Biomedical subjects

S Papa

Publications and source records attributed to S Papa.

At least 163 records · Page 9Linked to original sources

PubMed1992

[Membrane ATPase of Vibrio alginolyticus. Ion transport activity and homology with F0F1-ATPase from E. coli].

F0F1-ATPase has been isolated from the marine alkali-resistant bacterium Vibrio alginolyticus. The enzyme subunits cross-reacted with antibodies against subunits alpha, beta, gamma, epsilon, and b of E. coli ATPase. The purified ATPase was reconstituted into liposomes effecting an ATP-dependent uptake of H+. Proton transport was inhibited by the ATPase blockers DCCD, triphenyltin, and venturicidin. Na+ ions had no effect on ATP-dependent proton transport. No ATP-dependent transport of Na+ was detected in proteoliposomes.

Adenosine Triphosphatases↗

PubMed1991

Spectrophotometric determination of functional characteristics of protein kinases with coupled enzymatic assay.

The performance of enzyme-coupled spectrophotometric assay of protein kinases and its merits as compared with the radioisotopic method in characterizing functional activity of protein kinases are described. Kinetic parameters of protein kinase C and protein kinase A so obtained are presented. General application of the spectrophotometric assay in the study of protein kinases is recommended.

Animals↗

PubMed1991

Structural and functional characterization of subunits of the F0 sector of the mitochondrial F0F1-ATP synthase.

Proteolytic digestion of F1-depleted submitochondrial particles (USMP), reconstitution with isolated subunits and titration with inhibitors show that the nuclear-encoded PVP protein, previously identified as an intrinsic component of bovine heart F0 (F01) (Zanotti, F. et al. (1988) FEBS Lett. 237, 9-14), is critically involved in maintaining the proper H+ translocating configuration of this sector and its correct binding to the F1 catalytic moiety. Trypsin digestion of USMP, under conditions leading to cleavage of the carboxyl region of the PVP protein and partial inhibition of transmembrane H+ translocation, results in general loss of sensitivity of this process to F0 inhibitors. This is restored by addition of the isolated PVP protein. Trypsin digestion of USMP causes also loss of oligomycin sensitivity of the catalytic activity of membrane reconstituted soluble F1, which can be restored by the combined addition of PVP and OSCP, or PVP and F6. Amino acid sequence analysis shows that, in USMP, modification by [14C] N,N'-dicyclohexylcarbodiimide of subunit c of F0 induces the formation of a dimer of this protein, which retains the 14C-labelled group. Chemical modification of cysteine-64 of subunit c results in inhibition of H+ conduction by F0. The results indicate that proton conduction in mitochondrial F0 depends on interaction of subunit c with the PVP protein.

Adenosine Triphosphatases↗

PubMed1991

H+/e- stoichiometry of mitochondrial cytochrome complexes reconstituted in liposomes. Rate-dependent changes of the stoichiometry in the cytochrome c oxidase vesicles.

The H+/e- stoichiometry of protonmotive cytochrome c oxidase, isolated from bovine heart mitochondria and reconstituted in liposomes, has been determined by making use of direct spectrophotometric measurements of the initial rates of e- flow and H+ translocation. It is shown that the ----H+/e- ratio for redox-linked proton ejection by the oxidase varies from around 0 to a maximum of 1 as a function of the rate of overall electron flow in the complex.

Animals↗

PubMed1991

The cytochrome chain of mitochondria exhibits variable H+/e- stoichiometry.

A study is presented of the ----H+/e- stoichiometry for H+ pumping by the cytochrome chain in isolated rat liver mitochondria under level-flow and steady-state conditions. It is shown that the ----H+/e- stoichiometry for the cytochrome chain varies under the influence of the flow rate and transmembrane delta microH+. The rate-dependence is shown to be associated with cytochrome c oxidase, whose ----H+/e- ratio varies from 0 to 1, whilst the ----H+/e- ratio for the span covered by cytochrome c reductase is invariably 2.

