Biomedical subjects

M Brunori

Publications and source records attributed to M Brunori.

At least 73 records · Page 4Linked to original sources

PubMed1998

Cytochrome c oxidase does not catalyze the anaerobic reduction of NO.

A possible role of reduced cytochrome c oxidase in the metabolism of nitric oxide (NO) has been examined with amperometric and stopped-flow photometric techniques. Reduced purified cytochrome c oxidase and mitochondria showed no catalytic reaction with NO under anaerobic conditions within more than 30 minutes. Only fast binding of NO to the reduced enzyme in a 1:1 stoichiometric ratio was observed. The NO binding rate was strongly decreased in the presence of 1 mM cyanide. These data indicate that, contrary to previous proposals, cytochrome c oxidase in the absence of oxygen does not contribute to physiological NO metabolism.

Anaerobiosis↗

PubMed1998

Equilibrium unfolding of a small bacterial cytochrome, cytochrome c551 from Pseudomonas aeruginosa.

The unfolding of the small cytochrome c551 from the bacterium Pseudomonas aeruginosa has been characterized at equilibrium by circular dichroism (CD) and fluorescence spectroscopy. The process can be described by a two state mechanism and the thermodynamic stability of cytochrome c551 is found to be smaller than that of the larger horse cytochrome c (deltaGw = -8.2 vs. -9.7 kcal/mol); we propose that this finding is related to the absence of an 'omega' loop in the bacterial cytochrome. Cytochrome c551 loses most of its secondary structure at pH 1.5. The acid transition (pKA approximately 2) is highly cooperative (n > or =2); analysis of optical titrations and contact map suggests that (at least) His-16 (proximal Fe3+ ligand) and Glu-70 are both involved in the acid transition. The role of selected hydrophobic, electrostatic and conformational contributions to the overall stability has been investigated by protein engineering. The equilibrium characterization of wild-type and mutant cytochrome c551 supports the view that this small cytochrome is an interesting protein to analyze the thermodynamics and the kinetics of folding in comparison with the widely studied horse cytochrome c.

Acids↗

PubMed1998

Tryptophan 121 of subunit II is the electron entry site to cytochrome-c oxidase in Paracoccus denitrificans. Involvement of a hydrophobic patch in the docking reaction.

To investigate the contribution of hydrophobic residues to the molecular recognition of cytochrome c with cytochrome oxidase, we mutated several hydrophobic amino acids exposed on subunit II of the Paracoccus denitrificans oxidase. KM and kcat values and the bimolecular rate constant were determined under steady- or presteady-state conditions, respectively. We present evidence that Trp-121 which is surrounded by a hydrophobic patch is the electron entry site to oxidase. Mutations in this cluster do not affect the binding of cytochrome c as the KM remains largely unchanged. Rather, the kcat is reduced, proposing that these hydrophobic residues are required for a fine tuning of the redox partners in the initial collisional complex to obtain a configuration optimal for electron transfer.

Copper↗

PubMed1998

Cytochrome-c-binding site on cytochrome oxidase in Paracoccus denitrificans.

To monitor the docking site for cytochrome c on cytochrome oxidase from Paracoccus denitrificans, a series of site-directed mutants in acidic residues exposed on the three largest subunits was constructed, and the purified enzymes were assayed for their steady-state kinetic parameters, their ionic strength dependence, and their fast electron entry kinetics by stopped-flow measurements. Increasing the ionic strength, the maximum of the bell-shaped dependence of the steady-state rate observed for wild type shifts the maximum to lower ionic strength in most of the mutants. The Km determined in steady-state experiments under different conditions is largely increased for most of the subunit II and one of the subunit I mutants, giving evidence that binding is impaired, whereas subunit III residues do not seem to contribute significantly. In addition, the bimolecular rate constant for cytochrome c oxidation under pre-steady state conditions was measured using stopped flow spectroscopy. Taken together, the results demonstrate that the initial interaction of cytochrome c and oxidase is mediated through glutamates and aspartates mainly located in subunit II. The crystal structure of oxidase reveals that the participating residues are clustered, creating an extended, negatively charged patch. We propose this clustering to be a decisive factor in the recognition of positively charged patches on the surface of cytochrome c.

