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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mixed-sex adult stages of Brugia pahangi and Dipetalonema viteae, in the absence of exogenous substrate, consumed oxygen at rates of 4.18 +/- 0.38 and 2.12 +/- 0.20 ngatoms O2 min-1 mg-1 dry wt. respectively. When calculated on a unit dry weight basis the endogenous O2 consumption rates (E-QO2) of mature adult male macrofilariae of B. pahangi and D. viteae were significantly greater than those of mature females, although the E-QO2 calculated per individual worm was essentially similar irrespective of sex. When assayed as separate unisexual groups, the oxygen uptake of male and female macrofilariae of both species was inhibited by classical inhibitors of respiratory electron transport (RET), and showed classical substrate bypass phenomena in response to succinate and ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine with respect to the RET inhibitors rotenone (inhibitor of complex I) and antimycin A (inhibitor of complex III). Since male worms elicited similar responses to the classical RET inhibitors as did mixed-sex and/or adult female populations, the possibility that developmental stages contained within the female filariids were contributing in any significant manner to the overall responses observed with the RET inhibitors can be discounted. Such responses as observed with live-intact macrofilariae are normally elicited only by mitochondrial preparations and suggest that the cuticles of both species are permeable to rotenone, succinate, antimycin A, N,N,N',N'-tetramethyl-p-phenylenediamine, azide and cyanide. The uncoupler 2,4-dinitrophenol stimulated the endogenous rate of oxygen consumption (E-QO2) of intact B. pahangi at 33-160 microM, indicating the probable occurrence of RET-coupled oxidative phosphorylation. Higher concentrations of 2,4-dinitrophenol proved inhibitory. Respiratory studies on subcellular fractions substantiated the responses elicited by the intact parasites, suggesting the presence of antimycin A-sensitive and -insensitive RET pathways capable of utilising alpha-glycerophosphate, succinate, and malate as substrates. Both B. pahangi and D. viteae macrofilariae therefore probably possess branched RET-pathways bifurcating on the substrate side of RET-complex III. The rates of substrate oxidation in terms of QO2 mg-1 mitochondrial protein compare well with those observed with other nematode parasites.
Mol Biochem Parasitol 1985 Mar
PMID:Evidence for the occurrence of respiratory electron transport in adult Brugia pahangi and Dipetalonema viteae. 403 7

Treatment of intact Chinese hamster ovary cells with HgCl2 produced a rapid, concentration-dependent induction of DNA single-strand breaks (SSB) as revealed by alkaline elution analysis. Direct addition of HgCl2 to cell lysates did not result in DNA strand breaks. HgCl2 treatment of cells also caused a rapid leakage of superoxide radicals that were detected in their media by measurement of the reduction of exogenously added cytochrome c. There was a linear relationship between the production of radicals and the induction of DNA strand breaks, and there were also excellent temporal correlations in these parameters. Addition of oxygen radical scavengers, such as the enzymes superoxide dismutase and catalase, to the extracellular media significantly reduced the extent of DNA damage caused by HgCl2 without a similar attenuation of its uptake into cells, as did the autoclaved enzymes. Similarly, addition of radical scavengers such as glycerol or ascorbate inhibited the DNA damage but also reduced the uptake of the metal by almost the same degree. Thus, because of secondary effects on uptake of the metal, the radical scavenger experiments could not address the importance of oxygen radicals in the DNA damage caused by HgCl2. SSB were enhanced when cells were treated with HgCl2 and diethylmaleate or diethyldithiocarbamate, agents that deplete cellular reduced glutathione or inhibit the intracellular activity of superoxide dismutase, respectively. Thus, DNA damage in cells rendered sensitive to radicals was greater when these cultures were subsequently treated with HgCl2. The binding of 203HgCl2 to the DNA of intact Chinese hamster ovary cells was also studied. These studies were made possible by the relatively high stability of Hg(II) interaction with DNA and by utilizing a gentle method of DNA isolation that minimized redistribution of intracellular Hg(II) complexes after cells were lysed. The amount of Hg(II) bound to DNA varied from approximately 7 to 35 Hg atoms per 10(4) base pairs (bp) at concentrations of HgCl2 that have been previously shown to produce between 1 SSB/10(7) bp and 1 SSB/10(6) bp. The Hg(II)-DNA adducts were relatively stable complexes, since they resisted treatment with 0.1 M EDTA and 1 M NaCl and were stable to precipitation of the DNA with ethanol and trichloroacetic acid. However, the Hg(II) was released from the DNA when it was degraded enzymatically to mononucleosides, suggesting that the Hg(II)-DNA bonds formed in the cell were not truly covalent and that the strength of Hg(II) binding to DNA depended upon polynucleotide structure.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1984 Sep
PMID:Mechanism of HgCl2 cytotoxicity in cultured mammalian cells. 609 Aug 87

