RecA-ADP-Pi transition state. We conclude that the elementary reaction that switches RecA protein from a high affinity single-stranded DNA binding state to a low affinity single-stranded DNA binding state is not ATP hydrolysis per se but Pi release.     

Aluminum fluoride interactions with troponin C.

The increasing interest in the metal ion aluminum fluoride and beryllium fluoride complexes as phosphate analogs in the myosin ATPase reaction and in muscle fiber studies prompted the examination of their interactions with the regulatory system of troponin and tropomyosin. In this work, the effects of these metal ion analogs on the spectral properties of the Ca(2+)-binding subunit of troponin, troponin C (TnC), were examined. In contrast to beryllium fluoride which did not change the spectral properties of TnC, aluminum fluoride binding induced an increase in both the alpha-helicity and the tyrosine fluorescence of TnC and exposed a hydrophobic region on this protein for fluorescent probe binding. Aluminum fluoride also reduced the Ca2+ and/or Mg(2+)-induced changes on TnC. These results indicate a direct interaction of aluminum fluoride with TnC and merit consideration in designing muscle fiber experiments with this phosphate analog.     

Characterization of the aluminum and beryllium fluoride species bound to F-actin and microtubules at the site of the gamma-phosphate of the nucleotide

Aluminum fluoride and beryllium fluoride complexes have previously been shown to bind tightly to F-ADP-actin and GDP-microtubules in competition with Pi and to mimic the XDP-Pi transient state of the polymerization. The structure of the bound complexes is investigated here in further detail. Using a fluoride ion-specific electrode, the number of fluoride atoms per aluminum or beryllium atom in the bound complex could be determined. The results indicate that AIF-4 and either BeF2(OH)-.H2O or BeF3-.H2O are the tightly bound species in both F-actin and microtubules. The dependences of the binding on pF and pH are consistent with this conclusion. The possible geometries of aluminum and beryllium fluorides in the gamma-phosphate subsite of the nucleotide are discussed in correlation with the catalytic mechanism of nucleotide hydrolysis.     

Magnesium fluoride-dependent binding of small G proteins to their GTPase-activating proteins.

GTPase-activating proteins (GAPs) enhance the intrinsic GTPase activity of small G proteins, such as Ras and Rho, by contributing a catalytic arginine to the active site. An intramolecular arginine plays a similar role in heterotrimeric G proteins. Aluminum fluoride activates the GDP form of heterotrimeric G proteins, and enhances binding of the GDP form of small G proteins to their GAPs. The resultant complexes have been interpreted as analogues of the transition state of the hydrolytic reaction. Here, equilibrium binding has been measured using scintillation proximity assays to provide quantitative information on the fluoride-mediated interaction of Ras and Rho proteins with their respective GAPs, neurofibromin (NF1) and RhoGAP. High-affinity fluoride-mediated complex formation between Rho.GDP and RhoGAP occurred in the absence of aluminum; however, under these conditions, magnesium was required. Additionally, the novel observation was made of magnesium-dependent, fluoride-mediated binding of Ras.GDP to NF1 in the absence of aluminum. Aluminum was required for complex formation when the concentration of magnesium was low. Thus, either aluminum fluoride or magnesium fluoride can mediate the high-affinity binding of Rho. GDP or Ras.GDP to GAPs. It has been reported that magnesium fluoride can activate heterotrimeric G proteins. Thus, magnesium-dependent fluoride effects might be a general phenomenon with G proteins. Moreover, these data suggest that some protein.nucleotide complexes previously reported to contain aluminum fluoride may in fact contain magnesium fluoride.     

Characterization of the aluminum and beryllium fluoride species which activate transducin. Analysis of the binding and dissociation kinetics.