Binding Sites↗

PubMed1998

Unfolding of apomyoglobin from Aplysia limacina: the effect of salt and pH on the cooperativity of folding.

The equilibrium unfolding pathway of Aplysia apomyoglobin has been studied under various solvent conditions. The protein exhibits a single unfolding transition in acid in contrast to the two transitions observed for the mammalian apomyoglobins with which it shares a common fold but a low level of sequence identity (24%). This acid-unfolded species has considerable residual structure as evidenced by both tryptophan fluorescence and far-UV CD spectroscopy. It remains 40% alpha-helical under low salt conditions (2 mM citrate, 4 degrees C); the folded form is 65% helical. A similar species is observed for the mammalian globins in mild acid conditions. Titration with GdnHCl at pH 7 reveals two unfolding transitions, the first having common features with that observed in acid and the second resulting in a completely unfolded state. Under the same conditions, urea unfolds the protein completely in an apparently single cooperative transition. Assuming a simple three-state model (F<-->I<-->U), data from GdnHCl and urea titrations over a range of pH conditions were used to derive values for the apparent stability (delta Gw(app) and solvent accessibility (n(app)) of the folded (F) and intermediate (I) forms of the protein. Urea titrations were then repeated over a range of KCl concentrations in order to understand the contribution of Cl- to the different unfolding activity of GdnHCl. A three-state scheme is justified when changes in delta G(w(app)) occur without changes in n(app). The change in free energy of folding of I<-->F (delta Gw(F/I)) decreases to 0 at pH 4 as expected from the acid unfolding curve. delta Gw(I/U) reaches its maximum at pH 4.5, the isoelectric point of the protein. Variations of this value with pH and chloride are as much as 3 kcal mol-1 and correlate closely with changes in n(app) although there is no change in the alpha-helical content of I across the pH range. This observation is interpreted here as a deviation of the unfolding of the I state of Aplysia apomyoglobin from a cooperative behaviour.

Animals↗

PubMed1998

Kinetic control of internal electron transfer in cytochrome c oxidase.

Two alternative hypotheses have been proposed to account for the relatively slow (ms) internal eT observed in the oxidized cyt c oxidase. The thermodynamic control hypothesis states that eT between cyt a and a3 is very fast (microsecond), but the apparent reduction of cyt a3 is slow because thermodynamics favors reduced cyt a. Whereas the kinetic control hypothesis states that inter-heme eT is intrinsically slow (ms), for the oxidized binuclear center. Monitoring by stopped flow the anaerobic reduction of the oxidized enzyme by ruthenium hexamine in the absence and presence of CO or NO, used as "trapping" ligands for cyt a3(2+), we found that the rate of formation of the cyt a3(2+)-NO adduct (k' approximately 20-25 s-1) is independent of the concentration of ruthenium hexamine and NO. We conclude that in the oxidized enzyme the two hemes are not in very rapid redox equilibrium and internal eT is kinetically controlled.

Anaerobiosis↗

PubMed1998

The effect of monensin and chloroquine on the endocytosis and toxicity of chimeric toxins.

The toxicity of two conjugates containing ribosome-inactivating proteins (RIPs, i.e. saporin and ricin-A chain x-linked to transferrin) has been measured on a prostatic cancer line (PC3) naturally overexpressing the transferrin receptor, in the presence of monensin and chloroquine. This paper investigates whether the increased toxicity of Tf-RIPs induced by monensin and chloroquine may be due to alterations of the normal endocytotic pathway of the complexes mediated by the transferrin receptor. Monensin, besides inducing alkalinization of normally acid intracellular compartments, causes an accumulation of the receptor-bound Tf-RIP in a perinuclear region contiguous to the cisternae of the trans-Golgi network. Chloroquine, though increasing the intracellular pH, seems not to modify the endocytotic pathway of these chimeric molecules. We believe that the enhanced toxicity of the Tf-RIPs may be related to intracellular alkalinization (i.e., endosomal or lysosomal pH) rather than to the effects on the recycling of transferrin receptor-bound toxins. We conclude that the efficacy of chimeric toxins may be modulated not only by the carrier used for their engineering but also by addition of drugs able to influence the stability and activation of the toxins inside the cell.