Coupled phosphorylation was examined in liver, kidney and brain mitochondria from rats made thyrotoxic by injecting repeated doses of triiodothyronine. Liver and kidney mitochondria were maximally affected under these conditions, whereas effects on brain mitochondria were marginal. State-3 respiration rates with succinate decreased considerably in all the tissues, whereas glutamate oxidation increased in liver, but decreased in kidney and brain mitochondria. Oxidation rates of beta-hydroxybutyrate decreased in kidney and brain mitochondria but were not significantly affected in liver mitochondria. Oxidation of ascorbate + TMPD was not affected. State-4 respiration rates increased in general with all the substrates resulting in lowering of the RCI. The ADP/O ratios decreased in a site-specific manner in the mitochondria from the three tissues. The content of cytochrome b decreased in all three tissues, whereas the content of cytochrome c + c1 increased in liver and kidney but decreased in brain. The content of cytochrome a, however, was not significantly affected. Basal and Mg2+-stimulated ATPase activities increased in mitochondria of liver and kidney but not in those of brain; total ATPase activities, however, were not altered. The results imply that excessive levels of thyroid hormones over normal in the serum can lead to impairment of mitochondrial energy metabolism in a tissue-specific manner.
Mol Cell Endocrinol 1982 Oct
PMID:Effect of experimental thyrotoxicosis on oxidative phosphorylation in rat liver, kidney and brain mitochondria. 621 75

Cytochrome c is modified by covalent binding of pyridoxal phosphate (PLP) to lysine residues. One di-substituted [(PLP)2--C] and two mono-substituted derivatives [(PLP)--c and (PLP)''--c] were obtained and precisely purified. The peak at 695 nm and CD-spectra in 190--600 nm region show that all derivatives have native conformation. The differential UV-spectra of the derivatives against native protein show that in (PLP)2--c there is a contact dipole-dipole interaction between PLP chromophores. It is calculated that the N-atoms of the two PLP-substituted lysines must be at a distance less than or equal to 12 A. Analysing our and literature data, one may suppose that Lys-13 and Lys-87 are the most probable candidates for modification with PLP. (PLP)---c and (PLP)''--c behave differently during ion-exchange chromatography and when added to cytochrom c-depleted mitochondria. (PLP)''--c restores electron transfer at higher concentrations than (PLP)'--c. Both they restore fully succinate and ascorbate oxidation but at considerably higher concentrations than the native protein, i. e. modification of any one of the reactive towards PLP lysines descreases but does not exclude the interaction with its reductase and oxidase. The effective equilibrium constants of binding of modified derivatives to cytochrome c-depleted mitochondria are lower than the constant for native protein. Together with decrease in binding activity, Hill coefficients increase. From our results it may be supposed that probably the binding sites of cytochrome c for its reductase and oxidase partially overlap.
Mol Biol (Mosk)
PMID:[Pyridoxalphosphate-modified derivatives of cytochrome c. Mono- and disubstituted derivatives: characteristics and effect on electron transport in cytochrome c-depleted mitochondria]. 624 46

The fast and slow refolding reactions of iron(III) cytochrome c (Fe(III) cyt c), previously studied by Ikai et al. (Ikai, A., Fish, W. W., & Tanford, C. (1973) J. Mol. Biol. 73, 165--184), have been reinvestigated. The fast reaction has the major amplitude (78%) and is 100-fold faster than the slow reaction in these conditions (pH 7.2, 25 degrees C, 1.75 M guanidine hydrochloride). We show here that native cyt c is the product formed in the fast reaction as well as in the slow reaction. Two probes have been used to test for formation of native cyt c. absorbance in the 695-nm band and rate of reduction of by L-ascorbate. Different unfolded species (UF, US) give rise to the fast and slow refolding reactions, as shown both by refolding assays at different times after unfolding ("double-jump" experiments) and by the formation of native cyt c in each of the fast and slow refolding reactions. Thus the fast refolding reaction is UF leads to N and the slow refolding reaction is Us leads to N, where N is native cyt c, and there is a US in equilibrium UF equilibrium in unfolded cyt c. The results are consistent with the UF in equilibrium US reaction being proline isomerization, but this has not yet been tested in detail. Folding intermediates have been detected in both reactions. In the UF leads to N reaction, the Soret absorbance change precedes the recovery of the native 695-nm band spectrum, showing that Soret absorbance monitors the formation of a folding intermediate. In the US leads to N reaction an ascorbate-reducible intermediate has been found at an early stage in folding and the Soret absorbance change occurs together with the change at 695 nm as N is formed in the final stage of folding.
 ...
PMID:Nature of the fast and slow refolding reactions of iron(III) cytochrome c. 626 2

