Biomedical subjects

G Lippe

Publications and source records attributed to G Lippe.

At least 19 recordsLinked to original sources

PubMed2002

The nucleotide-independent Fe(III)-binding site is located on beta subunit of the mitochondrial F(1)-ATPase.

Upon separation of a crude preparation of beta subunit ("beta fraction") from mitochondrial F(1)-ATPase containing one equivalent of Fe(III) in the nucleotide-independent site (1Fe(III)-loaded MF(1)), Fe(III) is almost completely recovered. CD spectra show that "beta fraction" maintains the structural changes induced by Fe(III) in the whole enzyme. In accordance, EPR reveals that the Fe(III) site geometry is conserved in "beta fraction." Moreover, the EPR spectra of 1Fe(III)-loaded MF(1) and its "beta fraction" undergo similar changes of the line-shape upon Pi binding at the catalytic site, indicating that the Pi and Fe(III) are proximal on beta. Highly purified beta in nucleotide-free form binds 1mol of Fe(III)/mol of protein. MF(1) "freezed" by inhibitors with two beta in closed conformation and one beta in open or half-closed conformation binds 1mol of Fe(III)/mol of enzyme. Therefore, the Fe(III) site location in the unique beta subunit not adopting the closed conformation is proposed.

Animals↗

PubMed2001

Effects of Fe(III) binding to the nucleotide-independent site of F1-ATPase: enzyme thermostability and response to activating anions.

Mitochondrial F1-ATPase was induced in different conformations by binding of specific ligands, such as nucleotides. Then, Fourier transform infrared spectroscopy (FT-IR) and kinetic analyses were run to evaluate the structural and functional effects of Fe(III) binding to the nucleotide-independent site. Binding of one equivalent of Fe(III) induced a localised stabilising effect on the F1-ATPase structure destabilised by a high concentration of NaCl, through rearrangements of the ionic network essential for the maintenance of enzyme tertiary and/or quaternary structure. Concomitantly, a lower response of ATPase activity to activating anions was observed. Both FT-IR and kinetic data were in accordance with the hypothesis of the Fe(III) site location near one of the catalytic sites, i.e. at the alpha/beta subunit interface.

Anions↗

PubMed2001

Fe(III) binding to Bacillus PS3 F(1)ATPase, alphabeta subcomplexes and isolated alpha- and beta-subunits.

Isolated alpha- and beta-subunits of Thermophilic Bacillus PS3 F(1)ATPase (TF(1)) bind about 1 Fe(III) equivalent. Upon reassembling in the symmetric alpha(3)beta(3) hexamer, Fe(III) binding capacity decreases, as this complex binds about three Fe(III) equivalents. In accordance, when the hexamer is dissociated in the alpha(1)beta(1) heterodimer, each heterodimer binds about one Fe(III) equivalent. On the contrary, native TF(1) exhibits a single Fe(III) site. CD spectra in far UV indicate that upon Fe(III) binding both the whole complex and the isolated beta-subunit undergo structural modifications accompanied by decrease of alpha-helix content, while alpha-subunit doesn't. As in alpha(3)beta(3) and in the whole enzyme the number of bound Fe(III) equivalents is consistent with the number of beta-subunits in the "empty" conformation, it is inferred that the single Fe(III) site in TF(1) is probably located in beta(E).

Bacillus↗

PubMed1999

EPR detection of protein-derived radicals in the reaction of H(2)O(2) with Fe bound in mitochondrial F(1)ATPase.

A severe inactivation is obtained upon the addition of H(2)O(2) to bovine heart F(1)ATPase samples containing Fe(III) in the nucleotide-independent site, and Fe(II) in the ATP-dependent site. EPR spectra at 4.9 K of these samples indicate that H(2)O(2) produces the complete oxidation of Fe(II) to Fe(III) and the concomitant appearance of two protein-derived radical species. The two signals (g = 2.036 and g = 2.007) display a different temperature dependence and saturation behavior. The relaxation properties of the radical at g = 2.036 suggest magnetic interaction with one of the two iron centers. Such events are not observed when H(2)O(2) is added either to native F(1)ATPase containing a high amount of Fe(II) and low amount of Fe(III) or to F(1)ATPase deprived of endogenous Fe and subsequently loaded with only Fe(III) in both sites. It is hypothesized that in F(1)ATPase samples containing both Fe(III) and Fe(II), intramolecular long-range electron transfer may occur from Fe(II) to a high oxidation state species of Fe formed in the nucleotide-independent site upon oxidation of Fe(III) by H(2)O(2).

