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G M Blackburn

Publications and source records attributed to G M Blackburn.

At least 19 recordsLinked to original sources
PubMed2001

Uracil-DNA glycosylase acts by substrate autocatalysis.

In humans, uracil appears in DNA at the rate of several hundred bases per cell each day as a result of misincorporation of deoxyuridine (dU) or deamination of cytosine. Four enzymes that catalyse the hydrolysis of the glycosylic bond of dU in DNA to yield an apyridiminic site as the first step in base excision repair have been identified in the human genome. The most efficient and well characterized of these uracil-DNA glycosylases is UDG (also known as UNG and present in almost all known organisms), which excises U from single- or double-stranded DNA and is associated with DNA replication forks. We used a hybrid quantum-mechanical/molecular-mechanical (QM/MM) approach to determine the mechanism of catalysis by UDG. In contrast to the concerted associative mechanism proposed initially, we show here that the reaction proceeds in a stepwise dissociative manner. Cleavage of the glycosylic bond yields an intermediate comprising an oxocarbenium cation and a uracilate anion. Subsequent attack by a water molecule and transfer of a proton to D145 result in the products. Surprisingly, the primary contribution to lowering the activation energy comes from the substrate, rather than from the enzyme. This 'autocatalysis' derives from the burial and positioning of four phosphate groups that stabilize the rate-determining transition state. The importance of these phosphates explains the residual activity observed for mutants that lack key residues. A corresponding catalytic mechanism could apply to the DNA glycosylases TDG and SMUG1, which belong to the same structural superfamily as UDG.

Catalysis↗
PubMed2001

The 1.2 A structure of a novel quorum-sensing protein, Bacillus subtilis LuxS.

In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. The autoinducer-2 production protein LuxS, is involved in a novel quorum-sensing system and is thought to catalyse the degradation of S-ribosylhomocysteine to homocysteine and the autoinducer molecule 4,5-dihydroxy-2,3-pentadione. The crystal structure of Bacillus subtilis LuxS has been determined at 1.2 A resolution, together with the binary complexes of LuxS with S-ribosylhomocysteine and homocysteine to 2.2 and 2.3 A resolution, respectively. These structures show that LuxS is a homodimer with an apparently novel fold based on an eight-stranded beta-barrel, flanked by six alpha-helices. Each active site contains a zinc ion coordinated by the conserved residues His54, His58 and Cys126, and includes residues from both subunits. S-ribosylhomocysteine binds in a deep pocket with the ribose moiety adjacent to the enzyme-bound zinc ion. Access to the active site appears to be restricted and possibly requires conformational changes in the protein involving the movement of residues 125-129 and those at the N terminus. The structure contains an oxidised cysteine residue in the active site whose role in the biological process of LuxS has not been determined. The autoinducer-2 signalling pathway has been linked to aspects of bacterial virulence and pathogenicity. The structural data on LuxS will provide opportunities for targeting this enzyme for the rational design of new antibiotics.

Amino Acid Sequence↗
PubMed2001

Characterization of active-site residues in diadenosine tetraphosphate hydrolase from Lupinus angustifolius.

Site-directed mutagenesis has been used to characterize the functions of key amino acid residues in the catalytic site of the 'nudix' hydrolase, (asymmetrical) diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) hydrolase (EC 3.6.1.17) from Lupinus angustifolius, the three-dimensional solution structure of which has recently been solved. Residues within the nudix motif, Gly-(Xaa)5-Glu-(Xaa)7-Arg-Glu-Uaa-Xaa-(Glu)2-Xaa-Gly (where Xaa represents unspecified amino acids and Uaa represents the bulky aliphatic amino acids Ile, Leu or Val) conserved in 'nudix enzymes', and residues important for catalysis from elsewhere in the molecule, were mutated and the expressed proteins characterized. The results reveal a high degree of functional conservation between lupin asymmetric Ap4A hydrolase and the 8-oxo-dGTP hydrolase from Escherichia coli. Charged residues in positions equivalent to those that ligate an enzyme-bound metal ion in the E. coli 8-oxo-dGTP hydrolase [Harris, Wu, Massiah and Mildvan (2000) Biochemistry 39, 1655-1674] were shown to contribute to catalysis to similar extents in the lupin enzyme. Mutations E55Q, E59Q and E125Q all reduced kcat markedly, whereas mutations R54Q, E58Q and E122Q had smaller effects. None of the mutations produced a substantial change in the Km)for Ap4A, but several extensively modified the pH-dependence and fluoride-sensitivities of the hydrolase. It was concluded that the precisely positioned glutamate residues Glu-55, Glu-59 and Glu-125 are conserved as functionally significant components of the hydrolytic mechanism in both of these members of the nudix family of hydrolases.