ESR spectra of erythrocyte membranes labeled with a maleimide spin label (MSL) show two types of label environment: a weakly immobilized component and a strongly immobilized component. Chlorpromazine (CPZ) markedly altered the spectra: at pH 8.0, 3 mM CPZ reduced the amplitude of the spectrum by 40%, and the weakly immobilized component was almost completely removed. In order to clarify the mechanisms of these spectral changes the protein release from erythrocyte membranes induced by CPZ has been followed. CPZ had a weak solubilizing effect on erythrocyte membranes: less than 1% of the membrane protein was released, mainly Band 6. By comparison with the protein release induced by low-salt treatment it was found that the "detergent-like" property of CPZ cannot explain the alterations in the ESR spectra. The nature of the spectral changes induced by CPZ was different from that of changes induced by lowering the pH to 4.5; correlated with other data this shows that changes in organization or conformation of membrane protein cannot explain the CPZ-induced alterations in the ESR spectra. These spectral changes appeared to be due to the reduction by CPZ of the nitroxide free radical. This was documented by the marked reduction of spin concentration of the labeled ghosts in the presence of CPZ resulting in a decrease in amplitude of the ESR spectrum of MSL-labeled erythrocyte ghosts induced by CPZ. The reduction by CPZ of the nitroxide free radical was compared with that induced by ascorbate. It was found that CPZ preferentially reduces the mobile component of the ESR spectrum of MSL-labeled ghosts. The action of CPZ in reducing free radicals may have consequences for patients receiving long-term treatment with phenothiazine derivatives.
Mol Pharmacol 1983 May
PMID:Effect of chlorpromazine on proteins in human erythrocyte membranes as inferred from spin labeling and biochemical analyses. 630 35

The optical properties, copper content, catalytic activity and quaternary structure of many preparations of ascorbate oxidase purified with two different methods were examined. Fresh samples appeared identical and were characterized by optical ratios A280/A610 = 25 +/- 1 and A330/A610 = 0.8 +/- 0.05, by specific activity toward ascorbate of 3.48 +/- 0.05 mol g-1 min-1 and by a copper content of 8 +/- 0.3 mol/145 000 Mr. The enzyme is composed of two non-covalently linked subunits of slightly different molecular mass (75 000 and 72 000 respectively). These subunits cannot be further resolved by reduction of disulfide bonds. Proteolytic cleavage of the protein chains was observed during purification and storage in the absence of the protease inhibitor 6-amino caproic acid. Ascorbate oxidase exists as a monomer at neutral pH and undergoes reversible association into higher molecular weight species at slightly acid pH values. Association is not accompanied by spectroscopic or catalytic changes.
Mol Cell Biochem 1983
PMID:A reinvestigation on the quaternary structure of ascorbate oxidase from Cucurbita pepo medullosa. 664 10

The role of various enzymes and biological molecules on the activation and deactivation of the metabolites of phenol was investigated in vitro. Phenol, the major metabolite of benzene, is metabolized to hydroquinone and catechol. Activation of these metabolites and deactivation of their oxidized forms was assessed by the amount of covalent binding to microsomal protein. [14C]Phenol and NADPH were incubated with hepatic microsomes isolated from phenobarbital-pretreated guinea pigs, and 2.33 nmoles of hydroquinone and 0.12 nmole of catechol were formed per minute per milligram of microsomal protein. Covalent binding of the metabolites to microsomal protein incubated with microsomes isolated from guinea pigs pretreated with phenobarbital was 252 pmoles bound/min/mg; with microsomes from untreated guinea pigs, covalent binding was 146 pmoles bound/min/mg. Covalent binding was inhibited greater than 90% with the addition of N-octylamine, ascorbate, or GSH. The addition of superoxide dismutase inhibited covalent binding with microsomes isolated from phenobarbital-pretreated guinea pigs 35% but did not inhibit it with microsomes isolated from untreated animals. Partially purified guinea pig hepatic DT-diaphorase [NAD(P)H (quinone acceptor) oxidoreductase, EC 1.6.99.2] inhibited covalent binding 70%. This effect was reversed in the presence of dicumarol, a specific inhibitor of DT-diaphorase. DT-diaphorase present in the 10(5) X g supernatant fraction was also active in inhibiting covalent binding but only after the removal of endogenous reduced glutathione. This effect could also be reversed by dicumarol. The addition of diaphorase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) partially purified from Clostridium kluyveri inhibited covalent binding 86%. The addition of hydrogen peroxide and horseradish peroxidase (peroxidase, EC 1.11.17) or myeloperoxidase(s) increased covalent binding 30-fold and 6-fold, respectively. Ascorbate decreased this binding greater than 95%. These results indicate that hydroquinone, catechol, and phenol as well as their oxidized forms can be activated or deactivated by several of the above model systems. These systems may play a role in the myelotoxicity of benzene by modulating covalent binding.
Mol Pharmacol 1984 Jul
PMID:DT-diaphorase and peroxidase influence the covalent binding of the metabolites of phenol, the major metabolite of benzene. 674 27

