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Biomedical subjects

A Azzi

Publications and source records attributed to A Azzi.

At least 199 records · Page 11Linked to original sources
PubMed1983

Preparation of monomeric cytochrome C oxidase: its kinetics differ from those of the dimeric enzyme.

Bovine cytochrome c oxidase in 0.1% dodecylmaltoside, 50 mM KCl and 10 mM Tris-HCl, pH 7.4 is monodisperse with an apparent Mr 360,000 (dimer) as estimated by filtration on Ultrogel AcA 34. In the absence of added KCl the apparent Mr is 160,000 (monomer). The dimeric enzyme has a high and a low affinity site for cytochrome c; the monomeric, only the high affinity site. The results are consistent with the existence of one active site per monomer, having high affinity for cytochrome c. Since in a dimer the two sites are in close proximity, the binding of the first molecule of cytochrome c to the first site hinders the binding of the second molecule to the second site. The kinetic data fit with a model of homotropic negative cooperativity. The effect of salts on the cytochrome c oxidase kinetics is also present in isolated bovine heart mitochondria.

Animals↗
PubMed1983

An evaluation of the evidence for H+ pumping by reconstituted cytochrome c oxidase in the light of recent criticism.

We review the evidence for H+ translocation by reconstituted cytochrome c oxidase; attention is paid to those aspects which we feel most open to criticism. Possible alternative hypotheses are assessed, with regard to experiments carried out to test them directly and with regard to published data. We conclude that, whilst certain aspects of this system are worthy of further clarification, the reported observations are all consistent with proton translocation by reconstituted cytochrome c oxidase; most of these observations provide a positive indication of such an activity.

Animals↗
PubMed1983

Interaction between isolated cytochrome c1 and cytochrome c.

Cytochrome c1 from bovine heart mitochondria was isolated by a modification of the technique of König et al. [(1980) Biochim. Biophys. Acta 621, 283-295] which involved an affinity chromatography step on a gel with yeast cytochrome c as a ligand. Its spectra, electrophoretic pattern in presence of sodium dodecylsulfate, its reducibility by ascorbate and cytochrome c were characteristic of a native cytochrome, with a single polypeptide having an apparent molecular weight of 30 000. By using an arylazido derivative of cytochrome c, having the photoactive group bound to lysine 13, upon illumination a cross-link with the described preparation of cytochrome c1 was obtained. By pepsin digestion of the cross-linked complex a limiting fragment was obtained and partially sequenced. It allowed to identify the site of binding of cytochrome c near the sequence 167-174 of cytochrome c1.

Amino Acid Sequence↗
PubMed1983

Structural and functional properties of cytochrome c oxidase from Bacillus subtilis W23.

The terminal component of the electron transport chain, cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase) was purified from Bacillus subtilis W23. The enzyme was solubilized with alkyglucosides and purified to homogeneity by cytochrome c affinity chromatography. The enzyme showed absorption maxima at 414 nm and 598 nm in the oxidized form and at 443 nm and 601 nm in the reduced form. Upon reaction with carbon monoxide of the reduced purified enzyme the absorption maxima shifted to 431 nm and 598 nm. Sodium dodecylsulfate polyacrylamide gel electrophoresis indicated that the purified enzyme is composed out of three subunits with apparent molecular weights of 57 000, 37 000 and 21 000. This is the first report on a bacterial aa3-type oxidase containing three subunits. The functional properties of the enzyme are comparable with those of the other bacterial cytochrome c oxidases. The reaction catalyzed by this oxidase was strongly inhibited by cyanide, azide and monovalent salts. Furthermore a strong dependence of cytochrome c oxidase activity on negatively charged phospholipids was observed. Crossed immunoelectrophoresis experiments strongly indicated a transmembranal localization of cytochrome c oxidase.

Bacillus subtilis↗
PubMed1982

Purification and characterization of the cytochrome c oxidase from Rhodopseudomonas sphaeroides.

When grown aerobically in the dark, Rhodopseudomonas sphaeroides develops a respiratory chain similar to that in mitochondria and the photosynthetic apparatus is suppressed. The aa3-type cytochrome c oxidase from Rps. sphaeroides has been purified in Triton X-100 by affinity chromatography with Sepharose 4B coupled to yeast cytochrome c. The oxidase contains 14 nmol heme a/mg protein and is composed of three polypeptide subunits with relative molecular masses of 45000, 37000 and 35000. The enzyme is highly active in the presence of detergents, with a maximal velocity of 300 s-1/mol oxidase using either yeast or horse-heart cytochrome c. The Rps. sphaeroides oxidase is cross-reactive with antibodies directed against the oxidases from Paracoccus denitrificans and Saccharomyces cerevisiae. A particularly close relationship is indicated in the case of P. denitrificans. The Rps. sphaeroides oxidase has been incorporated into phospholipid vesicles. The resulting oxidase in these vesicles demonstrates high enzymatic activity and a respiratory control ratio of 5. Using these vesicles, no evidence for proton extrusion accompanying cytochrome c oxidation was observed. The data suggest that the Rps. sphaeroides oxidase does not function as a proton pump.