Animals↗

PubMed1998

Effect of inhibitor binding to beta subunits of F1ATPase on enzyme thermostability: a kinetic and FT-IR spectroscopic analysis.

FT-IR analysis shows that treatment of F1ATPase with the inhibitors DCCD and Nbf-Cl, in the presence of saturating concentrations of ADP and AMP-PNP and in the absence of Mg2+, does not modify the secondary structure of the enzyme, but significantly modifies its compactness and thermal stability, although to different extents. Nbf-Cl causes a significant increase in stabilisation, in addition to that induced by nucleotides, while DCCD is less effective in this regard. Determination by HPLC of the exchange rate, in the absence of Mg2+, of tightly bound nucleotides of F1ATPase treated with the two inhibitors shows that DCCD does not significantly affect the exchange rate of ADP with AMP-PNP and vice versa in catalytic and non-catalytic tight sites, while Nbf-Cl selectively reduces the enzyme's capacity to exchange ADP bound in the tight catalytic site. It is suggested that the effects of DCCD, unlike those of Nbf-Cl, are closely related to the presence or absence of Mg2+.

4-Chloro-7-nitrobenzofurazan↗

PubMed1998

Decrease of cytochrome c oxidase protein in heart mitochondria of copper-deficient rats.

Copper deficiency has been reported to be associated with decreased cytochrome c oxidase activity, which in turn may be responsible for the observed mitochondrial impairment and cardiac failure. We isolated mitochondria from hearts of copper-deficient rats: cytochrome c oxidase activity was found to be lower than in copper-adequate mitochondria. The residual activity paralleled copper content of mitochondria and also corresponded with the heme amount associated with cytochrome aa3. In fact, lower absorption in the alpha-band region of cytochrome aa3 was found for copper-deficient rat heart mitochondria. Gel electrophoresis of protein extracted from mitochondrial membranes allowed measurements of protein content of the complexes of oxidative phosphorylation, revealing a lower content of complex IV protein in copper-deficient rat heart mitochondria. The alterations caused by copper deficiency appear to be specific for cytochrome c oxidase. Changes were not observed for F0F1ATP synthase activity, for heme contents of cytochrome c and b, and for protein contents of complexes I, III and V. The present study demonstrates that the alteration of cytochrome c oxidase activity observed in copper deficiency is due to a diminished content of assembled protein and that shortness of copper impairs heme insertion into cytochrome c oxidase.

Animals↗

PubMed1998

Redox properties of iron in the binding site(s) of F1ATPase from mammalian mitochondria and thermophilic bacterium PS3: a comparative study.

Iron ions in the two iron centers of beef heart mitochondrial F1ATPase, which we have been recently characterized (FEBS Letters 1996, 379, 231-235), exhibit different redox properties. In fact, the ATP-dependent site is able to maintain iron in the redox state of Fe(II) even in the absence of reducing agents, whereas in the nucleotide-independent site iron is oxidized to Fe(III) upon removal of the reductant. Fe(III) ions in the two sites display different reactivity towards H2O2, because only Fe(III) bound in the nucleotide-independent site rapidly reacts with H2O2 thus mediating a 30% enzyme inactivation. Thermophilic bacterium PS3 bears one Fe(III) binding site, which takes up Fe(III) either in the absence or presence of nucleotides and is unable to maintain iron in the redox state of Fe(II) in the absence of ascorbate. Fe(III) bound in thermophilic F1ATPase in a molar ratio 1:1 rapidly reacts with H2O2 mediating a 30% enzyme inactivation. These results support the presence in mitochondrial and thermophilic F1ATPase of a conserved site involved in iron binding and in oxidative inactivation, in which iron exhibits similar redox properties. On the other hand, at variance with thermophilic F1ATPase, the mitochondrial enzyme has the possibility of maintaining one equivalent of Fe(II) in its peculiar ATP-dependent site, besides one equivalent of Fe(III) in the conserved nucleotide-independent site. In this case mitochondrial F1ATPase undergoes a higher inactivation (75%) upon exposure to H2O2. Under all conditions the inactivation is significantly prevented by PBN and DMSO but not by Cu, Zn superoxide dismutase, thus suggesting the formation of OH radicals as mediators of the oxidative damage. No dityrosines, carbonyls or oxidized thiols are formed. In addition, in any cases no protein fragmentation or aggregation is observed upon the treatment with H2O2.

Allosteric Site↗

PubMed1996

Characterization of the binding of Fe(III) to F1ATPase from bovine heart mitochondria.

