Biomedical subjects

A Baracca

Publications and source records attributed to A Baracca.

At least 19 recordsLinked to original sources

PubMed2004

Increased state 4 mitochondrial respiration and swelling in early post-ischemic reperfusion of rat heart.

Isolated rat hearts were exposed to 30 min ischemia or to 30 min ischemia followed by 2, 5 or 40 min reperfusion and mitochondria were isolated at these different time points. ADP-stimulated, succinate-dependent respiration rate (state 3) was not significantly changed at the different time points examined. In contrast, state 4 (non-ADP-stimulated) respiration rate was significantly increased after 30 min ischemia, and it increased further during the first post-ischemic reperfusion period. Mitochondrial swelling, as evaluated under conditions of the major controlled ion channels (i.e. permeability transition pore and ATP-dependent mitochondrial K(+) channel) closed, significantly increased in parallel. It is suggested that the inner mitochondrial membrane permeability is increased under exposure of the heart to ischemia and early reperfusion, and that the phenomenon is reversible upon subsequent long periods of reperfusion.

Adenosine Triphosphatases↗

PubMed2000

Catalytic activities of mitochondrial ATP synthase in patients with mitochondrial DNA T8993G mutation in the ATPase 6 gene encoding subunit a.

We investigated the biochemical phenotype of the mtDNA T8993G point mutation in the ATPase 6 gene, associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in three patients from two unrelated families. All three carried >80% mutant genome in platelets and were manifesting clinically various degrees of the NARP phenotype. Coupled submitochondrial particles prepared from platelets capable of succinate-sustained ATP synthesis were studied using very sensitive and rapid luminometric and fluorescence methods. A sharp decrease (>95%) in the succinate-sustained ATP synthesis rate of the particles was found, but both the ATP hydrolysis rate and ATP-driven proton translocation (when the protons flow from the matrix to the cytosol) were minimally affected. The T8993G mutation changes the highly conserved residue Leu(156) to Arg in the ATPase 6 subunit (subunit a). This subunit, together with subunit c, is thought to cooperatively catalyze proton translocation and rotate, one with respect to the other, during the catalytic cycle of the F(1)F(0) complex. Our results suggest that the T8993G mutation induces a structural defect in human F(1)F(0)-ATPase that causes a severe impairment of ATP synthesis. This is possibly due to a defect in either the vectorial proton transport from the cytosol to the mitochondrial matrix or the coupling of proton flow through F(0) to ATP synthesis in F(1). Whatever mechanism is involved, this leads to impaired ATP synthesis. On the other hand, ATP hydrolysis that involves proton flow from the matrix to the cytosol is essentially unaffected.

ATP Synthetase Complexes↗

PubMed2000

Effect of the oxidative stress induced by adriamycin on rat hepatocyte bioenergetics during ageing.

We have investigated the effect of ageing and of adriamycin treatment on the bioenergetics of isolated rat hepatocytes. Ageing per se, whilst being associated with a striking increase of hydrogen peroxide in the cells, induces only minor changes on mitochondrial functions. The adriamycin treatment induces a decrease of the mitochondrial membrane potential in situ and a consistent increase of the superoxide anion cellular content independently of the donor's age, whilst the hydrogen peroxide is significantly higher in aged than in adult rat hepatocytes. Kinetic studies in isolated mitochondria show that the mitochondrial respiratory chain activity (NADH --> O2) of 50 microM adriamycin-treated hepatocytes is lowered both in adult and aged rats. The same adriamycin concentration induces a slight decrease of the maximal rate of ATP hydrolysis in both young and aged rats, without affecting the Km for the substrate. However, at drug concentrations lower than 50 microM, both ATPase and NADH oxidation activities decrease significantly in aged rats only. The results suggest that free radicals increase during ageing in rat hepatocytes but are unable to induce major modifications of mitochondrial bioenergetics. This contrasts with the damaging effect of adriamycin, suggesting that some effects of the drug may be due to other reasons besides oxidative stress.

Adenosine Triphosphate↗

PubMed2000

Emerin presence in platelets.

