ATP Complex Research Articles

Nucleotide-binding properties of kinase-deficient epidermal-growth-factor-receptor mutants.

The nucleotide-binding properties of wild-type epidermal- growth-factor (EGF)-receptor protein tyrosine kinase (PTK) and EGF-receptor mutants with site-specific amino acid substitutions known to attenuate protein kinase activity were analysed by a fluorescence competition assay employing the nucleotide analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. Binding affinities for ATP and Mn.ATP complex were determined for the PTK domains of the wild-type and two mutant proteins. Surprisingly, mutation of the highly conserved Lys-721 residue in the nucleotide-binding site of the EGF- receptor PTK domain did not abolish ATP and Mn.ATP binding, although the binding affinity for the Mn.ATP complex was significantly reduced. A second kinase-inactivating mutation that targeted the highly conserved Asp-813 residue had little effect on the nucleotide-binding properties of the EGF-receptor PTK domain. These results indicated that the principle effect of these two kinase-inactivating amino acid substitutions is not to block nucleotide binding, but is instead an inhibition of the phospho-transfer reaction.

Characterization of the monovalent and divalent cation requirements for the xenobiotic carboxylic acid: CoA ligases of bovine liver mitochondria

The XL-I, XL-II and XL-III forms of xenobiotic/medium-chain fatty acid: CoA ligase were found to be inactive toward benzoate in the absence of either monovalent or divalent cations. The absolute requirement for monovalent cation was satisfied by either K +, Rb +, or NH + 4. Na + only supported a very low rate. Varying the nature of the anion had only a minor effect. For XL-I and XI-II, the optimum concentration of K + was 50 mM; higher (physiologic) concentrations led to a decrease in activity. K + did not inhibit XL-III. The absolute requirement for divalent cation was satisfied by Mg 2+ or Mn 2+, or to a lesser extent by Co 2+ or Fe 2+. For the XL-I and XL-II, excess uncomplexed Mg 2+ or Mn 2+ decreased the rate; the optimum concentration of Mn 2+ was approximately the same as the concentration of ATP in the assay, and the optimum concentration of Mg 2+ was approximately double the concentration of ATP in the assay. This is consistent with the concept that the divalent cation is required to complex with ATP and with the known stability constants for the ATP complexes of these two divalent cations. XL-III was not inhibited by uncomplexed divalent cations. Uncomplexed ATP was a moderate inhibitor of XL-I and XL-II, and a weak inhibitor of XL-III. The data indicate that in vivo benzoate conjugation is K + and Mg 2+ dependent, and that the cation effects are complex and differ for XL-I and XL-II as compared with XL-III.

DNA BINDING STUDIES OF MAGNESIUM(II), CALCIUM(II), BARIUM(II) AND ATP COMPLEXES

We have investigated the ways by which magnesium (II), calcium (II) and barium (II) and their ATP complexes bind to B-DNA. Effects on native calf thymus DNA conformation were studied and compared with those involving the uncomplexed metal ions and the polymer. It was found that MgATP−2, CaATP−2 and BaATP−2, interact with the phosphates of the polynucleotide; only BaATP −2 appears to bind also through the bases. ATP complexes cause transitions mainly to the B family. The C form is induced by small amounts of MgATP −2 or CaATP−2. At high complex concentrations (r = 2.5 or 5) the polynucleotide adopts the ψ conformation, while BaATP−2 induces this transition at r = 0.6. A contribution of the A form is observed in the case of CaATP-DNA at r = 0.6 and at the beginning of the reaction in the Ba-DNA system at 22°C, for r = 5. The alkaline earth metal ions induce a stabilization of the B conformation. Structural changes with magnesium (II) at r = 2.5 are interpreted in terms of a B to C transition. Calcium (II) at r = 5 produces the A form. In other cases the alkaline earth metal ions stabilize the B conformation. It was found that they all interact with the phosphates of the polynucleotide. The binding ratio, r, is an important factor in all structural changes.

