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PubMed1997

Activation of mitochondrial 2-oxoacid dehydrogenases by thioredoxin.

The regulation of mitochondrial dehydrogenases of 2-oxoacids by thioredoxin is established. It is found that at low NAD+ and saturating concentrations of 2-oxoacids and CoA, inactivation of 2-oxoacid dehydrogenase complexes takes place, preventing NAD+ reduction under such conditions. However, addition of oxidized E. coli thioredoxin to the reaction medium without dithiothreitol allows effective NAD+ reduction at this substrate ratio. Product accumulation curves show that thioredoxin activates the complexes by protecting them from the inactivation observed in the conditions when the complex-bound dihydrolipoate is accumulated. Disappearance of the activatory effect of thioredoxin after its treatment with SH-specific reagents indicates the involvement of the redox-active cysteine couple of thioredoxin in its activation of 2-oxoacid dehydrogenase complexes. The redox-inactive thioredoxin not only shows no activation, but in fact exerts an inhibitory effect. The inhibition manifests the complex formation between SH-modified thioredoxin and dehydrogenase systems, involving amino acid residues of thioredoxin other than cysteine. High efficiency of thioredoxin from E. coli as compared to chloroplast thioredoxin f and glutathione disulfide is revealed. This indicates the importance of specific protein structure also for the influence of the redox-active thioredoxin upon the 2-oxoacid dehydrogenase complexes. The results obtained suggest that these key enzyme systems of mitochondrial metabolism represent previously unidentified targets for the action of mitochondrial thioredoxin, which is known to resemble the E. coli counterpart studies in this work.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1983

Epidermal arachidonate lipoxygenase.

Guinea pig skin was found to display a high lipoxygenase activity, evidenced by the formation of a hydroxyeicosatetraenoic acid (HETE) from exogenous [14C]arachidonic acid. The lipoxygenase activity was localized to the epidermal layer of the skin, was completely inhibited by eicosatetraynoic acid (ETYA) and slightly enhanced by indomethacin. Susceptibility to inactivation by sulfhydryl-directed reagents indicated that an essential sulfhydryl is present in a hydrophobic region of the molecule. The enzyme exhibited a broad pH activity optimum and a Km of 2.48 . 10(-5) M. The cytosolic enzyme has been partly purified by ammonium sulfate precipitation and two steps of of column chromatography and exhibited an apparent high molecular weight. The lipoxygenase and hydroperoxidase activities were resolvable from one another. The physiological and pathophysiological roles of the enzyme remain to be elucidated.

Animals↗
PubMed1987

Ligand-induced asymmetry between active sites of cytoplasmic malate dehydrogenase: a chemical modification study.

The iodoacetate-dependent and iodoacetamide-dependent inhibition of cytoplasmic malate dehydrogenase (s-MDH) has been examined. We have confirmed previous reports that iodoacetate inhibits this dimeric enzyme by modifying a single active site methionine per s-MDH subunit. Time courses for the inactivation of the solution-state enzyme with both reagents indicate each s-MDH subunit is modified with equal rapidity in the absence of substrate or cofactor. However, the subunits react with distinctly different rates in the presence of cofactor or cofactor/substrate combinations, indicating some conformational asymmetry between subunits occurs when these ligands are bound. This is consistent with solution-state s-MDH behaving as a cooperative enzyme. Apo and holo crystalline s-MDH are also inhibited by iodoacetic acid. However, subunits of the crystalline enzyme are inhibited with different rates in the presence or absence of active site ligands. This suggests subunit conformations of the dimeric enzyme are not identical in crystalline s-MDH preparations regardless of ligand binding. Furthermore, by the criterion of inhibition rate constants, subunit conformations of the crystalline enzyme are not rigid but are perturbed by ligand binding. Comparisons of inactivation time courses for solution- and crystalline-state s-MDH suggest crystalline s-MDH exhibits at least some of the subunit asymmetry associated with the solution-state enzyme.

Amino Acids↗
PubMed1989

The uncoupling protein dimer can form a disulfide cross-link between the mobile C-terminal SH groups.