Emerin is an almost ubiquitous protein which is abnormal in X-linked Emery-Dreifuss muscular dystrophy (EMD), a syndrome characterized by muscle weakness, joint contractures and cardiac arrhythmia. Emerin is localized in the cells at the nuclear rim and its function is still unknown. In some models, emerin has also been described in the cytoplasm; however, its presence outside the nucleus is still matter of debate. We report the presence of emerin in circulating normal human platelets and its absence in platelets from X-linked EMD patients. Since platelets are cytoplasmic fragments derived from megakaryocytes, the presence of emerin in platelets confirms cytoplasmic localization of this protein, probably related to specific functions. We found also that emerin is present in the cytoplasm of megakaryocytes, while it is absent in circulating granulocytes.

Blood Platelets↗

PubMed1999

The effect of aging and an oxidative stress on peroxide levels and the mitochondrial membrane potential in isolated rat hepatocytes.

We have investigated the effect of ageing and of adriamycin treatment on the bioenergetics of isolated rat hepatocytes. Ageing per se, whilst being associated with a striking increase of hydrogen peroxide in the cells, induces only minor changes on the mitochondrial membrane potential. The adriamycin treatment induces a decrease of the mitochondrial membrane potential in situ and a consistent increase of the superoxide anion cellular content independently of the donor age. The hydrogen peroxide is significantly increased in both aged and adult rat hepatocytes, however, due to the high basal level in the aged cells, it is higher in aged rat cells not subjected to oxidative stress than that elicited by 50 microM adriamycin in young rat hepatocytes. The results suggest that a hydrogen peroxide increase in hepatocytes of aged rats is unable to induce major modifications of mitochondrial bioenergetics. This contrasts with the damaging effect of adriamycin, suggesting that the effects of the drug may be due to the concomitant high level of both superoxide and hydrogen peroxide.

Aging↗

PubMed1997

Modification of the mitochondrial F1-ATPase epsilon subunit, enhancement of the ATPase activity of the IF1-F1 complex and IF1-binding dependence of the conformation of the epsilon subunit.

Treatment of bovine heart submitochondrial particles with a low concentration of 2-hydroxy-5-nitrobenzyl bromide (HNB), a selective reagent for the Trp residue of the epsilon subunit [Baracca, Barogi, Lenaz and Solaini (1993) Int. J. Biochem. 25, 1269-1275], enhances the ATP hydrolytic activity of the particles exclusively when the natural inhibitor protein IF1 is present. Similarly, isolated F1 [the catalytic sector of the mitochondrial H+-ATPase complex (ATP synthase)] treated with the reagent has the ATPase activity enhanced exclusively if IF1 is bound to it. These experiments suggest that the modification of the epsilon subunit decreases the inhibitory activity of IF1, eliciting the search for a relationship between the epsilon subunit and the inhibitory protein. Certainly, a reverse relationship exists because HNB binds covalently to the isolated F1 exclusively when the inhibitory protein is present. This finding is consistent with the existence of the epsilon subunit in different conformational states depending on whether IF1 is bound to F1 or not. Support for this assertion is obtained by measurements of the intrinsic phosphorescence decay rate of F1, a probe of the Trp epsilon subunit conformation in situ [Solaini, Baracca, Parenti-Castelli and Strambini (1993) Eur. J. Biochem. 214, 729-734]. A significant difference in phosphorescence decay rate is detected when IF1 is added to preparations of F1 previously devoid of the inhibitory protein. These studies indicate that IF1 and the epsilon subunit of the mitochondrial F1-ATPase complex are related, suggesting a possible role of the epsilon subunit in the mechanism of regulation of the mitochondrial ATP synthase.

2-Hydroxy-5-nitrobenzyl Bromide↗

PubMed1997

Protonophoric activity of NADH coenzyme Q reductase and ATP synthase in coupled submitochondrial particles from horse platelets.

A method to prepare coupled submitochondrial particles from horse platelets is described. The method allowed us to study the protonophoric activities of both complex I and complex V following the fluorescence quenching of the monoamine 9-amino-6-chloro-2 methoxyacridine (ACMA), a probe highly sensitive to the generation of a transmembrane delta pH. We carried out a kinetic analysis of each enzyme complex studying the proton translocation and the electron transfer activities of complex I as well as the proton translocation and the ATP hydrolytic activities of complex V. A micromethod to prepare coupled submitochondrial particles from platelets might be useful to investigate cell bioenergetic damage occurring in mitochondrial diseases and ageing.

Adenosine Triphosphatases↗

PubMed1997

Structural mapping of the epsilon-subunit of mitochondrial H(+)-ATPase complex (F1).

