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Biomedical subjects

A Azzi

Publications and source records attributed to A Azzi.

At least 145 records · Page 8Linked to original sources
PubMed1990

The monocarboxylate carrier from rat liver mitochondria. Purification and kinetic characterization in a reconstituted system.

The monocarboxylate (pyruvate) carrier was extracted from rat liver mitochondria with Triton X-100 in the presence of asolectin and partially purified by chromatography on HTP. The HTP eluate reconstituted in liposomes was shown to catalyze active pyruvatein/acetoacetateout and acetoacetatein/pyruvateout counter-exchange. Kinetic characterization of the reconstituted pyruvate carrier was achieved by an original spectrophotometric method consisting of determination of substrate release from proteoliposomes with a coupled enzymatic assay.

Acetoacetates↗
PubMed1990

Detection of B19 parvovirus infections by a dot-blot hybridization assay using a digoxigenin-labelled probe.

A non-radioactive dot-blot hybridization assay for the detection of B19 parvovirus infections was developed using a digoxigenin-labelled probe both on nylon and nitrocellulose filters. A 700 bp BamHI HindIII fragment of B19 DNA was used to construct the probe. Probe labelling was carried out by incorporating deoxyuridine triphosphate labelled with digoxigenin. The dot-blot hybridization assay was visualized by an immunoenzymatic reaction using antidigoxigenin Fab fragments labelled with alkaline phosphatase. The specificity and sensitivity of digoxigenin-labelled B19 DNA probe was compared with the results obtained with 32P-labelled B19 DNA probe. Out of the 504 serum samples tested, 3 samples were positive in all the hybridization assays performed and 494 were negative, 7 serum samples gave a weak positive reaction when Dig-B19 probe was used on nitrocellulose filters. The 77 pharyngeal swabs tested were negative in all the hybridization assays performed. Our hybridization assay showed a high sensitivity and reproducibility and it appears to be a rapid, practical and reliable test for routine screening of B19 parvovirus DNA in large numbers of clinical specimens.

Adolescent↗
PubMed1989

A novel a-type terminal oxidase from Sulfolobus acidocaldarius with cytochrome c oxidase activity.

Cytochrome C oxidase was solubilized with a nonionic detergent n-decanoyl-N-methyl glucamide from the membranes of Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium, and was purified. The enzyme oxidized horse heart cytochrome C with a Vmax of 63 mumols/min/mg at 50 degrees C. The activity was sensitive to cyanide. The enzyme also catalyzed oxygen uptake detergent on N, N, N', N'-tetramethyl p-phenylene diamine. An apparent molecular mass was estimated to be 150 kDa. The enzyme is composed of three subunits of 37, 23 and 14 kDa. Spectral characteristics were similar to typical bacterial aa3 except for the presence of a novel 583 nm peak observed in reduced minus oxidized difference spectrum.

Archaea↗
PubMed1989

Suramin, an anti-cancer drug, inhibits protein kinase C and induces differentiation in neuroblastoma cell clone NB2A.

Protein kinase C purified from rat brain was found to be inhibited by suramin, a substance used originally in the therapy of antitrypanosomic infections and more recently proposed as antineoplastic agent. The inhibition of suramin was competitive with one of the substrates of the enzyme, ATP with a Ki of 10 microM. At concentrations adequate to inhibit the isolated enzyme, suramin was shown to slow the rate of proliferation of neuroblastoma NB2A cells in vitro and to induce their differentiation as evidenced by typical morphological changes.

Animals↗
PubMed1989

Monocarboxylate and alpha-ketoglutarate carriers from bovine heart mitochondria. Purification by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate.

2-Cyano-4-hydroxycinnamate was covalently linked, through a diazo bond, to Sepharose 4B, which had been elongated with a hydrophobic spacer. A Triton X-100 extract from bovine heart mitochondria was pre-purified by hydroxylapatite chromatography and passed through the 2-cyano-4-hydroxycinnamate affinity resin in the presence of 0.7% deoxycholate. At pH 6 and in the presence of 0.2 M sodium chloride, a single polypeptide with an Mr of 34,000 was eluted. Subsequently, at pH 8 and in the presence of 2-cyano-4-hydroxycinnamate, another single protein with an Mr of 31,500 was released. Both proteins were reconstituted into phospholipid vesicles and their transport activities were measured. High, delta pH-dependent, 2-cyanocinnamate-sensitive pyruvate uptake was measured in vesicles containing only the 34-kDa protein. alpha-Ketobutyrate and other alpha-ketomonocarboxylic acids were competitive inhibitors of the pyruvate uptake, whereas di- and tricarboxylates had only small effects. alpha-Ketoglutarate-alpha-ketoglutarate exchange could only be measured in vesicles containing the 31.5-kDa protein. The molecular weight of this protein and its functional properties were similar to those of the alpha-ketoglutarate carrier isolated by a different method (Bisaccia, Indiveri, C., and Palmieri, F. (1985) Biochim. Biophys. Acta 810, 362-369). 2-Cyano-4-hydroxycinnamate inhibited the alpha-ketoglutarate exchange in a noncompetitive manner with an apparent Ki of 0.7 mM. It is concluded that by the described affinity chromatography procedure, two mitochondrial carriers transporting alpha-ketoacids, i.e. the monocarboxylate and the alpha-ketoglutarate carrier, could be purified in a functionally active state.