Protein kinase activities in ripening mango, Mangifera indica L., fruit tissue : I: Purification and characterization of a calcium-stimulated casein kinase-I

A Ca 2+-stimulated protein kinase (PK-I), active with dephosphorylated casein as exogenous substrate, was purified from ripening mango fruit. The purification procedure involved 30–70% ammonium sulphate fractionation and sequential anion exchange-, affinity-, hydrophobic interaction- and gel filtration chromatography. PK-I was purified ca. 40-fold with an overall yield of <1%. The final specific activity in the presence of 0.1 mM Ca 2+ was 55 nmol min −1 mg −1. Analysis of the most highly purified preparations revealed a monomeric enzyme with an M r of 30.9 kDa and p I of 5.1. PK-I efficiently phosphorylated casein and phosvitin, but did not phosphorylate histone II-S, histone III-S, protamine sulphate or bovine serum albumin. PK-I activity was stimulated by micromolar concentrations of Ca 2+ and was dependent on millimolar Mg 2+ concentrations, which could not be substituted with Mn 2+. PK-I activity was stimulated by, but was not dependent on Ca 2+. Calmodulin and calmodulin inhibitors did not affect PK-I activity, but heparin and cAMP acted as inhibitors. The pH and temperature optima of the enzyme under standard reaction conditions were 6.5 and 35°C, respectively. The kinetic reaction mechanism of PK-I was studied by using casein as substrate. Initial velocity and product inhibition studies with ADP as product inhibitor best fit an ordered bi–bi kinetic mechanism with the Mg 2+–ATP complex binding first to the enzyme followed by binding of the protein substrate. The K mATP and K mcasein of PK-I were 9 μM and 0.26 mg ml −1, respectively. The K iADP of PK-I was 9 μM.

Plant lactate dehydrogenase: NADH kinetics and inhibition by ATP

Lactate dehydrogenase (LDH), isolated from either turnip ( Brassica rapa, Cruciferae) or from leek ( Allium porrum, Liliaceae) shows normal Michaelis-Menten kinetics with NAD + in the lactate dehydrogenase direction, but non-hyperbolic kinetics with NADH in the pyruvate reductase direction. These kinetics are not ‘sigmoidal’ and appear consistently as two intersecting straight lines in reciprocal plots, representing a sharp decline in the effectiveness of NADH as a substrate in its lower concentration range. The overall apparent affinity for NADH decreases with decreasing pH. Competitive inhibition by ATP, which is much stronger than that characteristic of mammalian LDH, also displays kinetics that seem to be biphasic, but the K i value does not vary significantly with pH in the physiological range. The counter-inhibitory effect of Mg 2+ ions is shown to be due to the formation of non-inhibitory MgATP complexes. The dissociation of this is strongly pH dependent and this results in most of the ATP being complexed, and therefore non-inhibitory at pH 7.4 (the normal pH in plant cytosol) but largely uncomplexed and inhibitory at pH 6.4 (probably a lower cytosolic pH limit). These factors, and particularly the last, combine to inhibit the enzyme more strongly as the pH drops (as a result of lactate-terminating anaerobic glycolysis) and may act as an effective negative feed-back mechanism in plant cells, allowing a pH-activated switchover to ethanol-terminating glycolysis while preventing ‘overshoot’ into harmful acidosis. © 1997 Elsevier Science Ltd. All rights reserved

Exploiting nucleotide thiophosphates to probe mechanistic aspects of Escherichia coli DNA gyrase.