Phosphorescence and fluorescence energy transfer measurements have been used to locate the epsilon-subunit within the know structural frame of the mitochondrial soluble part of F-type H(+)-ATPase complex (F1). The fluorescence probe 2'-O-(trinitrophenyl)adenosine-5'-triphosphate was bound to the nucleotide binding sites of the enzyme, whereas the probe 7-diethylamino-3'-(4'-maleimidylphenyl)-4-methylcoumarin was attached to the single sulfhydryl residue of isolated oligomycin sensitivity-conferring protein (OSCP), which was then reconstituted with F1. Fluorescence and phosphorescence resonance energy transfer yields from the lone tryptophan residue of F1 present in the epsilon-polypeptide and the fluorescence labels attached to the F1 complex established that tryptophan is separated by 3.7 nm from Cys-118 of OSCP in the reconstituted OSCP-F1 complex, by 4.9 nm from its closest catalytic site and by more than 6.4 nm from the two other catalytic sites, including the lowest affinity ATP site. These separations together with the crystallographic coordinates of the F1 complex (Abrahams, J.P., A. G. W. Leslie, R. Lutter, and J.E. Walker. 1994. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 370:621-628) place the epsilon-subunit in the stem region of the F1 molecule in a unique asymmetrical position relative to the catalytic sites of the enzyme.

Adenosine Triphosphatases↗

PubMed1995

Lack of major changes in ATPase activity in mitochondria from liver, heart, and skeletal muscle of rats upon ageing.

ATP hydrolase activity has been investigated in mitochondria from liver, heart, and skeletal muscle from adult (6 months) and aged (24 months) rats. No significant changes in total ATPase activity were observed in the three tissues, but the oligomycin sensitivity was slightly decreased in heart mitochondria of aged rats. The bicarbonate-induced stimulation of hydrolytic activity was somewhat decreased in mitochondria from aged rats, particularly in liver. No significant change was observed in ATPase activity after release of the endogenous inhibitor protein, IF1. It is concluded that no activity changes to be directly ascribed to the catalytic sector F1 of the enzyme occur upon ageing, but it cannot be excluded that changes in the membrane sector F0 occur as a consequence of mtDNA mutations.

Adenosine Triphosphatases↗

PubMed1995

Conformational changes of the mitochondrial F1-ATPase epsilon-subunit induced by nucleotide binding as observed by phosphorescence spectroscopy.

Changes in conformation of the epsilon-subunit of the bovine heart mitochondrial F1-ATPase complex as a result of nucleotide binding have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas nucleoside triphosphate binding to the enzyme in the presence of Mg2+ increases the flexibility of the protein structure surrounding the chromophore, nucleoside diphosphate acts in an opposite manner, enhancing the rigidity of this region of the macromolecule. Such changes in dynamic structure of the epsilon-subunit are evident at high ligand concentration added to both the nucleotide-depleted F1 (Nd-F1) and the F1 preparation containing the three tightly bound nucleotides (F1(2,1)). Since the effects observed are similar in both the F1 forms, the binding to the low affinity sites must be responsible for the conformational changes induced in the epsilon-subunit. This is partially supported by the observation that the Trp lifetime is not significantly affected by adding an equimolar concentration of adenine nucleotide to Nd-F1. The effects on protein structure of nucleotide binding to either catalytic or noncatalytic sites have been distinguished by studying the phosphorescence emission of the F1 complex prepared with the three noncatalytic sites filled and the three catalytic sites vacant (F1(3,0)). Phosphorescence lifetime measurements on this F1 form demonstrate that the binding of Mg-NTP to catalytic sites induces a slight enhancement of the rigidity of the epsilon-subunit. This implies that the binding to the vacant noncatalytic site of F1(2,1) must exert the opposite and larger effect of enhancing the flexibility of the protein structure observed in both Nd-F1 and F1(2,1). The observation that enhanced flexibility of the protein occurs upon addition of adenine nucleotides to F1(2,1) in the absence of Mg2+ provides direct support for this suggestion. The connection between changes in structure and the possible functional role of the epsilon-subunit is discussed.

Adenine Nucleotides↗

PubMed1995

A study of the mitochondrial F1-ATPase tryptophan phosphorescence at 273 K.