Aluminofluoride and beryllofluoride complexes can activate the heterotrimeric G-proteins by binding next to GDP in the nucleotide site of their G alpha subunit and acting as analogs of the gamma-phosphate of a GTP. However, the exact structures of the activatory complexes in solution as well as those of the bound complexes in the nucleotide site are still disputed. We have studied, by monitoring the activation-dependent tryptophan fluorescence of transducin T alpha subunit, the pF (-log[F-]) and pH dependencies of the kinetics of activation and deactivation of T alpha GDP in the presence of NaF and aluminum or beryllium salts. Comparisons were made with the calculated pF and pH dependencies of the distribution of the metallofluoride complexes, in order to identify the activating species. We observed that the contribution of a magnesium-dependent mechanism of activation by fluoride (Antonny, B., Bigay, J., and Chabre, M. (1990) FEBS Lett. 268, 277-280) and effects due to slow equilibration kinetics between various aluminofluoride complexes could give rise to puzzling kinetics that had caused misinterpretations of previous results. Once corrected for these effects, our results suggest that with aluminum AlF3(OH)- is, rather than AlF4-, the main activating species and that the bound form of the complex is tetracoordinated GDP-AlF3. Deactivation kinetics depend on the free fluoride concentration in the medium, suggesting that the simple bimolecular scheme: T alpha GDP-AlF3 in equilibrium with T alpha GDP+AlF3(OH) does not fully describe the interaction. Fluorides in the bound complexes can also exchange with free F- ions in solution. With beryllium, two complexes are activatory: BeF3-.H2O and BeF2(OH)-.H2O. In the nucleotide site these give two tetracoordinated complexes, GDP.BeF3 and GDP.BeF2(OH), as shown by their different dissociation rates.     

Colorimetric determination of aluminum in soil extracts using haematoxylin

Summary A method for the determination of small amounts of aluminum using haematoxylin has been developed for use with soil extracts. The effects of initial and final pH, time, variations in the amounts of haematoxylin and acetate buffer and interference of ions on colour development were studied. As compared with the standard aluminon method, this method is six times more sensitive. Moreover, aluminum in the soil extracts of complex forming reagents such as fluoride, oxalate, citrate or EDTA can be determined. The recovery of added aluminum in the fluoride extract of the soil was complete.     

Inhibition of Maize Root H+-ATPase by Fluoride and Fluoroaluminate Complexes

Vesicles derived from maize roots retain a membrane-bound H+-ATPase that is able to pump H+ at the expense of ATP hydrolysis. The H+ pumping and the ATPase activity of these vesicles are inhibited by lithium fluoride and by the complex formed between fluoride and aluminum. The inhibition promoted by lithium fluoride increases as the MgCl2 concentration in the medium is increased from 2 to 20 mM. The inhibitory activity of both lithium fluoride and aluminum fluoride increases as the temperature of the medium is increased from 20 to 35[deg]C. Inorganic phosphate (10-40 mM) inhibits the H+ -ATPase at pH 6.5 but not at pH 7.0, and at both pH values, it antagonizes the inhibition promoted by lithium fluoride and fluoroaluminate complexes.     

Crystal structure of a transition state mimic of the catalytic subunit of cAMP-dependent protein kinase

To understand the molecular mechanism underlying phosphoryl transfer of cAMP-dependent protein kinase, the structure of the catalytic subunit in complex with ADP, aluminum fluoride, Mg2+ ions and a substrate peptide was determined at 2.0 A resolution. Aluminum fluoride was modeled as AlF3 in a planar geometry; it is positioned 2.3 A from both the donor oxygen of ADP and the hydroxyl group of the recipient Ser residue. In this configuration, the aluminum atom forms a trigonal bipyramidal coordination with the oxygen atoms of the donor and recipient groups at the apical positions. This arrangement suggests that aluminum fluoride mimics the transition state and provides the first direct structural evidence for the in-line mechanism of phosphoryl transfer in a protein kinase.     