Acid Anhydride Hydrolases↗
PubMed2001

Coronary vasomotor and cardiac electrophysiologic effects of diadenosine polyphosphates and nonhydrolyzable analogs in the guinea pig.

Platelet activation in heart disease is important owing to the effects of platelet-derived compounds on myocardial perfusion and cardiac electrophysiology. Diadenosine polyphosphates are secreted from platelets and present in the myocardium, but their electrophysiologic and vasomotor effects are incompletely understood. We used isolated guinea-pig hearts to study the effects of diadenosine triphosphate (Ap3A), tetraphosphate (Ap4A), pentaphosphate (Ap5A), and hexaphosphate (Ap6A) (10 pM-0.1 mM), comparing their actions to those of adenosine, adenosine triphosphate, and non-hydrolyzable Ap4A and Ap5A analogs. Diadenosine polyphosphates (0.1 nM-0.1 microM) transiently reduced coronary perfusion pressure, which recovered during the continued presence of the compounds. At concentrations greater than 0.1 microM effects were maximal and sustained (perfusion pressure decreased from 36.5+/-3.4 to 18.6+/-2.5 mm Hg, p < 0.001, with 1 microM Ap4A). The changes in action potential duration and refractory period developed slowly but were maintained (0.1 nM-1 microM). With 1 nM Ap4A, action potential duration increased from 170.6+/-2.6 to 187.3+/-3.8 ms, p < 0.05, and refractory period increased from 138.5+/-1.6 to 147.9+/-2.0 ms, p < 0.05. Ap4A and its analog reduced QRS duration (from 24.7+/-1.1 to 13.9+/-1.6 ms with 1 microM Ap4A, p < 0.05). P2-purinergic (adenosine triphosphate) receptor antagonism (suramin) reduced perfusion pressure but was without electrophysiologic effect. Other changes in coronary perfusion pressure and electrophysiologic variables associated with Ap4A were not seen in the presence of suramin. P1-(adenosine) antagonism (8-[p-sulfophenyl]theophylline) attenuated the electrophysiologic effects only. Diadenosine polyphosphates have potent cardiac electrophysiologic and coronary vasomotor effects via purinergic receptors, suggesting an important role during platelet activation in acute coronary syndromes.

Action Potentials↗
PubMed2000

The three-dimensional structure of the Nudix enzyme diadenosine tetraphosphate hydrolase from Lupinus angustifolius L.

The solution structure of diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L., an enzyme of the Nudix family, has been determined by heteronuclear NMR, using a torsion angle dynamics/simulated annealing protocol based on approximately 12 interresidue NOEs per residue. The structure represents the first Ap4A hydrolase to be determined, and sequence homology suggests that other members will have the same fold. The family of structures shows a well-defined fold comprised of a central four-stranded mixed beta-sheet, a two-stranded antiparallel beta-sheet and three helices (alphaI, alphaIII, alphaIV). The root-mean-squared deviation for the backbone (C',O,N,Calpha) of the rigid parts (residues 9 to 75, 97 to 115, 125 to 160) of the protein is 0.32 A. Several regions, however, show lower definition, particularly an isolated helix (alphaII) that connects two strands of the central sheet. This poor definition is mainly due to a lack of long-range NOEs between alphaII and other parts of the protein. Mapping conserved residues outside of the Nudix signature and those sensitive to an Ap4A analogue suggests that the adenosine-ribose moiety of the substrate binds into a large cleft above the four-stranded beta-sheet. Four conserved glutamate residues (Glu55, Glu58, Glu59 and Glu125) form a cluster that most likely ligates an essential magnesium ion, however, Gly41 also an expected magnesium ligand, is distant from this cluster.

Acid Anhydride Hydrolases↗
PubMed2000

Uracil-DNA glycosylase-DNA substrate and product structures: conformational strain promotes catalytic efficiency by coupled stereoelectronic effects.