Isolated uncoupling protein (UCP) can be cross-linked, by various disulfide-forming reagents, to dimers. The best cross-linking is achieved with Cu2+-phenanthroline oxidation. Because cross-linking is independent of UCP concentration and prevented by SDS addition, a disulfide bridge must be formed between the two subunits of the native dimer. Cross-linking is prevented by SH reagent and reversed by SH-reducing reagents. In mitochondria, cross-linking of UCP with disulfide-forming agents is even more efficient than in isolated state. It proves that UCP is a dimer in mitochondria, before isolation. Disulfide-bridge formation does not inhibit GTP-binding to UCP. Cross-linked UCP re-incorporated in proteoliposomes either before or after cross-linking fully retains the H1-transport function. Rapid cross-linking by membrane impermeant reagents indicates a surface localization of the C-terminus in soluble UCP and projection to the outer surface in mitochondria. Intermolecular disulfide-bridge formation in a dimer requires juxtaposition of identical cysteines at the twofold symmetry axis. A rigid juxtaposition of cysteines is unlikely, unless intended for a native disulfide bridge. The absence of such a bridge in UCP suggests that juxtaposition of cysteines is generated by high mobility. In order to localize the cysteine involved, cross-linked UCP was cleaved by BrCN. The CB-7 C-terminal peptide, which contains cysteines at positions 287 and 304, disappears. Limited trypsinolytic cleavage, previously shown to occur at Lys-292, removed cross-linking in UCP both in the solubilized and mitochondrially bound state. The cleaved C-terminal peptide of 11 residues contains only cystein-304 which, thus, should be the only one (out of 7 cysteines in UCP) involved in the S-S bridge formation. Obviously, the C-terminal location of the cysteine, because of its high mobility, permits juxtapositioning for cross-linking. This agrees with predictions from hydrophobicity analysis that the last 14 residues in UCP protrude from the membrane.

Animals↗
PubMed1981

Purification and characterization of an immunosuppressive factor from ovarian cancer ascites fluid.

A nonspecific immunosuppressive factor present in malignant (ovarian carcinoma) ascites fluid has been purified by acid extraction from a high molecular weight (greater than 20000) complex followed by preparative isoelectric focusing on Bio-lyte media. It is an acidic protein (pI = 3.6) of mol. wt. 50000 to 52000 as estimated by gel filtration and composed of subunits of 25000 to 26000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing conditions. It inhibits the phytohemagglutinin-dependent mitogenic response of normal peripheral blood lymphocytes by 50% at 2 microgram/ml concentrations in vitro and suppresses 80% of the plaque-forming cell response to sheep erythrocytes at 100 microgram per mouse in vivo. Its chemical identity with any of the known plasma proteins has not been established. Its failure to stain with periodic acid Schiff's reagent indicates its minimal content of carbohydrates. It is susceptible to tryptic and pronase digestion but insensitive to deoxyribonuclease and ribonuclease digestion. A smaller suppressive factor identified in the same fluid appears to be a lymphotoxin; it differs from the acid-extracted nonspecific suppressive factor in its lack of susceptibility to trypsin.

Acids↗
PubMed2006

Frequency and resistance of CD95 (Fas/Apo-1) gene-transfected tumor cells to CD95-mediated apoptosis by the elimination and methylation of integrated DNA.