Chromaffin cells in primary culture take up [14C]ascorbic acid from the incubation medium. Cells, stimulated immediately after a short labeling period with [14C]ascorbate, secrete ascorbic acid concomitantly with catecholamines (CA) through a nicotinic receptor-mediated Ca2+-dependent process. A proportional release of CA and [14C]ascorbic acid was observed through a large range of secretion rates obtained by varying the concentration of nicotine or by changing the concentrations of Ca2+ and Na+ in the external medium. However, under the same conditions of stimulation, different cell preparations secrete 2-10 times more CA than [14C]ascorbate (as percentage of cell content). Furthermore, a different time course of secretion was observed for CA and [14C]ascorbate for each of several secretagogues. In addition, Ba2+ is a much more potent stimulus for CA secretion than for secretion of [14C]ascorbate, and Ca2+ channel blockers are more potent in inhibiting CA secretion than [14C]ascorbate secretion. These data suggested the possibility that newly taken up ascorbate was being secreted from a compartment altogether distinct from the chromaffin vesicle. This hypothesis was confirmed by subcellular distribution studies, where only a minor fraction of newly taken up [14C]ascorbate was found in the vesicular fraction (P2) from homogenates of chromaffin cells prepared after a short incubation with [14C]ascorbate. However, the subcellular distribution of [14C]ascorbate follows that of endogenous ascorbate when a short pulse with the label is chased by a prolonged equilibration period in the absence of ascorbate, indicating that a transfer has occurred from the extravesicular compartment(s) to the CA storage organelle. Endogenous ascorbate, which is found both inside and outside the chromaffin vesicle, was also found to be secreted from chromaffin cells, indicating that ascorbic acid could be released simultaneously from two different subcellular compartments.
Mol Pharmacol 1983 Mar
PMID:Secretion of newly taken up ascorbic acid by adrenomedullary chromaffin cells originates from a compartment different from the catecholamine storage vesicle. 683 2

Two factors must be present for primary avian tendon cells to commit 50% of their total protein production to procollagen: ascorbate and high cell density. Scorbutic primary avian tendon cells at high cell density (greater than 4 X 10(4) cells per cm2) responded to the addition of ascorbate by a sixfold increase in the rate of procollagen synthesis. The kinetics were biphasic, showing a slow increase during the first 12 h followed by a more rapid rise to a maximum after 36 to 48 h. In contrast, after ascorbate addition, the level of accumulated cytoplasmic procollagen mRNA (alpha 2) showed a 12-h lag followed by a slow linear increase requiring 60 to 72 h to reach full induction. At all stages of the induction process, the relative increase in the rate of procollagen synthesis over the uninduced state exceeded the relative increase in the accumulation of procollagen mRNA. A similar delay in mRNA induction was observed when the cells were grown in an ascorbate-containing medium but the cell density was allowed to increase. In all cases, the rate of procollagen synthesis peaked approximately 24 h before the maximum accumulation of procollagen mRNA. The kinetics for the increase in procollagen synthesis are not, therefore, in agreement with the simple model that mRNA levels are the rate-limiting factor in the collagen pathway. We propose that the primary control point is at a later step. Further support for this idea comes from inhibitor studies, using alpha, alpha'-dipyridyl to block ascorbate action. In the presence of 0.3 mM alpha, alpha'-dipyridyl there was a specific two- to threefold decrease in procollagen production after 4 h, but this was unaccompanied by a drop in procollagen mRNA levels. Therefore, inhibitor studies give further support to the idea that primary action of ascorbate is to release a post-translational block.
Mol Cell Biol 1983 Feb
PMID:Role of procollagen mRNA levels in controlling the rate of procollagen synthesis. 683 11


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