Animals↗

PubMed1991

F0F1-ATPase from Vibrio alginolyticus. Subunit composition and proton pumping activity.

An F0F1-ATPase was isolated from the membranes of the marine bacterium Vibrio alginolyticus. Homology between the subunits of the F0-complexes from E. coli and V. alginolyticus was found using antibodies against subunits a, b and c of the E. coli F0F1-ATPase. The F0F1-complex from V. alginolyticus was reconstituted into proteoliposomes, which were competent in ATP-dependent proton uptake. This process was inhibited by triphenyltin, DCCD, and venturicidin. Na+ did not affect proton translocation.

Adenosine Triphosphate↗

PubMed1991

Interaction of Zn2+ with the bovine-heart mitochondrial bc1 complex.

A study is presented of the effect of Zn2+ on the enzymatic properties of the bovine-heart cytochrome-bc1 complex. Micromolar concentrations of Zn2+ reversibly inhibit the cytochrome-c reductase activity of either the cholate-solubilized or liposome-reconstituted complex. Kinetic analysis of the redox reactions of the cytochromes indicate that Zn2+ affects the activity of the complex at the quinol oxidation site. The following have been determined: (a) Zn2+ inhibits the pre-steady-state reduction of cytochrome c1 by duroquinol either in the absence or in the presence of antimycin, (b) it does not inhibit the reduction of b cytochromes in the absence of antimycin or in the presence of myxothiazol, (c) it inhibits cytochrome-b reduction in the presence of antimycin. Furthermore Zn2+ inhibits the antimycin-promoted oxidant-induced extrareduction of b cytochromes. Addition of Zn2+ to reduced bc1 complex causes a red shift in the absorption spectrum of cytochrome b566 and a substantial decrease in the signal intensity of the EPR spectrum of the Fe-S protein. This is interpreted as an interaction of Zn2+ with the 2Fe-2S-cluster region of the Fe-S protein, thus giving rise to inhibition of the reductase activity and of the antimycin-insensitive reduction route of b cytochromes. A Scatchard-plot of 65Zn2+ binding to the native isolated complex gave a straight line from which a value of three binding sites and a single dissociation constant of 3 x 10(-6) M can be calculated, which is practically equal to the concentration causing 50% inhibition of electron flow.

Animals↗

PubMed1991

The effect of F0 inhibitors on the Vibrio alginolyticus membrane ATPase.

The inhibition of membrane ATPase from the marine alkalotolerant bacterium Vibrio alginolyticus by DCCD, triphenyltin and venturicidin was studied. DCCD proved to be an irreversible inhibitor, while venturicidin and triphenyltin produced a reversible inhibitory effect. The DCCD-binding proteolipid was identified in the membrane preparations. The effect of the inhibitors on ATPase activity and ATP-dependent Na(+)-transport in V. alginolyticus subcellular vesicles is discussed.

Adenosine Triphosphate↗

PubMed1991

Structural and functional characteristics of polypeptide subunits of the bovine heart ubiquinol--cytochrome-c reductase complex.

Structural and functional characteristics of subunits of bovine heart cytochrome-c reductase have been investigated by controlled digestion of soluble and membrane-reconstituted purified bc1 complex and direct amino acid sequencing of native and digested protein subunits. The results obtained show that the N-terminal segments of core protein II and the 14-kDa protein extend at the periphery of the complex, protruding into the inner matrix space. The Fe-S protein, located at the outer C-periphery of the complex, is shown to be anchored to other subunits of the complex by the amphipathic N-terminal region. Proteolytic cleavage of 7-11 residues from the N-terminal segment of the 14-kDa protein is apparently associated with decoupling of redox-linked proton pumping. Partial digestion of core protein II, the 6.4-kDa protein, and the C-terminal region of the 9.2-kDa protein, is without effect on the redox and proton-motive activity of the complex.

Amino Acid Sequence↗

PubMed1991

Natural killer function in flow cytometry: identification of human lymphoid subsets able to bind to the NK sensitive target K562.