It is important for more effective gene therapies to clarify the mechanisms by which cDNA integrated into cells can maintain or lose its function in vivo. We evaluated genetic and epigenetic events leading to alternation of the introduced CD95 (Fas/Apo-1) gene as a model of gene therapy. Solid tumors formed by CD95 cDNA-transfected hepatoma cells (F6b) were almost completely cured by a single treatment of anti-CD95 monoclonal antibody (mAb) but recurred in gld/gld lpr/lpr mice after initial complete response. Recurred tumors were resistant to repeated mAb treatment. The ratio of resistant cells in tumors was estimated as 4.2 cells per 10(6) cells. The CD95-resistant tumor contained CD95-vanished and CD95-decreased cells. CD95-vanished cells were due to the deletion of CD95cDNA. However, CD95-decreased cells retained CD95cDNA, which was highly methylated when determined with methylation-dependent enzymes and a demethylation reagent, indicating that DNA methylation was responsible for the reduced CD95 expression and resistance to mAb. CD95-decreased cells reduced the CD95 expression further but did not delete cDNA after a second in vivo treatment with anti-CD95 mAb, suggesting that the elimination of cDNA is not induced after its methylation and that cells containing methylated genes became more resistant by further methylation. Thus, the elimination and methylation of integrated cDNA appear to occur through different mechanisms. Our study of resistant tumor cells, which arose by both mutational and epigenetic modifications of the introduced CD95 plasmid, provides important and fundamental information about the fate of introduced cDNA, augmenting the efficiency of gene therapy.

Animals↗
PubMed1988

Human ovarian carcinoma lysis by cytotoxic T cells targeted by bispecific monoclonal antibodies: analysis of the antibody components.

In the perspective of in vivo therapeutic applications, the monoclonal antibody (MAb) MOv18 was selected for its restricted reactivity with human ovarian carcinoma. Using the pH 2.8 desorption assay, we found that the antigen recognized by MOv18 had a high stability on the cell membrane and poor internalization. Therefore, a therapeutic approach which does not require internalization, i.e., the re-targeting of cytotoxic T lymphocytes (CTL) by bispecific MAbs, was investigated. MOv18 and anti-CD3 MAbs were used to produce bispecific reagents, obtained either by chemical cross-linkage (hetero-conjugates) or by somatic hybridization techniques (hybrid MAbs). The maintenance of the binding reactivity and specificity of the bispecific MAbs was analyzed by solid-phase radioimmunoassay, immunofluorescence and cross-competition tests on the relevant target cells (ovarian carcinoma cell line OVCA 432 for MOv18 and PHA-stimulated peripheral blood mononuclear cells for anti-CD3 MAbs), and on 2 irrelevant tumor cell lines. Bv a 51Cr-release assay the bispecific MAbs were found to efficiently promote, at picomolar concentration, cell lysis by CTL clones, but the specificity pattern was wider than that predicted by the binding studies. The F(ab')2 fragment of one hybrid MAb mediated a lysis which was just as efficient as the entire MAb on the relevant target cells and allowed specific lysis to be distinguished from Fc-receptor-mediated lysis. Human immunoglobulins were unable to compete with the Fc receptor binding of the hybrid MAbs and therefore, in the perspective of in vivo applications, Fc fragment removal seems to be an essential step. Analysis of the bispecific reagents indicated that hybrid MAbs are superior to the heteroconjugate as far as storage stability is concerned.

Animals↗
PubMed1988

Anti-ovarian carcinoma anti-T3 heteroconjugates or hybrid antibodies induce tumor cell lysis by cytotoxic T-cells.

In the perspective of therapeutic in vivo targeting for T-cell attack, the monoclonal antibody (MAb) MOv18, selected for its restricted reactivity with human ovarian carcinoma, and an anti-T3 MAb were used to produce heteroconjugate or hybrid antibodies derived by fusion of relevant hybridomas. Specificity and activity of bispecific MAbs were analyzed by solid-phase RIA, immunofluorescence and a 51Cr-release assay on the ovarian carcinoma cell line OVCA 432, which expresses the relevant tumor-associated antigen, and on several irrelevant tumor cell lines. Both reagents efficiently promoted, at picomolar concentration, target cell lysis by cytotoxic T-cell (CTL) clones. Although the pattern of tumor cell lines which were lysed was wider than that predicted by binding studies, further studies using a double-determinant immunoradiometric assay confirmed the specificity of MAb targeting. Analysis of reagents indicated that the hybrid MAb was superior to the heteroaggregate as far as purification recovery and storage stability were concerned. Besides CTL clones, peripheral blood lymphocytes could also be used as cytolytic effectors, provided that a suitable in vitro activation scheme was used.