Effects of inhibitors on luminal opening of Ca2+ binding sites in an E2P-like complex of sarcoplasmic reticulum Ca22+-ATPase with Be22+-fluoride

We document here the intrinsic fluorescence and 45Ca2+ binding properties of putative "E2P-related" complexes of Ca2+-free ATPase with fluoride, formed in the presence of magnesium, aluminum, or beryllium. Intrinsic fluorescence measurements suggest that in the absence of inhibitors, the ATPase complex with beryllium fluoride (but not those with magnesium or aluminum fluoride) does constitute an appropriate analog of the "ADP-insensitive" phosphorylated form of Ca2+-ATPase, the so-called "E2P" state. 45Ca2+ binding measurements, performed in the presence of 100 mm KCl, 5 mm Mg2+, and 20% Me2SO at pH 8, demonstrate that this ATPase complex with beryllium fluoride (but again not those with magnesium or aluminum fluoride) has its Ca2+ binding sites accessible for rapid, low affinity (submillimolar) binding of Ca2+ from the luminal side of SR. In addition, we specifically demonstrate that in this E2P-like form of ATPase, the presence of thapsigargin, 2,5-di-tert-butyl-1,4-dihydroxybenzene, or cyclopiazonic acid prevents 45Ca2+ binding (i.e. presumably prevents opening of the 45Ca2+ binding sites on the SR luminal side). Since crystals of E2P-related forms of ATPase have up to now been described in the presence of thapsigargin only, these results suggest that crystallizing an inhibitor-free E2P-like form of ATPase (like its complex with beryllium fluoride) would be highly desirable, to unambiguously confirm previous predictions about the exit pathway from the ATPase transmembrane Ca2+ binding sites to the SR luminal medium.     

The effects on tubulin polymerization and associated guanosine triphosphate hydrolysis of aluminum ion, fluoride and fluoroaluminate species

The effects of aluminum ion, fluoride, and fluoroaluminate species on the assembly of tubulin in the presence of guanine nucleotides and the consequences of these ions on the associated GTPase of microtubules was investigated. Combinations of GDP and fluoroaluminate species were incapable of activating tubulin for polymerization, in contrast to other guanine nucleotide binding proteins, in which these species produce a functional GTP equivalent. Fluoride alone has an effect on GTP-magnesium-promoted microtubule assembly, causing an increased amount of polymer formation and a reduced rate of associated GTP hydrolysis. It is concluded that aluminum ion and fluoroaluminate species possess distinct mechanisms in inhibiting GTP hydrolysis of GTP-binding proteins and that subpopulations of GTP-binding proteins must exist based on differential sensitivities to these ions.     

Effect of cations on the tyrosine kinase activity of the insulin receptor: Inhibition by fluoride is magnesium dependent

We have recently reported that fluoride interacts directly with the insulin receptor, which causes inhibition of its phosphotransferase activity. The inhibitory effect of fluoride on phosphotransferase activity is not due to the formation of complexes with aluminium and occurs in the absence of alterations to the binding of ATP or insulin. In this report we substantiate that the tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle shows a strict requirement of Mg²⁺ ions (Ka near 11 mM). This effect of Mg²⁺ was inhibited in a competitive manner by Mn²⁺, which is compatible with competition of both divalent ions for binding sites. The inhibition of tyrosine kinase activity caused by fluoride was dependent on the concentration of Mg²⁺ in the medium and no inhibitory effect was detected at low concentrations of Mg²⁺. Moreover, the addition of increasing concentrations of Mn²⁺ in the presence of a constant high concentr rease in the inhibitory effect of fluoride. These results indicate that the Mg-insulin receptor complex is the major fluoride-susceptible form. Based on the characteristics of the inhibition of tyrosine kinase shown by fluoride it might be proposed that its action is exerted by the formation of multi-ionic MgF complexes analogous to Pi, which bind to the insulin receptor kinase.     

Synthesis and characterization of transition metal fluoride complexes with imidazole: X-ray crystal structure reveals short hydrogen bonds between lattice water and lattice fluoride

A new series of complexes of cobalt(II) fluoride, nickel(II) fluoride, copper(II) fluoride and zinc(II) fluoride with imidazole were synthesized and characterized by elemental analysis, molar conductance, magnetic moments, IR and electronic absorption measurements. Based on elemental and spectral data, the complexes were found to be of [M(im)₆]F₂ · XH₂O type, where M is Co(II), Ni(II), Cu(II) and Zn(II) and X 4-5. The magnetic moments and spectral data suggested that all the complexes possessed an octahedral geometry. The crystal structure of the nickel complex, [Ni(im)₆]F₂ · 5H₂O, is also reported in which nickel atom is surrounded by six nitrogen atoms of imidazole. Strong intra- and inter-molecular hydrogen bonding exists between fluoride ions (uncoordinated), nitrogen of imidazole and the -OH of water molecules.     