NK cells are a phenotypically, morphologically and functionally heterogeneous population. This has led to the current thought that the non-MHC restricted cytotoxicity is a cellular function that can be associated to different phenotypes. The recognition of the target cell and the conjugate formation is always the first step that eventually leads to the lysis of target. Characterization of the phenotypical pattern of the cells able to bind to K562 targets is the purpose of this study. A multi-parametric flow cytometry binding assay has been employed to identify the different K562-bound lymphocyte subsets. In particular, cells that coexpress the CD16 and CD8 antigens (CD16+8dim+) showed a significantly higher binding capacity than their CD16+8- counterpart. Moreover, the highest binding values have been found in cells that did not express the CD16 antigen at all, but still expressed the CD8dim antigen, such as the small CD8dim+3+ population. These data show that, the NK lytic function being dependent on binding, minor subpopulations must be considered among effector cells, which might correspond to different lytic activities. None of the previously published methodologies that analyze conjugates by flow cytometry or fluorescence microscopy were able to measure the binding capacity of small, double stained, lymphocyte subsets.

Adult↗

PubMed1991

Technical improvements for flow karyotyping by standard FACS 440 flow cytometer.

Chromosomal DNA measurements represent the most accurate methodology for the quantitative analysis of human karyotype. A modified FACS 440 signal collection is described here. The FSC signal voltage coming from the optical bench has been split in two equal parts by a T-connector placed at the preamplifier entrance. The side scatter channel that is not employed during chromosome analysis has been used for the half-FSC signal. This modification allowed the contemporary use of FSC signal both for gating and analysis leading to a better discrimination of background, and therefore to a more accurate definition of sorting windows.

Cell Line↗

PubMed1991

Characterization and cell cycle kinetics of hepatocytes during rat liver regeneration: in vivo BrdUrd incorporation analysed by flow cytometry and electron microscopy.

The incorporation of bromodeoxyuridine (BrdUrd) into newly synthesized DNA has been analysed during hepatocellular regeneration induced by partial hepatectomy in young rats. The kinetic state of the liver has been studied by flow cytometric analysis of the incorporated BrdUrd, while the fine localization of DNA replication sites through the cell cycle has been investigated at the ultrastructural level by the immunogold technique. Eighteen hours after partial hepatectomy flow cytometry revealed an early S phase distribution which corresponded to a specific staining of the interchromatin domains of the hepatocyte nucleus. Thirty-four hours after hepatectomy, on the other hand, when most cells were in late S, a specific staining of heterochromatin domains was observed. The effect of the BrdUrd technique on nuclear aggregation has also been analysed and discussed. The results demonstrate that specific patterns of DNA replication can be recognized during the cell cycle and that flow cytometry and electron microscopy appear to be complementary in the kinetic study of liver regeneration.

Animals↗

PubMed1991

Cell cycle analysis in flow cytometry: use of BrdU labelling and side scatter for the detection of the different cell cycle phases.

Cell cycle analysis in flow cytometry is based on the incorporation of labelled precursors in DNA. The use of BrdU versus SSC, in which side scatter substitutes PI fluorescence, has proved to be useful also for the distinction between G2 and Mitotic cells. Mitoses often produce an SSC decrease due to the morphological changes that happen in the nucleus during this phase of cell cycle. Moreover, DNA accessibility to PI varies during mitosis, as well. However, most of these variations, detectable by flow cytometry appear to be basically dependent on the cell line used.

Bromodeoxyuridine↗

PubMed1990

The gamma subunit of F1 and the PVP protein of F0 (F0I) are components of the gate of the mitochondrial F0F1 H(+)-ATP synthase.

The gamma subunit of the F1 moiety of the bovine mitochondrial H(+)-ATP synthase is shown to function as a component of the gate. Addition of purified gamma subunit to F0-liposomes inhibits transmembrane proton conduction. This inhibition can be removed by the bifunctional thiol reagent diamide. Immunoblot analysis shows that the diamide effect is likely due to disulphide bridging of the gamma subunit with the PVP protein of the F0 sector.