The bovine heart mitochondrial F1-ATPase complex exhibits an intrinsic tryptophan phosphorescence that can be used to monitor structural changes of the epsilon-subunit. The phosphorescence decay rate of F1 containing the tightly bound nucleotides increases upon addition of adenine nucleoside triphosphate in the presence of magnesium. The average phosphorescence lifetime of this enzyme preparation decreases from 10.2 to 7.8 ms upon Mg-ATP addition. Since increasing phosphorescence decay rate is related to increasing flexibility of proteins, Mg-ATP added to the F1-ATPase complex can enhance the flexibility of the protein structure surrounding the chromophore. Experiments carried out on F1 prepared with the three noncatalytic sites filled and the three catalytic sites vacant show a significant increase of the phosphorescence lifetime from 6.4 ms to 7.6 ms upon Mg-ATP addition. These results suggest that the mitochondrial F1-ATPase epsilon-subunit conformation senses differently the nucleoside triphosphate binding to catalytic or noncatalytic sites.

Adenosine Triphosphate↗

PubMed1994

Mitochondrial activities of rat heart during ageing.

Some analytical and functional parameters of rat heart mitochondrial have been investigated at six different periods of ageing from 2 to 26 months. The fatty acid composition of the mitochondrial membranes reveals a percentage increase of polyunsaturated fatty acids (20:4 n-6, 22:6 n-3) up to 12 months, followed by a decrease; however, fluorescence polarization of the membrane probe diphenylhexatriene is not changed, revealing that membrane fluidity is not significantly affected. No major change in ubiquinone-9 and in cytochrome content is apparent, indicating that the relative ratio of the respiratory chain components is unmodified. Nevertheless, significant changes in enzyme specific activities are detected: NADH cytochrome c reductase and cytochrome oxidase activities increase up to 12 months, then decrease at 18-26 months; ubiquinol cytochrome c reductase exhibits a peak at 18 months, followed by a decrease. All these activities follow a similar trend during the whole life span of the rat, even though the 'maximum' is different. No significant changes have been found in ATP synthase. Succinate-cytochrome c reductase steadily increases over the whole life span. The results, showing activity decreases in the respiratory enzymes having subunits encoded by mitochondrial DNA, are compatible with the 'mitochondrial' theory of ageing.

Aging↗

PubMed1994

Dietary lipids and 5'-nucleotidase activity of rat cell plasma membranes.

Three groups of adult rats were fed black currant or olive oils or a 1:1 mixture of the two for three months. Feeding black currant oil diets, high in 18:2 (n-6), 18:3 (n-6), 18:3 (n-3), increased the heart and liver plasma membrane content of linoleic and arachidonic acid with a concomitant decrease of oleic acid. PUFA, n-3 and n-6 content and the bilayer lipid fluidity as examined by measuring the fluorescence anisotropy of diphenylhexatriene were not significantly affected. On the contrary, the 5'-nucleotidase of liver membrane of rats fed black currant diets was lower than that observed in membranes of liver from olive oil fed rats. Therefore it is concluded that PUFA and n-3/n-6 ratio as well as membrane fluidity do not influence the 5'-nucleotidase activity. It is suggested that the activity is sensitive to the amount of a specific fatty acid of the membrane (i.e., oleic or arachidonic acid) and/or to lipid supplementation which can influence the eicosanoids metabolism.

5'-Nucleotidase↗

PubMed1994

The effect of dietary lipid changes on the fatty acid composition and function of liver, heart and brain mitochondria in the rat at different ages.

A correlation between dietary lipids and cellular enzyme activities is a problem that has only been partially addressed by nutritionists. Therefore, changes in the fatty acid composition and the activities of some key metabolic enzymes (ubiquinol-2-cytochrome c reductase (EC 1.10.2.2), cytochrome oxidase (EC 1.9.3.1) and ATPase (EC 3.6.1.3)) in the mitochondria of liver, heart and brain of rats fed on diets differing extensively in their polyunsaturated fatty acid compositions have been investigated. The results showed that fatty acid compositional changes brought about by the dietary differences were associated with extensive changes in the activities of these key enzymes in the mitochondria. The extent of the influence differed considerably with the period over which the diets were fed. The role of dietary lipids to effect changes through the preservation of membrane structural integrity is discussed.

Adenosine Triphosphatases↗

PubMed1993

Tryptophan phosphorescence as a structural probe of mitochondrial F1-ATPase epsilon-subunit.