Aluminum stimulates the proliferation and differentiation of osteoblasts in vitro by a mechanism that is different from fluoride

Summary Micromolar concentrations of aluminum sulfate consistently stimulated [³H]thymidine incorporation into DNA and increased cellular alkaline phosphatase activity (an osteoblastic differentiation marker) in osteoblast-line cells of chicken and human. The stimulations were highly reproducible, and were biphasic and dose-dependent with the maximal stimulatory dose varied from experiment to experiment. The mitogenic doses of aluminum ion also stimulated collagen synthesis in cultured human osteosarcoma TE-85 cells, suggesting that aluminum ion might stimulate bone formation in vitro. The effects of mitogenic doses of aluminum ion on basal osteocalcin secretion by normal human osteoblasts could not be determined since there was little, if any, basal secretion of osteocalcin by these cells. 1,25 Dihydroxyvitamin D₃ significantly stimulated the secretion of osteocalcin and the specific activity of cellular alkaline phosphatase in the human osteoblasts. Although mitogenic concentrations of aluminum ion potentiated the 1,25 dihydroxyvitamin D₃-dependent stimulation of osteocalcin secretion, they significantly inhibited the hormone-mediated activation of cellular alkaline phosphatase activity. Mitogenic concentrations of aluminum ion did not stimulate cAMP production in human osteosarcoma TE85 cells, indicating that the mechanism of aluminum ion does not involve cAMP. The mitogenic activity of aluminum ion is different from that of fluoride because (a) unlike fluoride, its mitogenic activity was unaffected by culture medium changes; (b) unlike fluoride, its mitogenic activity was nonspecific for bone cells; and (c) aluminum ion interacted with fluoride on the stimulation of the proliferation of osteoblastic-line cells, and did not share the same rate-limiting step(s) as that of fluoride. PTH interacted with and potentiated the bone cell mitogenic activity of aluminum ion, and thereby is consistent with the possibility that the in vivo osteogenic actions of aluminum ion might depend on PTH. In summary, low concentrations of aluminum ion could act directly on osteoblasts to stimulate their proliferation and differentiation by a mechanism that is different from fluoride.     

Aluminofluoride and beryllofluoride complexes: a new phosphate analogs in enzymology

The action of fluoride ions on G proteins as well as on various ATPases and phosphatases, is related to their complexation with traces of aluminium or beryllium. These fluorometallic complexes act as analogs of phosphate: they bind with high affinity, but reversibly, in phosphate sites or, concomitantly with nucleoside-diphosphate, in nucleoside-triphosphate sites. The beryllofluoride complexes are strictly tetrahedral; they cannot take on the pentavalent conformation adopted by phosphate in transition states hence they interfere with phospho-transfer reaction mechanisms.     

Fluoroaluminum and fluoroberyllium nucleoside diphosphate complexes as probes of the enzymatic mechanism of the mitochondrial F1-ATPase

The mechanism by which fluoride and aluminum or beryllium in combination with ADP inhibit beef heart mitochondrial F1-ATPase was investigated. The kinetics of inhibition depended on the nature of the anion present in the F1-ATPase assay medium. Inhibition required the presence of Mg2+ and developed more rapidly with sulfite and sulfate than with chloride, i.e., with anions which activate F1-ATPase activity. The ADP-fluorometal complexes were bound quasi-irreversibly to F1, and each mole of the inhibitory nucleotide-fluorometal complex was tightly associated with 1 mol of Mg2+. One mole of nucleotide-fluorometal complex was able to inhibit the activity of 1 mol of catalytic site in F1. Direct measurements of bound fluoride, aluminum, beryllium, and ADP indicated that the F1-bound ADP-fluorometal complexes are of the following types: ADP1A11F4, ADP1Be1F1, ADP1Be1F2, or ADP1Be1F3. Fluoroaluminates or fluoroberyllates are isomorphous to Pi, and the inhibitory nucleotide-fluorometal complexes mimicked transient intermediates of nucleotides that appeared in the course of ATP hydrolysis. On the other hand, each mole of fully inhibited F1, retained 2 mol of inhibitory complexes. The same stoichiometry was observed when ADP was replaced by GDP, a nucleotide which, unlike ADP, binds only to the catalytic sites of F1. These results are discussed in terms of a stochastic model in which the three cooperative catalytic sites of F1 function in interactive pairs.     