Bacterial Proteins↗
PubMed1982

Affinity chromatography purification of cytochrome c oxidase and b-c1 complex from beef heart mitochondria. Use of thiol-sepharose-bound Saccharomyces cerevisiae cytochrome c.

A method for simultaneous purification of cytochrome c reductase and cytochrome c oxidase using a cytochrome c affinity column is presented. Cytochrome c from Saccharomyces cerevisiae was linked to an activated thiol-Sepharose gel via its Cys-102 residue located far from the lysine residues on the front side of the molecule, responsible for the interaction with the reductase and oxidase. In previously reported affinity chromatography techniques these lysine residues most probably reacted with the column. Cytochrome c oxidase and reductase from bovine heart mitochondria bind specifically to the affinity column and can be recovered separately at different ionic strength in the elution buffer. The enzymes are highly pure and active.

Animals↗
PubMed1982

Affinity chromatography purification of cytochrome c binding enzymes.

An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.

Animals↗
PubMed1982

Studies on the transmembrane orientation of cytochrome c oxidase in phospholipid vesicles.

We report investigations into the direction of orientation of cytochrome c oxidase in reconstituted vesicles and the factors determining this. Measurement of the enzyme orientation employed two independent techniques: monitoring of the level of haem reduction by membrane-permeant and membrane-impermeant reagents and a kinetic analysis of the reduction of a spin label covalently bound to the oxidase surface. The method of preparation of the oxidase vesicles had a pronounced effect on the enzyme orientation and the two measurement techniques agreed in indicating that the proportion of mitochondrially oriented enzyme was approximately 85% and 50% for vesicles prepared by cholate dialysis and sonication respectively. Our results show that the membrane orientation of the oxidase is determined by interactions between the phospholipid bilayer and the portion of the enzyme embedded therein, as opposed to gross physical constraints. In particular, we demonstrate that the orientation of the oxidase is affected by the fluidity and surface charge of the membrane.

Electron Spin Resonance Spectroscopy↗
PubMed1981

Structural studies on the cytochrome c oxidase proton pump using a spin-label probe.

We report studies in which we have used N-(2,2,6,6-tetramethylpiperidyl-l-oxyl)-N' -cyclohexylcarbodiimide, a spinlabel analogue of N,N' -dicyclohexylcarbodiimide, to investigate the structural aspects of the cytochrome c oxidase proton pump. We establish that the spin label binds to the reconstituted enzyme at the same site as does N,N' -dicyclohexylcarbodiimide, i.e., within subunit III. ESR studies of the bound spin label indicate that its binding site is situated in an apolar region of the enzyme, though close to its surface. The binding of the spin label to the free oxidase is different form that with the reconstituted enzyme, leading to spin-spin exchange between the bound probe molecules. From this and the fact that N,N' -dicyclohexylcarbodiimide binds to subunits III and IV in the free oxidase, we conclude that these two subunits are at the most 20 A apart.

Binding Sites↗
PubMed1981

Studies on the molecular basis of H+ translocation by cytochrome c oxidase.

We report here studies which characterize further the interaction of N,N'-dicyclohexylcarbodiimide with cytochrome c oxidase leading to inhibition of H+ translocation by the enzyme. Further evidence is presented to show that the inhibition results from a real interaction of DCCD with the enzyme and cannot be accounted for by uncoupling and, contrary to recent criticisms, this interaction occurs specifically with subunit III of the enzyme even at relatively high inhibitor-to-enzyme stoichiometries. Use of a spin-label analogue of DCCD has enabled us to demonstrate that the carbodiimide-binding site is highly apolar and may not lie on the pathway of electron transfer.

Animals↗
PubMed1981

The estimation of extracellular protein production in detecting enterotoxigenic staphylococci.