The interaction of DNA gyrase with ATP has been probed using a range of thiophosphate ATP analogs. ATP gammaS is not detectably hydrolyzed by gyrase but can support limited, probably catalytic, DNA supercoiling. ATP gammaS is a good inhibitor of both ATP hydrolysis and ATP-supported supercoiling. In contrast, both ATP alphaS(Rp) and ATP betaS(Rp) have been shown to be good substrates for the ATPase reaction of gyrase and to support catalytic DNA supercoiling. The corresponding Sp diastereoisomers do not support significant levels of supercoiling and are not readily hydrolyzed, but are shown to be reasonable inhibitors of gyrase. For ATP alphaS(Rp), the supercoiling and ATPase activities appear to be tightly coupled with the thionucleotide being apparently a better substrate than ATP in terms of both DNA supercoiling and nucleotide hydrolysis. In the case of ATP betaS(Rp), DNA supercoiling and nucleotide hydrolysis appear to be uncoupled in that ATP betaS(Rp) is almost as good a substrate as ATP for the ATPase reaction of both intact gyrase and the 43 kDa GyrB fragment, whereas it only supports slow DNA supercoiling; the mechanistic consequences of these observations are discussed in terms of a new model for energy coupling in gyrase. DNA gyrase has been shown to be capable of catalyzing DNA supercoiling in the presence of Mg2+, Ca2+, and Mn2+ but not Zn2+, Co2+, Ni2+, or Cd2+. The pronounced diastereoselectivity seen in both the DNA supercoiling and ATPase activity with ATP alphaS and ATP betaS together with evidence from the X-ray structure of the 43 kDa GyrB-ADPNP-Mg complex is consistent with metal ion coordination at both of these sites, and probably to the gamma-phosphoryl center during turnover. Thus, the absolute configuration of the catalytically active Mg2+-ATP complex is likely to involve coordination to the pro-S oxygens at both P alpha and P beta, leading to the alpha,beta,gamma-tridentate Mg-ATP complex with the lambda-exo configuration.

Characterization of an RNP complex that assembles on the Rous sarcoma virus negative regulator of splicing element.

We have characterized an RNP complex that assembles in nuclear extracts on the negative regulator of splicing (NRS) element from Rous sarcoma virus. While no complex was detected by native gel electrophoresis under conditions that supported spliceosome assembly, gel filtration revealed a specific ATP-independent complex that rapidly assembled on NRS RNA. No complexes were formed on non-specific RNA. Unlike the non-specific H complex, factors required for NRS complex assembly are limiting in nuclear extract. The NRS complex was not detected in reactions containing ATP and pre-formed complexes were dissociated in the presence of ATP. In addition, the assembly process was sensitive to high salt but NRS complexes were salt stable once formed. Assembly of the NRS complex appears functionally significant since mutated NRS RNAs that fail to inhibit splicing in vivo are defective for NRS complex assembly in nuclear extract. The probable relationship of the NRS complex to spliceosomal complexes is discussed.

The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mt-Hsp70-Tim44 interaction driving mitochondrial protein import.

Import of preproteins into the mitochondrial matrix is driven by the ATP-dependent interaction of mt-Hsp70 with the peripheral inner membrane import protein Tim44 and the preprotein in transit. We show that Mge1p, a co-chaperone of mt-Hsp70, plays a key role in the ATP-dependent import reaction cycle in yeast. Our data suggest a cycle in which the mt-Hsp70-Tim44 complex forms with ATP: Mge1p promotes assembly of the complex in the presence of ATP. Hydrolysis of ATP by mt-Hsp70 occurs in complex with Tim44. Mge1p is then required for the dissociation of the ADP form of mt-Hsp70 from Tim44 after release of inorganic phosphate but before release of ADP. ATP hydrolysis and complex dissociation are accompanied by tight binding of mt-Hsp70 to the preprotein in transit. Subsequently, the release of mt-Hsp70 from the polypeptide chain is triggered by Mge1p which promotes release of ADP from mt-Hsp70. Rebinding of ATP to mt-Hsp70 completes the reaction cycle.