Animals↗
PubMed1989

Tricarboxylate carrier of bovine liver mitochondria. Purification and reconstitution.

The tricarboxylate carrier of bovine liver mitochondria has been solubilized by Triton X-114 and purified by chromatography on hydroxylapatite and Silica Gel 60. The purified carrier could be visualized as a single band in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with Mr 37,000-38,000. The carrier, after reconstitution in phospholipid vesicles, catalyzed the exchange of [14C]citrate against citrate, malate, and threo-D8-isocitrate and was inhibited by the specific tricarboxylate carrier inhibitor 1,2,3-benzenetricarboxylic acid.

Animals↗
PubMed1989

Ionic-strength-dependence of the oxidation of native and pyridoxal 5'-phosphate-modified cytochromes c by cytochrome c oxidase.

The ionic-strength-dependences of the rate constants (log k plotted versus square root of 1) for oxidation of native and pyridoxal 5'-phosphate-modified cytochromes c by three different preparations of cytochrome c oxidase have complex non-linear character, which may be explained on the basis of present knowledge of the structure of the oxidase and the monomer-dimer equilibrium of the enzyme. The wave-type curve (with a minimum and a maximum) for oxidation of native cytochrome c by purified cytochrome c oxidase depleted of phospholipids may reflect consecutively inhibition of oxidase monomers (initial descending part), competition between this inhibition and dimer formation, resulting in increased activity (second part with positive slope), and finally inhibition of oxidase dimers (last descending part of the curve). The dependence of oxidation of native cytochrome c by cytochrome c oxidase reconstituted into phospholipid vesicles is a curve with a maximum, without the initial descending part described above. This may reflect the lack of pure monomers in the vesicles, where equilibrium is shifted to dimers even at low ionic strength. Subunit-III-depleted cytochrome c oxidase does not exhibit the maximum seen with the other two enzyme preparations. This may mean that removal of subunit III hinders dimer formation. The charge interactions of each of the cytochromes c (native or modified) with the three cytochrome c oxidase preparations are similar, as judged by the similar slopes of the linear dependences at I values above the optimal one. This shows that subunit III and the phospholipid membrane do not seem to be involved in the specific charge interaction of cytochrome c oxidase with cytochrome c.

Cytochrome c Group↗
PubMed1989

Novel reconstitution and enzymatic assay of the mitochondrial tricarboxylate carrier.

The tricarboxylate carrier from beef liver mitochondria was reconstituted into liposomes using a protocol based on the absorption of Triton X-100 to hydrophobic Amberlite XAD-2 beads. The activity of the reconstituted carrier was determined spectroscopically by measuring the citrate/isocitrate exchange with an enzymatic assay. The Km for citrate obtained with this method was 35 microM and the Ki of 1,2,3-benzenetricarboxylate was 27 microM.

Animals↗
PubMed1988

Preparation of a one-subunit cytochrome oxidase from Paracoccus denitrificans: spectral analysis and enzymatic activity.

Cytochrome c oxidase was isolated from Paracoccus denitrificans as a two-subunit enzyme. Chymotrypsin-catalyzed proteolysis reduced the molecular weight of each subunit by about 8000. The spectral properties of this preparation, as well as its Km for cytochrome c(1.7 muM), remained unchanged with respect to the native enzyme. Vmax was reduced by about 55% when assayed in Triton X-100 or in Triton X-100 supplemented with asolectin. Following further proteolysis by Staphylococcus aureus V8 protease, subunit I remained unchanged as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas subunit II was split into small peptides. These were removed by ion-exchange high-performance liquid chromatography. The one-subunit enzyme had an apparent molecular weight of 43,000. The reduction of molecular weight was also confirmed by the diminution of the ultraviolet/Soret absorption ratio. This value was 1.8-2.1 for the native enzyme and 1.3-1.5 for the one-subunit enzyme. The spectral properties (including the spectrum CO reduced minus reduced) were not modified by the proteolytic treatment, indicating that cytochromes a and a3 were present in equal amounts. The lack of spectral alteration and the known close association of the copper B atom with cytochrome a3 suggest that copper B is also contained within the one-subunit enzyme. The Km of the one-subunit oxidase was similar to that of the two-subunit enzyme; Vmax was decreased by about 50%. The activity of the one-subunit oxidase had a salt-dependent maximum at 30 mM KCl, almost identical with that of the undigested enzyme, and was inhibited by micromolar concentrations of KCN.

Affinity Labels↗
PubMed1988

Subunit III of cytochrome c oxidase is expressed in Paracoccus denitrificans.