We report the detection of tryptophan phosphorescence emission from the sole residue in the epsilon-subunit of the bovine heart mitochondrial F1-ATPase complex. The phosphorescence spectrum, intensity and decay kinetics have been measured over the temperature range 160-273 K. The fine structure in the phosphorescence spectrum at low temperature, with the 0-0 vibrational band centered at 411 nm, reveals the hydrophobic nature of the chromophore's environment. Both the large width of the 0-0 vibrational band and the heterogeneous decay kinetics in fluid solution emphasize the existence of multiple conformations of the epsilon-subunit, structures which are rather stable as they do not interconvert in the millisecond time scale. Further, from the relatively long triplet lifetime at 273 K, it is possible to infer the existence of a tight, rigid core in the structure of the epsilon-subunit. Under subunit-dissociating conditions (6 M urea), the spectrum at 160 K undergoes a slight blue shift but since the phosphorescence lifetime, at all temperatures, is similar or longer than in the absence of dissociant, we conclude that dissociation does not lead to solvent exposure of the tryptophanyl side-chain. This conclusion is supported by the results obtained at 273 K by dissociating F1 in the presence of 0.3 M guanidine hydrochloride. Phosphorescence lifetimes indicate that 6 M urea leads to a more compact structure of the epsilon-subunit, whereas the opposite occurs when Mg-ATP is added to nucleotide-depleted F1. These spectroscopic changes establish unequivocally that the binding of the adenine nucleotide to the enzyme is accompanied by conformational changes involving the epsilon-subunit.

Adenosine Triphosphate↗

PubMed1993

Interactions and effects of 2-hydroxy-5-nitrobenzyl bromide on the bovine heart mitochondrial F1-ATPase.

1. The F1-ATPase from bovine heart mitochondria was shown to chemically react and to absorb 2-hydroxy-5-nitrobenzyl bromide (HNB) with changes in catalytic properties. 2. The treatment of the enzyme with HNB at concentrations below 0.5 mM resulted in an increase of Vm and in an unchanged Km. Above 0.5 mM HNB elicited a concentration-dependent inhibition of F1. 3. HNB was found tightly bound to the enzyme epsilon-subunit whose tryptophan residue resulted modified. 4. The F1 activation appears the consequence of the covalent binding of the reagent to the enzyme, whilst inhibition results from non-covalent, reversible binding. 5. The possibility that the epsilon-subunit of mitochondrial F1-ATPase may influence the functional or regulating domain of the enzyme is discussed.

2-Hydroxy-5-nitrobenzyl Bromide↗

PubMed1993

Diffusion limited component of mitochondrial F1-ATPase.

1. The possibility that the rate of ATP hydrolysis by F1-ATPase approaches the diffusion-controlled limits was investigated by measuring the values of kcat and kl (kcat/Km) as a function of increasing viscosity. 2. The values of kcat/Km decrease significantly with increasing viscosity; further such decrease was lower when Fl-ATPase hydrolyzed poor substrate such as Ca- and Mg-ITP or when the hydrolysis rates were measured at temperatures below 20 degrees C. 3. Viscosity also decreases kcat, but only at high concentrations of viscosogenic agents. 4. These results suggest that ATP hydrolysis is at least partly diffusion-controlled, although a general nonspecific perturbation in the enzyme structure is also effected by viscosity.

Adenosine Triphosphate↗

PubMed1992

Cytochrome b of fish mitochondria is strongly resistant to funiculosin, a powerful inhibitor of respiration.

We report here some unusual properties of ubiquinol: cytochrome c reductase of eel and other fish mitochondria. The turnover rate of the reductase is clearly higher than in mammalian mitochondria and the binding constant for ubiquinone seems to be larger than in other vertebrates. Additionally, the reductase activity of fish mitochondria is resistant to some powerful inhibitors that bind to cytochrome b, in particular to funiculosin. After sequencing most of the gene of eel cytochrome b and comparing the deduced amino acid sequence with that of other fish and animals, we hypothesize that the decreased binding of funiculosin could be due to a few amino acid replacements in the third and fourth transmembrane helix of the protein. In particular, the presence of methionine instead of alanine at position 125 seems to be largely responsible for the strong resistance to funiculosin and also to the partial resistance to myxothiazol in all fish mitochondria. Correlations between some residue substitutions in cytochrome b and the different effects of funiculosin in different species are also considered.

Amino Acid Sequence↗