Search PubMed⌕ Search

Biomedical subjects

A Azzi

Publications and source records attributed to A Azzi.

At least 163 records · Page 9Linked to original sources
PubMed1987

Purification by affinity chromatography of the dicarboxylate carrier from bovine heart mitochondria.

Submitochondrial particles were prepared from bovine heart mitochondria, solubilized with Triton X-114 in the presence of lipids and submitted to hydroxylapatite chromatography. The eluate obtained, containing a mixture of mitochondrial carriers, was processed further by affinity chromatography using as ligand p-aminophenylsuccinate coupled via a diazo bond to aminohexyl-Sepharose 4B. The activity of the dicarboxylate exchanger was measured after reconstitution into asolectin vesicles at each step of the purification procedure. All samples studied were found to display substrate and inhibitor specificity similar to those described for the dicarboxylate carrier in mitochondria. The specific activity of the final material eluted from the affinity column was found to be about 1000-times higher than that of the Triton X-114 extract of submitochondrial particles. SDS-polyacrylamide gel electrophoresis analysis of the affinity chromatography eluate showed the presence of only two polypeptides.

Animals↗
PubMed1987

The synthesis of fluorescent chlorotriazinylaminofluorescein-concanavalin A and its use as a glycoprotein stain on sodium dodecyl sulphate/polyacrylamide gels.

Concanavalin A, a specific glycoprotein probe, was optimally labelled to a maximum stoichiometry of 0.4 mol of chlorotriazinylaminofluorescein (CTAF)/mol of concanavalin A monomer under mild reaction conditions (pH 8.0, 6 h), and under these conditions the CTAF concanavalin A preparation retains its carbohydrate-binding ability and is able to penetrate SDS/7.5-15%-polyacrylamide gradient gels. CTAF-concanavalin A gives fluorescent bands for the glycoproteins transferrin, fetuin and deoxyribonuclease and shows no fluorescent response for the non-glycoproteins bovine serum albumin and soya-bean trypsin inhibitor. The detection limit of sensitivity for CTAF-concanavalin A, which is similar to that of fluorescein isothiocyanate-concanavalin A, is in the range 5-25 micrograms of glycoprotein. CTAF-concanavalin A is a suitable probe for the detection of glycoproteins in higher-percentage (greater than or equal to 10%) SDS/polyacrylamide gels, and will probably have other applications in, for example, fluorescent energy transfer and other structure-function studies.

Chemical Phenomena↗
PubMed1987

Fluorescent and photoactive probes for the study of protein kinase C.

A fluorescent diacylglycerol, 2-(12-N-dansylaminododecanoyl)-1-myristoyl-sn-glycerol (dansyl-DAG) and a photoactive diacylglycerol, 2-(12-[N-(4-azido-2-nitrophenyl)] aminododecanoyl)-1-myristoyl-sn-glycerol (azido-DAG) have been synthesized. Both have been shown to bind to protein kinase C by inhibition of phorbol dibutyrate binding. Dansyl-DAG was able to activate protein kinase C at low calcium concentrations. Stimulation of neutrophils with dansyl-DAG resulted in a large release of superoxide radicals from the cells. The physicochemical properties of dansyl-DAG and azido-DAG may allow one to label and follow specifically changes in the location of protein kinase C and to understand some aspects of its function and regulation.

Affinity Labels↗
PubMed1987

Isolation and partial characterization of the cytochrome c oxidase of Micrococcus luteus (lysodeikticus).

The cell membrane of Micrococcus luteus (lysodeikticus) contains a respiratory chain composed of hemes a, b, and c, which contain 171, 457, and 407 pmol/mg protein, respectively. Cytochrome c oxidase, the heme a containing component, has been purified after solubilization in Triton X-100, by gel filtration on Sepharose 4B-CL ammonium sulfate precipitation and ion-exchange and affinity chromatographies on a yeast cytochrome c-Sepharose 4B column. The purified complex, which contains three polypeptides of apparent Mr 47,000, 31,000, and 19,000, has CN-sensitive ferrocytochrome c oxidase activity (Ki = 0.35 microM) and a characteristic absorption spectrum with maxima in the oxidized form at 595 and 426 nm and in the reduced form at 601 and 444 nm. The purified enzyme contains 17.4 nmol/mg protein and its copper content is 23.2 nmol/mg protein. The enzyme was purified about 100-fold with respect to its content in crude membranes. The total heme a yield, also with respect to crude membranes content, was 6.8%.

Chromatography, Ion Exchange↗
PubMed1986

The monocarboxylate carrier from bovine heart mitochondria: partial purification and its substrate-transporting properties in a reconstituted system.