294 strains of staphylococci isolated from feces, throat and nasal swabs were assayed for production of enterotoxin, coagulase, thermostable deoxyribonuclease (thermonuclease) and high levels of global extracellular protein (ECP). The relationship observed among these biochemical features suggested that the determination of ECP can be an useful test in detecting enterotoxigenic staphylococcal strains which do not exhibit coagulase and thermonuclease production. In fact, among 17 strains which produced large amounts of ECP, but were coagulase- and thermonuclease-negative, 4 were found to be enterotoxigenic, while of 95 strains which produced low levels of ECP and were coagulase and thermonuclease-negative, none was enterotoxigenic.

Bacterial Proteins↗
PubMed1980

The proteolipid subunit of the chloroplast adenosine triphosphatase complex. Reconstitution and demonstration of proton-conductive properties.

The dicyclohexylcarbodiimide (DCCD)-binding proteolipid of the chloroplast ATPase complex was solubilized in 1-butanol, isolated by ion exchange chromatography, and reconstituted in a liposomal system. Proton-conducting activity was monitored by fluorometry with 9-aminoacridine as an indicator of delta pH in K+-loaded liposomes suspended in a K+-free medium. Addition of valinomycin served to create a membrane potential. Proton mediation was further followed potentiometrically with a pH electrode. Reconstituted chloroplast DCCD-binding proteolipid rapidly catalyzed passive proton movement as measured by the quenching of 9-aminoacridine fluorescence upon addition of valinomycin to K+-loaded vesicles. Proton translocation was inhibited to approximately 80% by hydrophobic dicyclohexylcarbodiimide but not its water-soluble analog (1-ethyl-3[3-dimethylaminopropyl]carbodiimide). Maximal inhibition by DCCD occurred after 60 min of incubation with 20 to 40 nmol of DCCD/20 nmol of proteolipid/10 mumol of lipid. The velocity of proton conduction increased when the external proton concentration in the medium was increased. Similar data were obtained using a pH electrode to follow proton movement. Subjection of proteolipid liposomes to tetranitromethane, resulting in nitration of tyrosine, markedly reduced proton conduction (62% inhibition). In a similar fashion preincubation of reconstituted vesicles with hydrophobic phenylisothiocyanate at neutral pH resulted in a decreased rate of proton movement (50% inhibition) while hydrophilic p-sulfophenylisothiocyanate appeared to have no effect.

Adenosine Triphosphatases↗
PubMed1980

Interaction of cytochrome c with cytochrome bc1 complex of the mitochondrial respiratory chain.

The binding of cytochrome c to the cytochrome bc1 complex of bovine heart mitochondria was studied. Cytochrome c derivatives, arylazido-labeled at lysine 13 or lysine 22, were prepared and their properties as electron acceptors from the bc1 complex were measured. Mixtures of bc1 complex with cytochrome c derivatives were illuminated with ultraviolet light and afterwards subjected to polyacrylamide gel electrophoresis. The gels were analysed using dual-wavelength scanning at 280 minus 300 and 400 minus 430 nm. It was found that illumination with ultraviolet light in the presence of the lysine 12 derivative produced a diminution of the polypeptide of the bc1 coplex having molecular weight 30 000 (band IV) and formation of a new polypeptide composed of band IV and cytochrome c. Band IV was identified as cytochrome c1, and it was concluded that this hemoprotein interacts with cytochrome c and contains its binding site in complex III of the mitochondrial respiratory chain. Illumination of the bc1 complex in presence of the lysine 22 derivative did not produce changes of the polypeptide pattern.

Animals↗
PubMed1980

Dicyclohexylcarbodiimide binds specifically and covalently to cytochrome c oxidase while inhibiting its H+-translocating activity.

We have investigated the covalent binding of dicyclohexylcarbodiimide (DCCD) to cytochrome c oxidase in relation to its inhibition of ferrocytochrome c-induced H+ translocation by the enzyme reconstituted in lipid vesicles. DCCD bound to the reconstituted oxidase in a time- and concentration-dependent manner which appeared to correlate with its inhibition of H+ translocation. In both reconstituted vesicles and intact beef heart mitochondria, the DCCD-binding site was located in subunit III of the oxidase. The apolar nature of DCCD and relatively minor effects of the hydrophilic carbodiimide, 1-ethyl-(3-dimethylaminopropyl)-carbodiimide, on H+ translocation by the oxidase indicate that the site of action of DCCD is hydrophobic. DCCD also bound to isolated cytochrome c oxidase, though in this case subunits III and IV were labeled. The maximal overall stoichiometries of DCCD molecules bound per cytochrome c oxidase molecule were 1 and 1.6 for the reconstituted and isolated enzymes, respectively. These findings point to subunit III of cytochrome c oxidase having an important role in H+ translocation by the enzyme and indicate that DCCD may prove a useful tool in elucidating the mechanism of H+ pumping.

Animals↗