Antibodies, Heterophile↗
PubMed1987

Sequence-specific positioning of nucleosomes over the steroid-inducible MMTV promoter.

We have investigated chromatin organization over the MMTV LTR, a promoter regulated by steroid hormones. The studies were performed on cell lines containing BPV-based episomal constructs. Nucleosome positioning was determined by localization of sites sensitive to the enzyme micrococcal nuclease, or to the chemical MPE-Fe(II). Experiments with both reagents indicate that nucleosomes are specifically positioned in MMTV LTR chromatin. In the absence of hormone a regular cutting pattern is obtained, with cleavage sites at +136, -60, -250, -444, -651, -826 and -1019 relative to the Cap site. In the presence of hormone the cutting pattern is unchanged, except for a region between -60 and -250 that becomes hypersensitive to MPE-Fe(II). This region contains the DNA sequences to which steroid receptor complexes bind during transcriptional activation. Our results indicate that this region is associated in chromatin from uninduced cells with a macromolecular complex (probably a nucleosome core), and this complex is displaced (or modified) upon binding of activated receptor.

Base Sequence↗
PubMed2001

Toxic effect of L-2-chloropropionate on cultured rat cerebellar granule cells is ameliorated after inhibition of reactive oxygen species formation.

Oral administration of rats to L-2-chloropropionate (L-CPA) causes selective necrosis to the granule cell layer of the cerebellum in vivo and to cultured rat cerebellar granule cells in vitro. The present study was conducted to characterize the involvement of reactive oxygen species (ROS) in cell death of L-CPA to rat cerebellar granule cells in vitro. Exposure to L-CPA (0.625-10 mM) produced a concentration dependent increase in formation of 2,7-dichlorofluorescein (DCF) as a measure of formation of ROS. The elevation of ROS was inhibited after incubation of the cells with the ERK-type of MAP kinases inhibitor U0126, the mitochondrial permeability transition pore inhibitor cyclosporin A (CSA), the antioxidant vitamin E, and the spin trap N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN). Measurements of nitrite (NO(2)) in the cell culture supernatant using the Griess reagent indicate generation of nitric oxide (NO) after exposure to L-CPA. Incubation with L-CPA (10 mM) for 48 hr lead to cell death (90%). When the granule cells were incubated with L-CPA in combination with the inhibitors of free radical production, the cell death was ameliorated. The results show that L-CPA is toxic to granular cells by production of ROS.

Animals↗
PubMed1997

Electrochemical assay of proteinase inhibitors using polycation-sensitive membrane electrode detection.

A simple and sensitive electrochemical method suitable for real time detection of trypsin-like proteinase inhibitors is described. The method is based on utilizing a protamine (a polycationic substrate for trypsin-like proteinases)-sensitive membrane electrode to monitor, potentiometrically, the initial rate of protamine decomposition upon the addition of a proteinase-antiproteinase test solution. In the presence of proteinase inhibitors, the initial rate of change in electromotive force is dependent on the concentration of proteinase inhibitor in the sample solution. The feasibility of this new assay method is demonstrated by detecting four inhibitors of trypsin-like proteinases: alpha1-antiproteinase inhibitor, alpha2-macroglobulin, aprotinin, and soybean inhibitor, using trypsin as the indicator proteinase. The efficacy of inhibition by each species, as expressed by I50 values (concentration of the inhibitor that induces 50% of the maximum proteinase inhibition), is shown to correlate well with literature values for the association constant of the proteinase-antiproteinase complex (k[assoc]). The proposed electrochemical assay for aprotinin is examined further using trypsin, plasmin, and kallikrein as the proteinase indicator reagents. It is found that the trypsin-aprotinin system offers the highest sensitivity and lowest detection limit for aprotinin detection. Application of the proposed method for measuring aprotinin in pretreated plasma samples is also reported.

Aprotinin↗
PubMed1997

Analysis of the Morgan-Elson chromogens by high-performance liquid chromatography.