Chloroquine↗

PubMed1998

Investigating the mechanism of electron transfer to the binuclear center in Cu-heme oxidases.

Novel experimental evidence is presented further supporting the hypothesis that, starting with resting oxidized cytochrome c oxidase, the internal electron transfer to the oxygen binding site is kinetically controlled. The reduction of the enzyme was followed spectroscopically and in the presence of NO or CO, used as trapping ligands for reduced cytochrome a3; ruthenium hexamine was used as a spectroscopically silent electron donor. Consistent with the high combination rate constant for reduced cytochrome a3, NO proved to be a very efficient trapping ligand, while CO did not. The results are discussed in view of two alternative (thermodynamic and kinetic) hypotheses of control of electron transfer to the binuclear (cyt.a3-CuB) center. Fulfilling the prediction of the kinetic control hypothesis: i) the reduction of cytochrome a3 and ligation are synchronous and proceed at the intrinsic rate of cytochrome a3 reduction, ii) the measured rate of formation of the nitrosyl derivative is independent of the concentration of both the reductant and NO.

Animals↗

PubMed1998

Paroxysmal atrial fibrillation after high-dose melphalan in five patients autotransplanted with blood progenitor cells.

Among the drugs used in conditioning regimens for stem cell transplantation, high-dose melphalan (HDM) plays an important role for both its strong myeloablative effect and for its favourable dose-response ratio. Here we report five cases of high frequency atrial fibrillation (AF) developing after HDM. Duration of the arrhythmia was always very short, beginning at variable intervals after the administration of HDM, in the absence of other factors potentially able to trigger AF. In all patients sinus rhythm was restored within 72 h and the follow-up did not show any cardiac damage. To the best of our knowledge, this side-effect has never been reported to occur after HDM.

Antineoplastic Agents, Alkylating↗

PubMed1998

Expression and potential role of Fsrg1, a murine bromodomain-containing homologue of the Drosophila gene female sterile homeotic.

We have isolated a cDNA which is a murine homologue of the Drosophila gene female sterile homeotic (fsh). This homologue, which we have designated Fsrg1*, contains two bromodomains and an ET motif characteristic of the Fsh sub-class of bromodomain-containing proteins. Northern blot hybridization analysis of adult tissues revealed that Fsrg1 was expressed at low levels rather ubiquitously, but most abundantly in the testis and ovary. Polyclonal antibodies raised against an Fsrg1 fusion protein were used to characterize the Fsrg1 gene product in tissues. Constructs were also generated in which the Fsrg1 cDNA was tagged with epitopes for hemaglutinin and used in transfection experiments. Immunoblot analysis revealed that the Fsrg1 protein migrates with a relative molecular mass of approximately 110 kDa, although the cDNA sequence would predict a protein of approximately 88 kDa. The migration at approximately 110 kDa was observed for both in vivo protein and protein produced in cultured cells. The Fsrg1 protein was localized to the nucleus when expressed in cultured cells, consistent with the presence of a nuclear localization signal motif in the Fsrg1 sequence. No kinase activity was detected for this nuclear protein as assessed in either autokinase or specific substrate assays. In situ hybridization analysis revealed strikingly high expression of Fsrg1 in granulosa cells of growing follicles in the adult ovary and suggested its possible involvement in folliculogenesis. Additional clues to its potential function were provided by the demonstration of its high level of expression in epithelia of tissues which undergo hormonally-modulated remodeling.

3T3 Cells↗

PubMed1998

Factors affecting hemopoietic recovery after high-dose therapy and autologous peripheral blood progenitor cell transplantation: a single center experience.