Enzymatic transformations of macromolecular substrates such as DNA repair enzyme/DNA transformations are commonly interpreted primarily by active-site functional-group chemistry that ignores their extensive interfaces. Yet human uracil-DNA glycosylase (UDG), an archetypical enzyme that initiates DNA base-excision repair, efficiently excises the damaged base uracil resulting from cytosine deamination even when active-site functional groups are deleted by mutagenesis. The 1.8-A resolution substrate analogue and 2.0-A resolution cleaved product cocrystal structures of UDG bound to double-stranded DNA suggest enzyme-DNA substrate-binding energy from the macromolecular interface is funneled into catalytic power at the active site. The architecturally stabilized closing of UDG enforces distortions of the uracil and deoxyribose in the flipped-out nucleotide substrate that are relieved by glycosylic bond cleavage in the product complex. This experimentally defined substrate stereochemistry implies the enzyme alters the orientation of three orthogonal electron orbitals to favor electron transpositions for glycosylic bond cleavage. By revealing the coupling of this anomeric effect to a delocalization of the glycosylic bond electrons into the uracil aromatic system, this structurally implicated mechanism resolves apparent paradoxes concerning the transpositions of electrons among orthogonal orbitals and the retention of catalytic efficiency despite mutational removal of active-site functional groups. These UDG/DNA structures and their implied dissociative excision chemistry suggest biology favors a chemistry for base-excision repair initiation that optimizes pathway coordination by product binding to avoid the release of cytotoxic and mutagenic intermediates. Similar excision chemistry may apply to other biological reaction pathways requiring the coordination of complex multistep chemical transformations.

Amino Acid Sequence↗
PubMed2000

Regioselective hydroxylation of debrisoquine by cytochrome P4502D6: implications for active site modelling.

1. Debrisoquine, a prototypic probe substrate for human cytochrome P4502D6 (CYP2D6), is hydroxylated at the alicyclic C4-position by this enzyme. Phenolic metabolites of debrisoquine (5-, 6-, 7- and 8-hydroxydebrisoquine) have also been reported as in vivo metabolites, but the role of CYP2D6 in their formation is unclear. 2. As part of studies to develop a predictive model of the active site of CYP2D6 using pharmacophore and homology modelling techniques, it became important to determine the precise regioselective hydroxylation of debrisoquine by CYP2D6. 3. Data from studies with human liver microsomes and yeast microsomes containing cDNA-derived CYP2D6 demonstrated unequivocally that debrisoquine was hydroxylated by CYP2D6 at each aromatic site in the molecule, as well as at the alicyclic 4-position. The four phenolic metabolites amounted to > 60% of the total identified products and the pattern of regioselective hydroxylation (4-HD > 7-HD > 6-HD > 8-HD > 5-HD) was similar in both in vitro systems. 4. A pharmacophore model for CYP2D6 indicated that while the hydroxylation of debrisoquine at alternative positions could arise from the substrate adopting multiple binding orientations, the energy constraints for the aromatic hydroxylations were unfavourable. An alternative proposal involving essentially a single binding orientation and a mechanism of hydroxylation based on benzylic radical spin delocalization could satisfactorily rationalize all the hydroxylations of debrisoquine. 5. This latter proposal demonstrates the need to consider the mechanism of oxidation as well as the spatial orientation of the substrate in the development of a predictive model of the active site of CYP2D6.

Binding Sites↗
PubMed1998

Highly potent bisphosphonate ligands for phosphoglycerate kinase.

We have synthesized a series of novel analogs of 1, 3-bisphospho-D-glyceric acid, 1,3-BPG,3 and evaluated their binding to phosphoglycerate kinase, PGK (EC 2.7.2.3). Nonscissile methanephosphonic acids replace the two phosphate monoesters of 1, 3-BPG and lead to several stable, tight-binding mimics of this intermediate species in glycolysis. Multiple fluorine substitution for hydrogen in the alpha-methylene groups of the phosphonic acid 1, 3-BPG analogs markedly improves their binding to PGK as determined by NMR analysis. The best ligands bind some 50-100 times more strongly than does the substrate 3-phospho-D-glyceric acid and show a requirement for pKa3 to be generally below 6.0, while the presence of a beta-carbonyl group seems to be of secondary importance.

Diphosphoglyceric Acids↗
PubMed1998

Synthesis and binding of stable bisubstrate ligands for phosphoglycerate kinase.