Polyclonal antibodies have been obtained against a synthetic dodecapeptide identical to the aminoacid sequence 120-131 DSPIKDGVWPPE (inferred from its DNA sequence) of Paracoccus denitrificans cytochrome c oxidase subunit III. The antibodies had a titer higher than 1:10000 when tested against the antigen. These antibodies have been used to produce immunological evidence that, despite the fact that subunit III is not isolated with cytochrome c oxidase, it exists in Paracoccus denitrificans lysates. The antibodies did not show reactivity with bovine heart cytochrome c oxidase either by ELISA or immunoblotting. It was also shown that the antibodies react with a single polypeptide present in Paracoccus denitrificans cell lysates, having an apparent molecular weight close to that of subunit III of bovine heart oxidase.

Animals↗
PubMed1988

The effect of non-esterified fatty acids on the proton-pumping cytochrome c oxidase reconstituted into liposomes.

Bovine heart cytochrome c oxidase was reconstituted in phospholipid vesicles, and the effect of different non-esterified fatty acids (NEFA) was studied on its proton pump and on the proton permeability of the vesicles. Neither parameter appeared to be affected by concentrations of NEFA known to uncouple oxidative phosphorylation (10 microM). Also the permeability for K+ was not affected by them. The fatty acids caused an increase in the rate of electron transfer in the absence, but not in the presence, of uncoupler and/or valinomycin [diminution of the respiratory-control index (RCI)]. The RCI of 8.7-7.5 was decreased to about 4.5 in the presence of 0.27-10 microM-NEFA. Oleic acid was not effective at the above concentrations. Subunit III-depleted enzyme preparations gave vesicles with an RCI of about 5.5, which was decreased to 4.5 in the presence of NEFA. With both native and subunit III-depleted oxidase the RCI was never decreased to the value of 1 by NEFA, as happens with classical protonophores.

Animals↗
PubMed1988

Vitamin E inhibits protein kinase C activity.

Vitamin E (dl-alpha-tocopherol) has been found to inhibit in vitro brain protein kinase c with a half inhibitory concentration of 450 microM. The known plasma concentrations of vitamin E are one order of magnitude lower than the protein kinase c half-inhibitory concentration but it is also known that, at the membrane level where the active protein kinase c is located, the lipophilic vitamin E is more concentrated (Burton, G.W., Joyce, A. and Ingold, K.U. and Locke, S. (1983) Arch. Biochem. Biophys. 221, 281-290). It appears that vitamin E, in addition to its antioxidant function, may play a role in regulating the activity of protein kinase c.

Animals↗
PubMed1988

Human parvovirus infection in haemophiliacs first infused with treated clotting factor concentrates.

A group of 27 first infused haemophiliacs was studied for association between heat-treated clotting factor concentrates and transmission of human parvovirus B19. The prevalence rate of B19 antibody, detected by the Immunoelectroosmophoresis (IEOP) reaction, was 55.5% in this group of first infused subjects, significantly higher than the 29.3% of the control group of 58 healthy blood donors but lower than the 93.3% of antibody positive subjects in a group of 30 haemophiliacs multitreated with unheated products. Five of 17 B19 antibody negative patients produced human parvovirus IgM, detectable by radioimmunoassay, after the first treatment with heated concentrates; two of them developed viraemia 6 and 10 days, respectively, after the first infusion dose. These results lead to the conclusion that human parvovirus is transmissible by blood derivatives even when they have been exposed to steam- or dry-heat treatment.

Adolescent↗
PubMed1988

Cytochrome c oxidase from Paracoccus denitrificans: both hemes are located in subunit I.

The two-subunit cytochrome c oxidase from Paracoccus denitrificans has been sequentially digested with chymotrypsin and Staphylococcus aureus V8 protease. The smaller subunit of the enzyme (apparent Mr 32,000) was split into numerous peptides that were removed by anion-exchange HPLC. The larger subunit was only digested to a limited extent (from an apparent Mr 45,000 to Mr 43,000), and the spectral properties were preserved relative to the native enzyme (a reduced minus oxidized difference spectrum with maxima at 447 and 607 nm in the Soret and alpha region, respectively). As judged from CO-reduced spectra this proteolytically digested, one-fragment oxidase was found to contain an equal amount of cytochromes a and a3. The enzymatic activity with reduced cytochrome c as substrate in the presence of Triton X-100 proceeded with equal affinity (apparent Km = 0.5-1.0 microM) and with a Vmax of approximately 20% (40 s-1) of that found with the native enzyme (200 s-1). When the assay system was supplemented with soybean phospholipids, the Km became 2 microM for both enzymes and the Vmax became 730 and 170 s-1 for the native and the digested enzyme, respectively. Thus subunit I of P. denitrificans oxidase, and most probably of the other cytochrome c oxidases as well, contains both hemes and at least one Cu atom and has significant enzymatic activity.

Chymotrypsin↗