Regulation of cADP-ribose-induced Ca2+ Release by Mg2+ and Inorganic Phosphate

cADP-ribose (cADPr) has recently been shown to release Ca2+ from an intracellular store of permeabilized T lymphocyte cell lines (Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu, G. A., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359). Using permeabilized Jurkat and HPB. ALL T lymphocytes, the effects of varying concentrations of inorganic phosphate and Mg2+ on cADPr-induced Ca2+ release were investigated. cADPr-induced Ca2+ release was dependent on the concentration of inorganic phosphate, showing very low Ca2+ release activity between 0.5 and 2 mM inorganic phosphate. At 4 to 5 mM inorganic phosphate, the cADPr-induced Ca2+ release was much more pronounced, reaching maximal values at 10 mM inorganic phosphate. The underlying mechanism for this stimulatory effect was an increased loading of the cADPr-sensitive Ca2+ store, which was demonstrated by enhanced resequestration of Ca2+ selectively into the cADPr-sensitive Ca2+ store. The free Mg2+ concentration also influenced cADPr-induced Ca2+ release in permeabilized cells: at 0 and 8.58 mM the release was nearly completely abolished, whereas at 1.06 mM maximal Ca2+ release by cADPr was observed. High performance liquid chromatographic analysis of exogenously added cADPr revealed that the catabolism of cADPr at varying Mg2+ and Pi concentrations had only minor relevance for the modulatory effects observed. To correlate the effects of inorganic phosphate and Mg2+ on cADPr-induced Ca2+ release observed in the permeabilized cell preparations, measurements of these ions in intact Jurkat T lymphocytes were carried out. Intact Jurkat T cells stimulated via the T cell receptor middle dotCD3 complex did not respond with significant elevation of the free intracellular Mg2+ concentration. In contrast, stimulation via the T cell receptor middle dotCD3 complex resulted in an increase in the intracellular inorganic phosphate concentration. These data indicate a role for the intracellular inorganic phosphate concentration in the regulation of cADPr-mediated Ca2+ release in T lymphocytes.

Crystal Structure of the Pertussis Toxin–ATP Complex: A Molecular Sensor

Pertussis toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. The protein is a hexamer containing a catalytic subunit (S1) that is tightly associated with a pentameric cell-binding component (B-oligomer). In vitroexperiments have shown that ATP and a number of detergents and phospholipids assist in activating the holotoxin by destabilizing the interaction between S1 and the B-oligomer. Similar processes may play a role in the activation of pertussis toxin in vivo. In this paper we present the crystal structure of the pertussis toxin–ATP complex and discuss the structural basis for the ATP-induced activation. In addition, we propose a physiological role for the ATP effect in the process by which the toxin enters the cytoplasm of eukaryotic cells. The key features of this proposal are that ATP binding signals the arrival of the toxin in the endoplasmic reticulum and, at the same time, triggers dissociation of the holotoxin prior to membrane translocation.

Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase.

The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl] -4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33 angstroms resolution and refined to an Rfactor 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2.

Intramolecular interligand interaction in zinc(II) and cadmium(II) mixed ligand complexes of ATP and aminoacids or dipeptides or phenolic compounds

The stability constants of (MATPL) complexes in aqueous medium have been determined potentiometrically, at 25°C and at an ionic strength of 0.7;2 M (NaClO4), by using the SCOGS computer program, where M=Zn(II) or Cd(II), ATP=adenosine 5′-triphosphate,l=α-alanine (Ala), phenylalanine (Phe),l-tyrosine (Tyr),l-tryptophan (Trp), glycyl glycine (gg), glycyll-alanine (ga), catechol (cat), orthoaminophenol (oap) and 2,3-dihydroxy naphthalene (dhn). The stability of these ternary complexes is discussed in terms of Δlogk values. Probable reasons for the stabilization or destabilization of the ternary complexes are suggested, with reference to the tetrahedral geometry of the complexes, a point of relevance in the environment around Zn(II) in the metalloenzyme.

In vivo assessment of free magnesium concentration in human brain by 31P MRS. A new calibration curve based on a mathematical algorithm.

Free cytosolic [Mg2+] can be assessed in vivo by 31P MRS from the chemical shift of beta-ATP which in turn depends on the fraction of total ATP complexed to Mg2+ ions. The reliability of these in vivo measurements depends on the availability of an appropriate in vitro calibration to determine the limits of chemical shifts of unbound ATP and Mg-ATP complexes, using solutions that mimic the in vivo cytosolic conditions as far as possible. We used an algorithm and software to allow a quantitative definition of the Mg(2+)-binding molecules to build a semi-empirical equation that correlates the chemical shift of the beta-ATP signal to the [Mg2+] taking into account the amount of Mg2+ bound to all other constituents in solution. Our experiments resulted in a simple and reliable equation directly usable to assess in vivo the free cytosolic magnesium concentration of human brain by 31P MRS. Our method is also flexible enough to make it suitable for in vivo measurements of [Mg2+] in other organs and tissues.