Stable bisubstrate ligands of phosphoglycerate kinase (PGK) have been synthesized with AMP or ADP conjugated to hydrolytically-stable, symmetrical analogues of 1,3-bisphosphoglycerate and their binding to yeast PGK evaluated. Their Kds decrease with net negative charge, with a penta-anionic analogue 7 showing highest affinity-in accordance with its approximation to the transition state for the reaction catalysed by PGK.

Adenosine Diphosphate↗
PubMed1998

A bisubstrate analog induces unexpected conformational changes in phosphoglycerate kinase from Trypanosoma brucei.

The glycolytic enzyme phosphoglycerate kinase (PGK) catalyzes phosphoryl transfer between 1,3-bis-phosphoglycerate and ADP to form 3-phosphoglycerate and ATP. During catalysis, a major hinge bending motion occurs which brings the N and C-terminal enzyme domains and their bound substrates together and in-line for phosphoryl transfer. We have crystallized Trypanosoma brucei PGK in the presence of the bisubstrate analog, adenylyl 1,1,5,5-tetrafluoropentane-1, 5-bisphosphonate, and solved the structure of this complex in two different crystal forms at 1.6 and 2.0 A resolution, obtained from PEG 8000 and ammonium phosphate solutions, respectively. These high resolution structures of PGK:inhibitor complexes are of particular interest for drug design since Trypanosoma brucei, the causative agent of African sleeping sickness, relies on glycolysis as its sole energy source. In both structures, the inhibitor is bound in a fully extended conformation with its adenosine moiety assuming exactly the same position as in ADP:PGK complexes and with its 5' phosphonate group occupying part of the 1,3-bis-phosphoglycerate binding site. The bisubstrate analog forces PGK to assume a novel, "inhibited" conformation, intermediate in hinge angle between the native structures of open and closed form PGK. These structures of enzyme-inhibitor complexes demonstrate that PGK has two distinct hinge points that can each be independently activated. In the "PEG" structure, the C-terminal hinge is partially activated while the N-terminal hinge point remains in an open state. In the "phosphate" structure, closure of the N-terminal hinge point is also evident. Finally and most unexpectedly, both complex structures also contain a 3 A shift of a helix that lies outside the flexible hinge region. We propose that a transient shift of this helix is a required element of PGK hinge closure during catalysis.

Animals↗
PubMed1998

Genetic, biochemical, and crystallographic characterization of Fhit-substrate complexes as the active signaling form of Fhit.

Alterations in the FHIT gene at 3p14.2 occur as early and frequent events in the development of several common human cancers. The ability of human Fhit-negative cells to form tumors in nude mice is suppressed by stable reexpression of Fhit protein. Fhit protein is a diadenosine P1,P3-triphosphate (ApppA) hydrolase whose fungal and animal homologs form a branch of the histidine triad (HIT) superfamily of nucleotide-binding proteins. Because the His-96 --> Asn substitution of Fhit, which retards ApppA hydrolase activity by seven orders of magnitude, did not block tumor-suppressor activity in vivo, we determined whether this mutation affected ApppA binding or particular steps in the ApppA catalytic cycle. Evidence is presented that His-96 --> Asn protein binds ApppA well and forms an enzyme-AMP intermediate extremely poorly, suggesting that Fhit-substrate complexes are the likely signaling form of the enzyme. The cocrystal structure of Fhit bound to Ado-p-CH2-p-ps-Ado (IB2), a nonhydrolyzable ApppA analog, was refined to 3.1 A, and the structure of His-96 --> Asn Fhit with IB2 was refined to 2.6 A, revealing that two ApppA molecules bind per Fhit dimer; identifying two additional adenosine-binding sites on the dimer surface; and illustrating that His-98 is positioned to donate a hydrogen bond to the scissile bridging oxygen of ApppA substrates. The form of Fhit bound to two ApppA substrates would present to the cell a dramatically phosphorylated surface, prominently displaying six phosphate groups and two adenosine moieties in place of a deep cavity lined with histidines, arginines, and glutamines.

Acid Anhydride Hydrolases↗
PubMed1998

Novel bisphosphonate inhibitors of phosphoglycerate kinase.

A series of novel, conformationally-restrained bisphosphonate analogues of 1,3-bisphosphoglyceric acid 1 have been synthesised and evaluated as inhibitors of 3-PGK. They are competitive inhibitors of the human enzyme and, especially for certain alpha-halophosphonic acid analogues, both Ki and IC50 values extend into the submicromolar range.