BACKGROUND AND OBJECTIVE: While the minimum number of CD34+ cells required for complete and long-lasting engraftment is quite well established, there is not general agreement about the optimal number of CD34+ per kg needed in order to obtain engraftment as rapidly as possible. In the present study we assess factors affecting hemopoietic recovery and the optimal peripheral blood progenitor cell (PBPC) number for rapid engraftment in patients treated with high-dose therapy. DESIGN AND METHODS: We enrolled 80 consecutive patients affected by hematologic and non-hematologic malignancies treated with a median of 10 chemotherapy courses (range 3-38). PBPC collection was performed after mobilization with high-dose chemotherapy and G-CSF 5 micrograms/kg/day. The circulating and harvested CD34+ cells were recognized in the cytofluorimetric CD45+/CD14- lymphocyte gate. After myeloablative therapy, PBPC infusion was followed by G-CSF 5 micrograms/kg/day from day +5 until WBC > or = 5.0 x 10(9)/L. Univariate and multivariate Cox analyses were performed to investigate factors affecting hemopoietic recovery. The Kaplan-Meier probabilities of hemopoietic reconstitution were compared by log-rank test to assess the optimal CD34+ cell number for rapid engraftment. RESULTS: We performed a median of two apheresis (range 1-4) per patient and we infused a median of 6.1 x 10(6) CD34+ cells/kg (range 0.5-30.5). Absolute neutrophil count (ANC) > 0.5 x 10(9)/L was reached after 11 days (range 8-15). The only factor affecting granulocyte recovery proved to be the CD34+ cell number; 5.0 to 7.8 x 10(6) CD34+ cells/kg allowed a significantly faster granulocyte recovery than < 2.5 x 10(6) CD34+ cells/kg (p = 0.0312). Platelet transfusion independence (> 20 x 10(9)/L) and 50 x 10(9)/L platelets were reached after 12 (range 8-24) and 15 days (range 9-40), respectively. The CD34+ cell number was also the only factor affecting platelet recovery; the number of 5.0 to 7.8 CD34+ cells/kg allowed a significantly faster platelet recovery than the lower dose, whereas a higher number did not. No late graft failures were observed. Patients receiving 5.0 to 7.8 x 10(9) CD34+ cells/kg had a significantly shorter duration of neutropenia, fewer platelet transfusions and less time spent in hospital than those receiving lower number did, whereas patients transplanted with a higher number had no advantage. INTERPRETATION AND CONCLUSIONS: When G-CSF is employed both for PBPC mobilization and after PBPC transplantation, the CD34+ cell number is the only factor that affects hemopoietic recovery. Moreover, > 5.0 x 10(6) CD34+ cells/kg is the optimal number for obtaining rapid platelet recovery and reducing the costs of HDT but there is no advantage exceeding the threshold of 7.8 x 10(6) CD34+ cells/kg.

Adolescent↗

PubMed1997

Covalent complex of microperoxidase with a 21-residue synthetic peptide as a maquette for low-molecular-mass redox proteins.

Here we report the structural and functional characterization of a covalent complex (MKP) obtained by cross-linking microperoxidase (Mp), the haem-undecapeptide obtained by the peptic digestion of cytochrome c, with a 21-residue synthetic peptide (P21) analogous to the S-peptide of the RNase A. The covalent complex has been prepared by introducing a disulphide bond between Cys-1 of P21 and Lys-13 of Mp, previously modified with a thiol-containing reagent. On formation of the complex (which is a monomer), the helical content of P21 increases significantly. The results obtained indicate that His-13 of P21 co-ordinates to the sixth co-ordination position of the haem iron, thus leading to the formation of a complex characterized by an equilibrium between an 'open' and a 'closed' structure, as confirmed by molecular dynamics simulations. Under acidic pH conditions, where His-13 of P21 is loosely bound to the haem iron ('open' conformation), MKP displays appreciable, quasi-reversible electrochemical activity; in contrast, at neutral pH ('closed' conformation) electrochemical behaviour is negligible, indicating that P21 interferes with the electron-transfer properties typical of Mp. On the whole, MKP is a suitable starting material for building a miniature haem system, with interesting potential for application to biosensor technology.

Amino Acid Sequence↗

PubMed1997

Functional properties of the quinol oxidase from Acidianus ambivalens and the possible catalytic role of its electron donor--studies on the membrane-integrated and purified enzyme.