The binding Fe(III) to F1ATPase purified from beef heart mitochondria has been characterized by chemical analyses and EPR spectroscopy. F1ATPase binds 2 mol of Fe(III)/mol of protein selectively in the presence of saturating concentrations of ATP. In the absence of nucleotides or in the presence of either saturating ADP or limiting ATP concentrations, the enzyme binds 1 equivalent of Fe(III). F1ATPase pretreated with 5'-p- fluorosulfonylbenzoyladenosine, that selectively modifies the non-catalytic sites, binds only 1 mol of Fe(III)/mol of protein in the presence of either saturating ATP or ADP, Fe(III)-loaded F1ATPase containing either 1 or 2 equivalents of Fe(III) show identical EPR signals at g=4.3. The signals are not perturbed by the binding of nucleotides to the enzyme while they are altered by phosphate addition. These results indicate that F1ATPase contains two distinct Fe(III)-binding sites, which differ from nucleotide-binding sites, and that one of these sites is opened up for Fe(III) uptake by conformational changes induced by binding of ATP to the loose non-catalytic site.

Adenosine Diphosphate↗

PubMed1995

Influence of ADP, AMP-PNP and of depletion of nucleotides on the structural properties of F1ATPase: a Fourier transform infrared spectroscopic study.

Mitochondrial F1ATPase from beef heart was treated with different buffers in order to modulate the nucleotide content of the enzyme and then analysed by FT-IR spectroscopy. Treatment of F1ATPase with a buffer lacking nucleotides and glycerol led to the formation of two fractions consisting of an inactive aggregated enzyme deprived almost completely of bound nucleotides and of an active enzyme containing ATP only in the tight sites and having a structure largely accessible to the solvent and a low thermal stability. Treatment of F1ATPase with saturating ADP, which induced the hysteretic inhibition during turnover, or AMP-PNP did not affect remarkably the secondary structure of the enzyme complex but significantly increased its compactness and thermal stability. It was hypothesised that the formation of the inactive aggregated enzyme was mainly due to the destabilisation of the alpha-subunits of F1ATPase and that the induction of the hysteretic inhibition is related to a particular conformation of the enzyme, which during turnover becomes unable to sustain catalysis.

Adenosine Diphosphate↗

PubMed1994

Differentiation potentiates oxidant injury to mitochondria by hydrogen peroxide in Friend's erythroleukemia cells.

Oxidative damage to mitochondrial functions was investigated upon non-lethal treatment with H2O2 of Friend's erythroleukemia cells induced to differentiate, in comparison with the parental cell line. Both respiration and maximal ATP synthase capacity were more severely diminished by H2O2 in induced cells. The effects were mediated by intracellular redox-active iron and OH. radicals. Specifically, the mechanisms of the selective oxidant injury to F0F1 ATP synthase observed in differentiating cells likely involved impairment of F0-F1 coupling sensitive to oligomycin. We suggest a Fenton-like reaction of H2O2 with iron ions, more available in the differentiating cells, as occurring at the surface and/or in the lipid bulk phase of the inner mitochondrial membrane, thus injuring subunits responsible for the coupling of F0F1 ATP synthase through generation in situ of the actual damaging species. Besides, we propose heme iron as the most likely candidate for such reaction in induced cells actively synthesizing heme. In accordance, pretreatment of uninduced cells with hemin made H2O2-damage qualitatively identical.

Acetamides↗

PubMed1993

Effect of neutral and acidic phospholipids on mitochondrial ATP synthase secondary structure.

The secondary structure of delipidated and egg phosphatidylcholine or asolectin reconstituted mitochondrial ATP synthase complex from beef heart was investigated by Fourier transform infrared spectroscopy. Upon reconstitution, the infrared spectra of ATP synthase revealed an increase in turns and a concomitant decrease in beta-sheet content which occurred to a larger extent in the presence of asolectin rather than in the presence of egg phosphatidylcholine. These data correlate with kinetic data showing a higher ATPase activity of the asolectin reconstituted enzyme protein than the egg phosphatidylcholine reconstituted or delipidated enzyme complexes.

Animals↗

PubMed1993

H2O2-induced damage to beef heart mitochondria F0F1 ATP synthase complex: differential sensitivity of the F1 and F0 moieties.