The monocarboxylate (pyruvate) carrier from bovine heart mitochondria was extracted from submitochondrial particles with Triton X-114 in the presence of cardiolipin. By a single hydroxylapatite chromatography step a 125-fold purification of the carrier protein could be achieved. High pyruvate/pyruvate-exchange activity was recovered, when the protein was reconstituted into phospholipid vesicles. No transport activity was observed, when the isolation occurred in the absence of phospholipids. The 2-cyano-4-hydroxycinnamate sensitive pyruvate exchange reaction was strongly temperature sensitive and dependent on the amount of protein reconstituted. Other 2-ketoacids caused competitive inhibition of the pyruvate uptake. Inhibitors of other mitochondrial carries, however, had very low or no effect on the monocarboxylate exchange. The influence of different -SH group reagents on the measured pyruvate/pyruvate-exchange in the reconstituted system was similar to the one observed with intact mitochondria. It is concluded that the described procedures for extraction, purification and reconstitution of the mitochondrial monocarboxylate carrier conserved the functional properties of the protein.

Animals↗
PubMed1986

Extraction, partial purification and functional reconstitution of two mitochondrial carriers transporting keto acids: 2-oxoglutarate and pyruvate.

Bovine heart submitochondrial particles were treated with a medium containing Triton X-114 and cardiolipin. The extract was subjected to hydroxyapatite chromatography. Only a few major polypeptides of similar molecular masses were found in the eluate, as shown by electrophoresis in an SDS-polyacrylamide gel stained with silver. The eluate was reconstituted into liposomes and was shown to catalyse two different transport activities: 2-oxoglutarate-2-oxoglutarate exchange sensitive to phthalonate and phenylsuccinate and pyruvate-pyruvate exchange sensitive to 2-cyano-4-hydroxycinnamate. Since both activities were found to have characteristics similar to those described for intact mitochondria, it was concluded that at least two of the polypeptides found in the hydroxyapatite eluate correspond to the two mitochondrial carriers.

Animals↗
PubMed1986

Cytochrome c oxidase from Paracoccus denitrificans in Triton X-100: aggregation state and kinetics.

Cytochrome c oxidase from Paracoccus denitrificans was homogeneously dispersed in Triton X-100. Using gel exclusion chromatography and sucrose gradient centrifugation analysis a molecular weight of the detergent-protein complex of 155,000 was determined. After subtraction of the bound detergent (111 mol/mol heme aa3) a molecular weight of 85,000 resulted, which agreed well with the model of a monomer containing two subunits. This monomer showed high cytochrome c oxidase activity when measured spectrophotometrically in the presence of Triton X-100 (Vmax = 85 s-1). The molecular activity, plotted according to Eadie-Hofstee, was monophasic as a function of the cytochrome c concentration. A Km of 3.6 X 10(-6) M was evaluated, similar to the Km observed in the presence of dodecyl maltoside [Nałecz et al. (1985).

Electron Transport↗
PubMed1986

The role of cytochrome a in the proton pump of cytochrome-c oxidase.

Cytochrome-c oxidase of bovine heart mitochondria was depleted of copper A by dialysis against 1 M KCN in the presence of dodecylmaltoside. There was no difference of the pH-dependence of the midpoint potential between the intact and the copper-depleted enzyme. Oxidation of reduced cytochrome a2+a3(3+).CN complex released about 1 proton/electron in the medium at pH 7.6. This release was inhibited by N,N'-dicyclohexylcarbodiimide. Again there was no difference between the intact and Cu-depleted enzyme. This limits the role of copper A in the mechanism of the proton pump. On the other hand, these experiments showed that cytochrome a could be a component of the proton pump.

Animals↗
PubMed1985

Functional characterization of the mitochondrial cytochrome b-c1 complex: steady-state kinetics of the monomeric and dimeric forms.

The QH2:cytochrome c oxidoreductase activity of the isolated bovine heart cytochrome b-c1 complex resolved into monomeric and dimeric form was titrated with three different inhibitors of electron transfer, antimycin, myxothiazol, and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). In all cases one inhibitor molecule per cytochrome c1 was found necessary to block completely the activity of both molecular forms of the enzyme. The antimycin-sensitive cytochrome c reduction catalyzed by the b-c1 complex was also studied as a function of increasing concentrations of either cytochrome c or quinol. Double-reciprocal plots of the activity of the monomeric enzyme were found linear either when the concentration of cytochrome c or of quinol derivatives, 2,3-dimetoxy-5-methyl-6-decyl-1,4-benzoquinol (DBH), and 2-methyl-3-undecyl-1,4-naphthoquinol (UNH), was changed. Cytochrome c reductase activity of the dimeric b-c1 complex also showed a linear Lineweaver-Burk plot as a function of cytochrome c concentrations. In contrast to the monomeric enzyme, however, dimers of the b-c1 complex express a clear nonlinear kinetic behavior toward quinol derivatives, with two apparent Km values differing approximately by one order of magnitude (about 3-4 and about 20-30 microM). At saturating quinol concentrations the activity of the dimeric enzyme becomes two to three times higher than that of monomers. The nonlinear kinetic plots were found to be the same at different temperatures and different cytochrome c concentrations. The data suggest that although the monomer of the b-c1 complex appears to be the functional unit of the enzyme, the dimer is more active. A regulatory role of the dimerization process resulting in an increase of the electrons flux through the enzyme is postulated.

Animals↗