High-resolution crystal structures of human cyclin-dependent kinase 2 with and without ATP: bound waters and natural ligand as guides for inhibitor design.

Inhibition of the cell cycle is widely considered as a new approach toward treatment for diseases caused by unregulated cell proliferation, including cancer. Since cyclin-dependent kinases (CDKs) are key enzymes of cell cycle control, they are promissing targets for the design and discovery of drugs with antiproliferative activity. The detailed structural analysis of CDK2 can provide valuable information for the design of new ligands that can bind in the ATP binding pocket and inhibit CDK2 activity. For this objective, the crystal structures of human CDK2 apoenzyme and its ATP complex were refined to 1.8 and 1.9 A, respectively. The high-resolution refinement reveals 12 ordered water molecules in the ATP binding pocket of the apoenzyme and five ordered waters in that of the ATP complex. Despite a large number of hydrogen bonds between ATP-phosphates and CDK2, binding studies of cyclic AMP-dependent protein kinase with ATP analogues show that the triphosphate moiety contributes little and the adenine ring is most important for binding affinity. Our analysis of CDK2 structural data, hydration of residues in the binding pocket of the apoenzyme, flexibility of the ligand, and structural differences between the apoenzyme and CDK2-ATP complex provide an explanation for the results of earlier binding studies with ATP analogues and a basis for future inhibitor design.

Structural basis for chemical inhibition of CDK2.

The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for discovering small inhibitory molecules that can modify the degree of cell proliferation. The three-dimensional structure of CDK2 provides a structural foundation for understanding the mechanisms of activation and inhibition of CDK2 and for the discovery of inhibitors. In this article five structures of human CDK2 are summarised: apoprotein, ATP complex, olomoucine complex, isopentenyladenine complex, and des-chloro-flavopiridol complex.

Refolding and Reassembly of Active Chaperonin GroEL After Denaturation

Conditions are reported that, for the first time, permit the folding and assembly of active chaperonin, GroEL, following denaturation in 8 m urea. The folding could be achieved by dilution or dialysis, and the best yields required the simultaneous presence of ammonium sulfate and the Mg2+ complexes of ATP or ADP. Ammonium sulfate was the key to this particular protocol, since there was a small recovery of oligomer in its presence, but no detectable recovery was induced by ATP or ADP without ammonium sulfate. The refolded/reassembled GroEL could arrest the spontaneous folding of rhodanese, and it could participate in the chaperonin-assisted refolding of rhodanese as effectively as GroEL that had never been unfolded. The results demonstrate that the primary sequence of GroEL contains the information required for its folding, assembly, and function. Thus, in contrast to previous studies, although chaperonins may facilitate GroEL folding, they are not necessary for the acquisition of the functional oligomeric state of this chaperone. This ability to fold denatured GroEL in vitro will facilitate studies of the influences that determine the interesting folding pattern adopted by the native protein.

Calcium influx but not pH or ATP level mediates glutamate-induced changes in intracellular magnesium in cortical neurons.