Exposure of purified mitochondrial F0F1 ATP synthase to H2O2 resulted in a marked inhibition of the ATPase activity, irrespective of the purification procedure used and of the incorporation of the enzyme complex into phospholipid vesicles. The inactivation appeared consequent to oxidative modifications of the F1 moiety, whereas damage to the F0 sector, leading to low enzyme activity through impaired binding with F1, seemed not to occur. In fact, when H2O2-inactivated complex was deprived of F1, no loss of the capacity of the F0 sector thus obtained to properly reassemble with untreated purified F1 was apparent, because the resulting enzyme complex showed full activity and oligomycin sensitivity. On the contrary, the exposure of the isolated components F1 or F0 to H2O2, followed by reassembly with untreated F0 and F1 respectively, resulted in both cases in lower catalytic activity of the reconstituted complexes, whereas low oligomycin sensitivity was exhibited only in the case of F0 treatment, suggesting the inactivation in this case as due to oxidative modifications leading to impaired binding with F1.

Animals↗

PubMed1991

The inactivation of mitochondrial F1 ATPase by H2O2 is mediated by iron ions not tightly bound in the protein.

Exposure to purified mitochondrial F1 ATPase to continuous flux of H2O2 resulted in significant loss (up to 60%) of the ATP hydrolytic activity. The presence of chelating agents including desferrioxamine or previous selective removal of the iron ions not tightly bound in the protein completely prevented the inactivation, whereas re-loading of the enzyme with F3+ restored the sensitivity to H2O2. A marked protective effect was provided as well by mannitol or by Cu,Zn superoxide dismutase. The results indicated the decomposition of H2O2 by redox-active iron-protein adducts as responsible for the enzyme inactivation, probably through site-directed generation of more highly reactive oxygen species. A possible role for iron associated to F1 component in the oxidation, aging and turnover of ATP synthase complex in vivo may be suggested on the basis on these results.

Adenosine Triphosphate↗

PubMed1991

Structural and functional modifications induced by diamide on the F0 sector of the mammalian ATP synthase.

In this report data are presented which firmly establish that by treating isolated F0 with the thiol reagent diamide, two 25 kDa F0 subunits react to form a dimer of 45 kDa apparent molecular mass. This dimerising effect is correlated to the impairment of the binding of F1 to F0, both at microM and mM diamide concentrations. Under the latter condition, modification of other F0 subunits also occurs. Passive proton conductance through F0, as well as its sensitivity to N,N'-dicyclohexylcarbodiimide, are affected at low diamide concentration. Thus perturbation of the cysteine residue of the 25 kDa F0 subunit is sufficient for altering the ATP synthase proton channel.

Animals↗

PubMed1988

ATP synthase complex from beef heart mitochondria. Role of the thiol group of the 25-kDa subunit of Fo in the coupling mechanism between Fo and F1.

In order to assess the role of thiol groups in the Fo part of the ATP synthase in the coupling mechanism of ATP synthase, we have treated isolated Fo, extracted from beef heart Complex V with urea, with thiol reagents, primarily with diazenedicarboxylic acid bis-(dimethylamide) (diamide) but also with Cd2+ and N-ethylmaleimide. FoF1 ATP synthase was reconstituted by adding isolated F1 and the oligomycin-sensitivity-conferring-protein (OSCP) to Fo. The efficiency of reconstitution was assessed by determining the sensitivity to oligomycin of the ATP hydrolytic activity of the reconstituted enzyme. Contrary to Cd2+, incubation of diamide with Fo, before the addition of F1 and OSCP, induced a severe loss of oligomycin sensitivity, due to an inhibited binding of F1 to Fo. This effect was reversed by dithiothreitol. Conversely, if F1 and OSCP were added to Fo before diamide, no effect could be detected. These results show that F1 (and/or OSCP) protects Fo thiols from diamide and are substantiated by the finding that the oligomycin sensitivity of ATP hydrolysis activity of isolated Complex V was also unaltered by diamide. Gel electrophoresis of FoF1 ATP synthase, reconstituted with diamide-treated Fo, revealed that the loss of oligomycin sensitivity was directly correlated with diminution of band Fo 1 (or subunit b). Concomitantly a band appeared of approximately twice the molecular weight of subunit Fo 1. As this protein contains only 1 cysteine residue (Walker, J. E., Runswick, M. J., and Poulter, L. (1987) J. Mol. Biol. 197, 89-100), the effect of diamide is attributed to the formation of a disulfide bridge between two of these subunits. These results offer further evidence for the proposal, based on aminoacid sequence and structural analysis, that subunit Fo 1 of mammalian Fo is involved in the binding with F1 (Walker et al. (1987]. N-Ethylmaleimide affects oligomycin sensitivity to a lesser extent than diamide, suggesting that the mode of action of these reagents (and the structural changes induced in Fo) is different.