茶树铝、氟富集研究进展

茶树是铝、氟超富集植物,过量铝、氟累积于叶片严重威胁了人类健康,了解铝、氟在茶树体内的代谢机理对降低茶叶中的铝、氟含量很有必要。本文系统阐述了茶树对铝、氟吸收、转运、累积和解毒的最新研究进展,推测了茶树对铝、氟吸收、转运及解毒的机制,提出了今后茶树铝、氟富集研究的方向。     

Aluminum fluoride inhibits phospholipase D activation by a GTP-binding protein-independent mechanism

Aluminum fluoride (AlF4-) inhibited guanine nucleotide-activated phospholipase D (PLD) in rat submandibular gland cell-free lysates in a concentration-dependent response. This effect was consistent in permeabilized cells with endogenous phospholipid PLD substrates. Inhibition was not caused by either fluoride or aluminum alone and was reversed by aluminum chelation. Inhibition of PLD by aluminum fluoride was not mediated by cAMP, phosphatases 1, 2A or 2B, or phosphatidate phosphohydrolase. AlF4- had a similar inhibitory effect on rArf-stimulated PLD, but did not block the translocation of Arf from cytosol to membranes, indicating a post-GTP-binding-protein site of action. Oleate-sensitive PLD, which is not guanine nucleotide-dependent, was also inhibited by AlF4-, supporting a G protein-independent mechanism of action. A submandibular Golgi-enriched membrane preparation had high PLD activity which was also potently inhibited by AlF4-, leading to speculation that the known fluoride inhibition of Golgi vesicle transport may be PLD-mediated. It is proposed that aluminum fluoride inhibits different forms of PLD by a mechanism that is independent of GTP-binding proteins and that acts via a membrane-associated target which may be the enzyme itself.     

Comaprative binding study of aluminum and chromium to human transferrin

The characteristics of aluminum and chromium binding to apotransferrin (apo-tf) have been investigated and compared. Both metal ions were taken up by human transferrin forming complexes with the maximum absorbances at 405 nm for chromium-transferrin (cr-tf) and 240 nm for aluminum-transferrin (Al-tf). In the presence of citric acid, chromium binding to transferrin is five times more than aluminum. The binding of aluminum or chromium to apo-transferrin was reduced by 18 and 22% in the presence of 200 ng/mL of iron. The binding of both metals to apo-tf appears to be pH dependent. In acidic pHs, less chromium and more aluminum binding occurred.     

Formation and characterization of kinesin.ADP.fluorometal complexes

Recent crystallographic studies of motor proteins showed that the structure of the motor domains of myosin and kinesin are highly conserved. Thus, these motor proteins, which are important for motility, may share a common mechanism for generating energy from ATP hydrolysis. We have previously demonstrated that, in the presence of ADP, myosin forms stable ternary complexes with new phosphate analogues of aluminum fluoride (AlF(4)(-)) and beryllium fluoride (BeF(n)), and these stable complexes mimic the transient state along the ATPase kinetic pathway [Maruta et al. (1993) J. Biol. Chem. 268, 7093-7100]. In this study, we examined the formation of kinesin.ADP.fluorometals ternary complexes and analyzed their characteristics using the fluorescent ATP analogue NBD-ATP (2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl]-ADP). Our results suggest that these ternary complexes may mimic transient state intermediates in the kinesin ATPase cycle. Thus, the kinesin.ADP.AlF(4)(-) complex resembles the kinesin.ADP state, and the kinesin.ADP.BeF(n) complex mimics the kinesin.ADP.P(i) state.