Diphosphonates↗
PubMed1997

Adenosine 5'-tetraphosphate phosphohydrolase from yellow lupin seeds: purification to homogeneity and some properties.

Adenosine 5'-tetraphosphate phosphohydrolase (EC 3.6.1.14) has been purified to homogeneity from the meal of yellow lupin (Lupinus luteus) seeds. The enzyme is a single polypeptide chain of 25+/-1 kDa. It catalyses the hydrolysis of a nucleoside 5'-tetraphosphate to a nucleoside triphosphate and orthophosphate, and hydrolysis of tripolyphosphate but neither pyrophosphate nor tetraphosphate. A divalent cation, Mg2+, Co2+, Ni2+ or Mn2+, is required for these reactions. The pH optimum for hydrolysis of adenosine 5'-tetraphosphate (p4A) is 8.2, Vmax is 21+/-1.7 micromol/min per mg of protein and the Km for p4A is 3+/-0.6 microM. At saturating p4A concentrations, the rate constant for the reaction is 8.5+/-0.7 s-1 [at 30 degrees C, in 50 mM Hepes/KOH (pH8.2)/5 mM MgCl2/0.1 mM dithiothreitol]. p4A and guanosine 5'-tetraphosphate are hydrolysed at the same rate. Adenosine 5'-pentaphosphate (p5A) is degraded 1/200 as fast and is converted into ATP and two molecules of orthophosphate, which are liberated sequentially. This contrasts with the cleavage of p5A by the lupin diadenosine tetraphosphate hydrolase (EC 3.6.1.17), which gives ATP and pyrophosphate. Zn2+, F- and Ca2+ ions inhibit the hydrolysis of p4A with I50 values of 0.1, 0.12 and 0.2 mM respectively.

Acid Anhydride Hydrolases↗
PubMed1997

Diadenosine polyphosphates inhibit adenosine kinase activity but decrease levels of endogenous adenosine in rat brain.

Findings in peripheral tissues that diadenosine polyphosphates (Ap(n)As) activate 5'-nucleotidase activity and inhibit adenosine kinase activity in vitro led us to test the hypothesis that Ap(n)As and analogues thereof, through such actions on purine enzymes, increase brain levels of endogenous adenosine in vivo. Accordingly, we tested Ap(n)As for their effects on the in vitro activities of adenosine kinase, adenosine deaminase, AMP deaminase and 5'-nucleotidase and, following unilateral microinjections in rat striatum, on in vivo levels of endogenous adenosine. Adenosine kinase activity was not affected significantly by 5',5'''-P1,P2-diadenosine pyrophosphate (Ap2A) or by 5',5'''-P1,P3-diadenosine triphosphate (Ap3A), but was inhibited by 5',5'''-P1,P4-diadenosine tetraphosphate (Ap4A), 5',5'''-P1,P5-diadenosine pentaphosphate (Ap5A) and 5',5'''-P1,P6-diadenosine hexaphosphate (Ap6A); apparent IC50 values were 5.0, 3.3 and 500 microM, respectively. Inhibition of adenosine kinase activity by Ap4A and the four metabolically stable analogues of Ap4A tested was uncompetitive. Following unilateral intrastriatal injections, adenosine levels, relative to uninjected contralateral striatum, were decreased significantly (P < 0.05) by 48% with Ap4A and by 37% with AppCH2ppA, a metabolically stable analogue of Ap4A. Striatal levels of adenosine were not affected significantly by Ap5A or Ap6A. Cytosolic, but not particulate 5'-nucleotidase activity was inhibited and AMP deaminase activity was increased by some Ap(n)As. Although adenosine kinase inhibitors increase levels of endogenous adenosine and we showed here that Ap(n)As were potent inhibitors of this enzyme, these particular actions of Ap(n)As were not consistent with their effects on levels of endogenous adenosine.

5'-Nucleotidase↗
PubMed1997

P1,P4-dithio-P2,P3-monochloromethylene diadenosine 5',5'''-P1,P4-tetraphosphate: a novel antiplatelet agent.