Adenosine Triphosphatases↗

PubMed1988

Kinetics of the release of the mitochondrial inhibitor protein. Correlation with synthesis and hydrolysis of ATP.

(1) The kinetics of the release of the mitochondrial inhibitor protein (IF1) is studied in bovine heart submitochondrial vesicles supplemented with 125I-labelled IF1, using a method for rapidly 'freezing' the state of F1-IF1 interaction. It is shown that generation of a protonmotive force leads to release of IF1 from F1 into solution, following an exponential process. (2) In one set of experiments the rate of IF1 release, in IF1 supplemented vesicles generating a protonmotive force, is correlated with the induction of ATP hydrolytic capacity. It is found that, even under different metabolic states (phosphorylating and non-phosphorylating conditions), both processes follow the same time-course (half-time of around 40 s) and that there is a direct correlation between induced ATPase capacity and IF1 released. This finding rules out the possibility of a non-inhibitory binding site for IF1 on the membrane. (3) In a second set of experiments, also using IF1 supplemented vesicles, the induction of the ATP hydrolytic capacity after energisation is correlated with the induction of the ATP synthetic capacity. Initial rates of both processes are monitored using firefly luciferase, keeping the assay systems as similar as possible. It is shown that the induction of each capacity follows an exponential time-course, with a half-time of around 40 s. This is in good agreement with the half-times obtained for the induction of ATP hydrolytic capacity and the rate of IF1 release, using the quench-stop method. (4) If the induction of ATP hydrolytic and synthetic capacities is followed in untreated submitochondrial vesicles, i.e., vesicles not supplemented with IF1, the extent and time-course of the change in both hydrolytic and synthetic capacities remain correlated, but the half-time of the transient falls to around 10 s. It is suggested that the length of the transient, observed in IF1 supplemented vesicles, results from partial loss of coupling during repeated centrifugations. (5) These results demonstrate that energy-dependent release of IF1 from F1 into solution results in a concomitant increase in both ATP synthetic and hydrolytic capacities of the ATP synthase complex, and that the time-course of this process is sensitive to the degree of coupling of the vesicles.

Adenosine Triphosphate↗

PubMed1988

The binding and release of the inhibitor protein are governed independently by ATP and membrane potential in ox-heart submitochondrial vesicles.

(1) The effects of membrane potential (delta psi) and nucleotides on the interaction between the F1-ATP synthase and its natural inhibitor protein (IF1) are studied in ox-heart submitochondrial vesicles. (2) Membrane potential causes displacement of IF1 from submitochondrial vesicles, as shown by measuring both delta psi-dependent stimulation of ATPase capacity and release of 125I-labelled IF1 from the vesicles. These effects are abolished if ATP is included in the incubation. (3) There is a linear increase in the steady-state ATPase capacity of oxidising vesicles as delta psi is increased from 100 mV to 135 mV. Increasing delta psi above 140 mV leads to no further change. (4) At a constant membrane potential, ATP suppresses the increase in ATPase capacity, with a concentration for half maximal effect of 140 microM. This value is close to the Km for ATP hydrolysis by membrane-bound F1. This suppression is related to ATP concentration rather than to delta Gp or ATP/ADP ratio. (5) The unidirectional on- and off-rates of IF1 were measured separately. The off-rate of IF1 is increased by membrane potential but unaffected by ATP. The on-rate, conversely, is increased by ATP. Thus, the suppression of the potential-dependent net release of IF1 from submitochondrial vesicles by ATP results from an increase of the IF1 on-rate above the off-rate.

Adenosine Triphosphate↗

PubMed1987

3-Hydroxy-3-methylglutaric, adipic, and 2-oxoglutaric acids measured by HPLC in the plasma from diabetic patients.

A method for the measurement of organic acids in human plasma is presented. The analytical procedure consists of plasma protein precipitation with acetonitrile, acid extraction by chromatography through a DEAE-cellulose column eluted with 100 mM perchloric acid, HPLC by cation-exchange column Aminex HPX-87 eluted with 6.5 mM sulfuric acid. Adipic, 3-hydroxy-3-methylglutaric, 2-oxoglutaric, and citric acids were determined in the plasma of diabetic patients. The concentrations of all the measured acids, but particularly those of adipic and 3-hydroxy-3-methylglutaric acids, were significantly higher than those of healthy controls. These results suggest that in diabetics the omega-oxidation of fatty acids is enhanced.

Adipates↗