The aa3 quinol oxidase has been purified from the thermoacidophilic archaea Acidianus ambivalens as a three-redox-centers enzyme. The functional properties of this oxidase both as purified and in its most integral form (i.e. in native membranes and in intact cells) were investigated by stopped-flow spectrophotometry. The results suggest that the enzyme interacts in vivo with a redox-active molecule, which favours the electron entry via heme a and provides the fourth electron demanded for catalysis. We observe that the purified enzyme has two hemes with apparent redox potentials 215 +/- 20 mV and 415 +/- 20 mV at pH 5.4, showing redox-Bohr effect, and a heme a3-CuB center with an affinity for carbon monoxide (Ka = 5.7 x 10(4) M(-1) at 35 degrees C) much lower than that reported for the mammalian enzyme (Ka = 4 x 10(6) M(-1) at 20 degrees C). The reduction by dithionite is fast and monophasic when the quinol oxidase is in the native membranes, whereas it is slow and biphasic in the purified enzyme (with heme a3 being reduced faster than heme a). The oxygen reaction of the reduced purified enzyme is fast (few milliseconds), but yields an intermediate (likely ferryl) clearly different from the fully oxidized enzyme. In contrast, the same reaction performed in intact cells leads to the fully oxidized enzyme. We postulate that caldariella quinol, the physiological electron donor, is in vivo tightly bound to the enzyme, providing the fourth redox active center lacking in the purified enzyme.

Dithionite↗

PubMed1997

N-terminal arm exchange is observed in the 2.15 A crystal structure of oxidized nitrite reductase from Pseudomonas aeruginosa.

BACKGROUND: Nitrite reductase from Pseudomonas aeruginosa (NiR-Pa) is a dimer consisting of two identical 60 kDa subunits, each of which contains one c and one d1 heme group. This enzyme, a soluble component of the electron-transfer chain that uses nitrate as a source of energy, can be induced by the addition of nitrate to the bacterial growth medium. NiR-Pa catalyzes the reduction of nitrite (NO2-) to nitric oxide (NO); in vitro, both cytochrome c551 and azurin are efficient electron donors in this reaction. NiR is a key denitrification enzyme, which controls the rate of the production of toxic nitric oxide (NO) and ultimately regulates the release of NO into the atmosphere. RESULTS: The structure of the orthorhombic form (P2(1)2(1)2) of oxidized NiR-Pa was solved at 2.15 A resolution, using molecular replacement with the coordinates of the NiR from Thiosphaera pantotropha (NiR-Tp) as the starting model. Although the d1-heme domains are almost identical in both enzyme structures, the c domain of NiR-Pa is more like the classical class I cytochrome-c fold because it has His51 and Met88 as heme ligands, instead of His17 and His69 present in NiR-Tp. In addition, the methionine-bearing loop, which was displaced by His17 of the NiR-Tp N-terminal segment, is back to normal in our structure. The N-terminal residues (5/6-30) of NiR-Pa and NiR-Tp have little sequence identity. In Nir-Pa, this N-terminal segment of one monomer crosses the dimer interface and wraps itself around the other monomer. Tyr10 of this segment is hydrogen bonded to an hydroxide ion--the sixth ligand of the d1-heme Fe, whereas the equivalent residue in NiR-Tp, Tyr25, is directly bound to the Fe. CONCLUSIONS: Two ligands of hemes c and d1 differ between the two known NiR structures, which accounts for the fact that they have quite different spectroscopic and kinetic features. The unexpected domain-crossing by the N-terminal segment of NiR-Pa is comparable to that of 'domain swapping' or 'arm exchange' previously observed in other systems and may explain the observed cooperativity between monomers of dimeric NiR-Pa. In spite of having similar sequence and fold, the different kinetic behaviour and the spectral features of NiR-Pa and NiR-Tp are tuned by the N-terminal stretch of residues. A further example of this may come from another NiR, from Pseudomonas stutzeri, which has an N terminus very different from that of the two above mentioned NiRs.

Amino Acid Sequence↗

PubMed1997

Internal electron transfer in Cu-heme oxidases. Thermodynamic or kinetic control?