1. We have recently shown that glutamate increases [Mg2+]i in cultured rat cortical neurons. However, the mechanism of this increase in [Mg2+]i is not well understood. We used fluorescence microscopic methods to measure [Mg2+]i, [Ca2+]i, and pHi in single neurons. Intracellular ATP analysis was performed by high-performance liquid chromatography (HPLC). 2. A 25-mM NH4Cl pulse followed by Na(+)-free wash rapidly acidified the cytosol. In 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded neurons, the pHi was reduced by 2.46 units, and in magfura-2-loaded neurons the [Mg2+]i was increased by 0.62 mM. Five-minute treatment with 100 microM glutamate, on the other hand, reduced the cytosolic pH by 0.73 units and increased the [Mg2+]i by 7.24 mM in rat cortical neurons. These results indicate that change in pHi does not play a significant role in the glutamate-induced [Mg2+]i elevation. 3. The metabolic inhibition (5 mM KCN and 1 mM iodoacetate) for 30 min significantly reduced the intracellular ATP levels. However, 5-min treatment with 100 microM glutamate did not significantly deplete intracellular ATP in cultured cortical neurons. When tested under similar conditions in magfura-2-loaded neurons, glutamate increased [Mg2+]i to a significantly larger extent than metabolic inhibition. This suggests that ATP depletion and subsequent release of Mg2+ from Mg(2+)-ATP complex is not the primary source of [Mg2+]i elevation observed during glutamate stimulation. 4. To further study the role of glutamate-induced Ca2+ influx in subsequent [Mg2+]i elevation, extracellular Ca2+ was elevated from 1.4 to 3.0 mM during glutamate application in magfura-2-loaded neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex.

The crystal structure of the human cyclinA-cyclin-dependent kinase2 (CDK2)-ATP complex has been determined at 2.3 A resolution. CyclinA binds to one side of CDK2's catalytic cleft, inducing large conformational changes in its PSTAIRE helix and T-loop. These changes activate the kinase by realigning active site residues and relieving the steric blockade at the entrance of the catalytic cleft.

Complexation studies on inositol-phosphates, VI. Al 3+ complexes of DL- myo-inositol 1,4,5-triphosphate and D- myo-inositol 1,2,6-triphosphate

Aluminum complexes of D- myo-inositol 1,2,6-triphosphate (Ins(1,2,6)P 3) and DL- myo-inositol 1,4,5-triphosphate (Ins(1,4,5)P 3) have been studied by potentiometry at 25°C in a 0.1 M tetrabutylammonium bromide medium. Both ligands form AlHL, AlL, and AlOHL species whereas Ins(1,2,6)P 3 forms, in addition, an Al 2L complex and Ins(1,4,5)P 3 an Al(OH) 2L species. In an attempt to assess the biological significance of the Al 3+ binding to Ins(1,4,5)P 3, the results were compared to the Al 3+-ATP complexes that have been found in similar medium conditions. Taking into account the relative stability of the complexes of both systems, it appears likely that the intracellular second messenger system involving Ins(1,4,5)P 3 may be disturbed by the presence of the aluminum cation.

Characteristics of Fe(II)ATP complex-induced damage to the rat liver mitochondrial membrane.

It is well established that several iron complexes can induce oxidative damage in hepatic mitochondrial membranes by catalyzing the formation of OH radicals and/or by promoting lipid peroxidation. This is a relevant process for the molecular basis of iron overload diseases. The present work demonstrates that Fe(II)ATP complexes (5-50 microM) promote an oxygen consumption burst in a suspension of isolated rat liver mitochondria (either in the absence or presence of Antimycin A), caused mainly by lipid peroxidation. Fe(II)ATP alone induced small levels of oxygen uptake but no burst. The time course of Fe(II)ATP oxidation to Fe(II)ATP in the extramitochondrial media also reveals a simultaneous 'burst phase'. The iron chelator Desferal (DFO) or the chain-break antioxidant butylated hydroxytoluene (BHT) fully prevented both lipid peroxidation (quantified as oxygen uptake burst) and mitochondrial swelling. DFO and BHT were capable of stopping the ongoing process of peroxidation at any point of their addition to the mitochondrial suspension. Conversely, DFO and BHT only halted the Fe(II)ATP-induced mitochondrial swelling at the onset of the process. Fe(II)ATP could also cause the collapse of mitochondrial potential, which was protected by BHT if added at the onset of the damaging process. These results, as well as correlation studies between peroxidation and mitochondrial swelling, suggest that a two phase process is occurring during Fe(II)ATP-induced mitochondrial damage: one dependent and another independent of lipid peroxidation. The involvement of lipid peroxidation in the overall process of mitochondrial membrane injury is discussed.