Animals↗

PubMed1990

Activation of a complex of ATPase with the natural protein inhibitor in submitochondrial particles.

Almost all ATPase molecules in submitochondrial particles, isolated from beef heart mitochondria in the presence of MgATP, are in an active complex with the natural protein inhibitor (IF1). In de-energized particles at high ionic strength a slow and irreversible ATPase activation is found to occur due to a dissociation of the enzyme-inhibitor complex. The pH-dependence of this process points out that deprotonation of IF1 molecule is an essential step in the dissociation of the complex. Zn2+ sharply accelerates ATPase activation, probably via binding with the deprotonated form of IF1. ATPase activation is completely prevented by MgATP, indicating the formation of a transient enzyme-inhibitor complex retaining ATPase activity.

Adenosine Triphosphatases↗

PubMed1990

Effect of ATP on the activity of bovine heart mitochondrial b-c1 complex.

The effect of ATP on the reductase activity of purified bovine heart b-c1 complex was studied. ATP stimulates the steady-state activity and the antimycin-insensitive pre-steady-state reduction of b and c1 cytochromes, also causing changes of kinetic properties of the enzyme. There is no absolute specificity for ATP since other polyvalent anions such as EDTA and EGTA produce similar effects in the micromolar range. It is proposed that ATP stimulates the activity of the b-c1 complex, chelating inhibitory cation(s), exerting a modulatory action on the enzyme.

Adenosine Triphosphate↗

PubMed1990

Protonmotive activity of cytochrome c oxidase: control of oxidoreduction of the heme centers by the protonmotive force in the reconstituted beef heart enzyme.

This paper contributes to the characterization of partial steps of electron and proton transfer in mitochondrial cytochrome c oxidase with respect to their membrane arrangement and involvement in energy-linked protonmotive activity. It is shown that delta psi controls electron flow from cytochrome c to heme a is consistent with the view that the latter center is buried in the membrane in a central position. The pressure exerted by delta psi on oxidation of heme alpha 3 by O2 indicates also that this center is buried in the membrane at some distance from the inner side and is consistent with observations showing that protons consumed in the reduction of O2 to H2O derive from the inner space. Electron flow from heme alpha to heme alpha 3 is shown to be specifically controlled by delta pH and in particular by the pH of the inner phase. Analysis of the effect of DCCD treatment of oxidase vesicles reveals that concentrations of this reagent which result in selective modification of subunit III (Prochaska et al., 1981) produce inhibition of redox-linked proton release. Higher concentrations of DCCD which result also in modification of subunits II and IV (Prochaska et al., 1981) cause inhibition of the pH-dependent electron-transfer step from heme alpha to heme alpha 3.

Animals↗

PubMed1990

Effect of chemical modification of lysine amino groups on redox and protonmotive activity of bovine heart cytochrome c oxidase reconstituted in phospholipid membranes.

A study is presented of the effect of chemical modification of lysine amino groups on the redox and protonmotive activity of bovine heart cytochrome c oxidase. Treatment of soluble oxidase with succinic acid anhydride resulted in succinylation of lysines in all the subunits of the enzyme. The consequent change of surface charges from positive to negative resulted in inversion of the orientation of the reconstituted enzyme from right-side-out to inside-out. Reconstitution of the oxidase in phospholipid vesicles prevented succinylation of subunits III and Vb and depressed that of other subunits with the exception of subunits II and IV which were predominantly labeled in a concentration-dependent manner by succinic acid anhydride. This modification of lysines produced a decoupling effect on redox-linked proton ejection, which was associated with a decrease of the respiratory control exerted by the delta pH component of PMF. The decoupling effect was directly shown to be exerted at the level of the pH-dependent rate-limiting step in intramolecular electron flow located on the oxygen side of heme a.

Animals↗