We present novel experimental evidence that, starting with the oxidized enzyme, the internal electron transfer in cytochrome c oxidase is kinetically controlled. The anaerobic reduction of the oxidized enzyme by ruthenium hexamine has been followed in the absence and presence of CO or NO, used as trapping ligands for reduced cytochrome a3. In the presence of NO, the rate of formation of the cytochrome a32+-NO adduct is independent of the concentration of ruthenium hexamine and of NO, indicating that in the oxidized enzyme cytochrome a and a3 are not in very rapid redox equilibrium; on the other hand, CO proved to be a poor "trapping" ligand. We conclude that the intrinsic rate constant for a --> a3 electron transfer in the oxidized enzyme is 25 s-1. These data are discussed with reference to a model (Verkhovsky, M. I., Morgan, J. E., and Wikström, M. (1995) Biochemistry 34, 7483-7491) in which H+ diffusion and/or binding at the binuclear site is the rate-limiting step in the reduction of cytochrome a3 in the oxidized enzyme.

Binding Sites↗

PubMed1997

Mutagenesis of nitrite reductase from Pseudomonas aeruginosa: tyrosine-10 in the c heme domain is not involved in catalysis.

In Pseudomonas aeruginosa, conversion of nitrite to NO in dissimilatory denitrification is catalyzed by the enzyme nitrite reductase (NiR), a homodimer containing a covalently bound c heme and a d1 heme per subunit. We report the purification and characterization of the first single mutant of P. aeruginosa cd1 NiR in which Tyr10 has been replaced by Phe; this amino acid was chosen as a possibly important residue in the catalytic mechanism of this enzyme based on the proposal (Fulop, V., Moir, J.W.B., Ferguson, S.J. and Hajdu, J. (1995) Cell 81, 369-377) that the topologically homologous Tyr25 plays a crucial role in controlling the activity of the cd1 NiR from Thiosphaera pantotropha. Our results show that in P. aeruginosa NiR substitution of Tyr10 with Phe has no effect on the activity, optical spectroscopy and electron transfer kinetics of the enzyme, indicating that distal coordination of the Fe3+ of the d1 heme is provided by different side-chains in different species.

Amino Acid Sequence↗

PubMed1997

Electron-transfer properties of Pseudomonas aeruginosa [Lys44, Glu64]azurin.

In the hydrophobic patch of azurin from Pseudomonas aeruginosa, an electric dipole was created by changing Met44 into Lys and Met64 into Glu. The effect of this dipole on the electron-transfer properties of azurin was investigated. From a spectroscopic characterization (NMR, EPR and ultraviolet-visible) it was found that both the copper site and the overall structure of the [Lys44, Glu64]azurin were not disturbed by the two mutations. A small perturbation of the active site at high pH, similar to that observed for [Lys44]azurin, occurs in the double mutant. At neutral pH the electron-self-exchange rate constant of the double mutant shows a decrease of three orders of magnitude compared with the wild-type value. The possible reasons for this decrease are discussed. Electron transfer with the proposed physiological redox partners cytochrome c551 and nitrite reductase have been investigated and the data analyzed in the Marcus framework. From this analysis it is confirmed that the hydrophobic patch of azurin is the interaction site with both partners, and that cytochrome c551 uses its hydrophobic patch and nitrite reductase a negatively charged surface area for the electron transfer.

Azurin↗

PubMed1997

The unusual stability of saporin, a candidate for the synthesis of immunotoxins.

Saporin, a monomeric protein extracted from the seeds of Saponaria officinalis, is an enzyme capable of specific depurination of the eukaryotic ribosomes. Because of its toxicity, saporin proved useful for the synthesis of immunotoxins, chimeric conjugates of a toxin and an antibody specifically directed against cancer cells or other targets. In this paper we report a study of the structural properties of saporin in the presence of denaturing agents and/or proteolytic enzymes. We found that saporin is extremely resistant to denaturation by urea or guanidine (up to 4 M), even at relatively high temperature (up to 55 degrees C). Moreover a structural change detected as a reduction of the fluorescence emission of the single Trp residue is reversible and is not paralleled by changes of the far UV CD spectrum, suggesting that even under harsh experimental conditions unfolding is limited. In good agreement with these results, guanidine-treated saporin is not attacked by proteolytic enzymes. The remarkable resistance of saporin to denaturation and proteolysis suggests this protein as an ideal candidate for biotechnological applications.

Circular Dichroism↗