We have previously demonstrated in a series of searches for antithrombotic agents that diadenosine 5',5"'-P1,P4-tetraphosphate (AppppA) and its analogues are competitive inhibitors of ADP-induced platelet aggregation. Among various analogues, the P2,P3-monochloromethylene analog of AppppA (AppCHClppA) is superior to unmodified AppppA in its antiplatelet and antithrombotic effects. In this communication, we compare the antiplatelet potency of five newly synthesized agents with that of AppCHClppA. The five new agents include four diadenosine polyphosphate analogues [Ap(s)pCHClpp(s)A (p(s) indicates a thiophosphate), dAppCHClppdA, dAp,pCHClpp(s)dA, and AppCHClpCHClppA], and an adenosine tetraphosphate analogue (AppCHClpCHClp). When tested for their inhibitory effects on platelet aggregation by ADP, the most promising agent among them was Ap(s)pCHClpp(s)A. Both molecular and functional integrity of this compound proved to be stable in blood at 37 degrees C for at least 3 h. It also showed an excellent heat stability. This agent inhibits a number of aspects of ADP-induced platelet activation-e.g., release reaction, cytoplasmic calcium mobilization, thromboxane production, fibrinogen binding sites, and platelet factor 3 activity. Moreover, platelet aggregation induced by agonists other than ADP-e.g., arachidonic acid, collagen, and epinephrine-was inhibited partially by Ap(s)pCHClpp(s)A. It is concluded that (i) Ap(s)pCHClpp(s)A is a promising antiplatelet agent; (ii) it is resistant to blood phosphodiesterases and stable to heat treatment; (iii) platelet aggregation induced by collagen, epinephrine, or arachidonic acid is also inhibited in part by this agent; and (iv) specificity of the inhibitory effects is presented by unmodified adenosine moieties of the agent. Resistance to phosphodiesterases raises the possibility of oral administration.

Blood Platelets↗
PubMed1997

Purification and crystallization of complexes modeling the active state of the fragile histidine triad protein.

Fragile histidine triad protein (Fhit) is a diadenosine triphosphate (ApppA) hydrolase encoded at the human chromosome 3 fragile site which is frequently disrupted in tumors. Reintroduction of FHIT coding sequences to cancer cell lines with FHIT deletions suppressed the ability of these cell lines to form tumors in nude mice even when the reintroduced FHIT gene had been mutated to allow ApppA binding but not hydrolysis. Because this suggested that the tumor suppressor activity of Fhit protein depends on substrate-dependent signaling rather than ApppA catabolism, we prepared two crystalline forms of Fhit protein that are expected to model its biologically active, substrate-bound state. Wild-type and the His96Asn forms of Fhit were overexpressed in Escherichia coli, purified to homogeneity and crystallized in the presence and absence of ApppA and an ApppA analog. Single crystals obtained by vapor diffusion against ammonium sulfate diffracted X-rays to beyond 2.75 A resolution. High quality native synchrotron X-ray data were collected for an orthorhombic and a hexagonal crystal form.

Acid Anhydride Hydrolases↗
PubMed1997

Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, by mammalian cells in vitro.

Clodronate, alendronate, and other bisphosphonates are widely used in the treatment of bone diseases characterized by excessive osteoclastic bone resorption. The exact mechanisms of action of bisphosphonates have not been identified but may involve a toxic effect on mature osteoclasts due to the induction of apoptosis. Clodronate encapsulated in liposomes is also toxic to macrophages in vivo and may therefore be of use in the treatment of inflammatory diseases. It is generally believed that bisphosphonates are not metabolized. However, we have found that mammalian cells in vitro (murine J774 macrophage-like cells and human MG63 osteosarcoma cells) can metabolize clodronate (dichloromethylenebisphosphonate) to a nonhydrolyzable adenosine triphosphate (ATP) analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, which could be detected in cell extracts by using fast protein liquid chromatography. J774 cells could also metabolize liposome-encapsulated clodronate to the same ATP analog. Liposome-encapsulated adenosine 5'-(beta, gamma-dichloromethylene) triphosphate was more potent than liposome-encapsulated clodronate at reducing the viability of cultures of J774 cells and caused both necrotic and apoptotic cell death. Neither alendronate nor liposome-encapsulated alendronate were metabolized. These results demonstrate that the toxic effect of clodronate on J774 macrophages, and probably on osteoclasts, is due to the metabolism of clodronate to a nonhydrolyzable ATP analog. Alendronate appears to act by a different mechanism.

Adenosine Triphosphate↗