The Morgan-Elson method for quantitative N-acetylhexosamine analysis is a two-step procedure comprising alkali treatment of the sugar and subsequent condensation of the resulting chromogens with p-dimethylaminobenzaldehyde (Ehrlich's reagent) to yield a colored product. In the present investigation, the products formed in the first step of the procedure were analyzed by high-performance liquid chromatography (HPLC) on a reversed-phase (C18) column, which was eluted with a water-methanol gradient; the absorbance of the effluent was monitored at 229 nm. The profile generated from alkali-treated N-acetylglucosamine exhibited two major peaks, in a ratio of approximately 2.5:1, which accounted for 94% of the total peak area. A third peak, accounting for 3% of the peak area, was eluted in an intermediate position, and several smaller peaks were also observed. The three predominant components, isolated by preparative HPLC, all gave a purple color on addition of Ehrlich's reagent, indicating that they were Morgan-Elson chromogens. The HPLC profile of alkali-treated N-acetylmannosamine was identical to that of the products generated from N-actylglucosamine, as was expected because of the elimination of the asymmetry at C-2 during formation of the chromogens. N-Acetylgalactosamine yielded two major peaks, which were eluted in the same positions as the two major products formed from N-acetylglucosamine, but the intermediate peak seen in the N-acetylglucosamine pattern was absent. The HPLC procedure allowed detection of as little as approximately 25 ng of N-acetylglucosamine and may therefore be of value as an alternative to the complete Morgan-Elson procedure when only small amounts of sample are available for quantitative analysis.

Acetylgalactosamine↗
PubMed1996

Disulfide bonds are necessary for structure and activity in Aspergillus ficuum phytase.

The function of disulfide bonds in Aspergillus ficuum phytase was elucidated by unfolding studies, using guanidinium hydrochloride (Gu.HCl) as denaturant. Although the enzyme is totally inactivated by 0.8 M Gu.HCl, at pH 5.0, the active conformation is instantaneously restored by 0.6 M Gu.HCl, at pH 5.0. Conditions which would permit refolding of phytase are completely negated by 10 mM beta-mercaptoethanol and causes its catalytic demise at pH 7.5. Assay of free thiols using Ellman's reagent indicates that none of the thiols in the ten cysteines in phytase are free; five disulfide bonds were predicted for the enzyme. Sequence comparison of mold phytases and yeast acid phosphatases indicates four conserved cysteines. Thus, disulfide bonds play an important role in the folding of fungal phytase; any perturbation of the process of its formation causes an altered three-dimensional structure that is inconsistent with catalytic activity.

6-Phytase↗
PubMed1981

An ultrastructural assessment of mitochondrial in the gastric parietal cell with the high iron diamine methods.

The ultrastructural densification of mitochondria by the high iron diamine (HID) method has been investigated by staining gastric parietal cells with each component of the HID reagents, alone or in combination, or with an otherwise modified HID solution. The effect of certain chemical treatments prior to staining has also been assessed. These tests provided evidence for at least three cytochemically distinguishable constituents in the mitochondrial matrix. In addition, the results from these tests and observation obtained with a newly introduced diaminobenzidine (DAB)-FeCl3 staining reagent indicated that the probable mode of action of the HID method in imparting density to mitochondrial matrix entails combination between a complex of iron with polymerized or aggregated m- and p-diamine and some matrix constituent. Assessment of the cytochemical staining in varied fixation condition revealed that the lucent population of mitochondria recently described in HID-stained parietal cells reflects a failure to stain with the method because of inadequate fixation. The possible nature of the HID-reactive substance in the mitochondrial matrix is discussed in the light of these observations and other cytochemical reactivities.

Animals↗
PubMed2006

Role of the conserved glutamine 291 in the rat gamma-aminobutyric acid transporter rGAT-1.

We investigated the role of the Q291 glutamine residue in the functioning of the rat gamma-aminobutyric acid (GABA) transporter GAT-1. Q291 mutants cannot transport GABA or give rise to transient, leak and transport-coupled currents even though they are targeted to the plasma membrane. Coexpression experiments of wild-type and Q291 mutants suggest that GAT-1 is a functional monomer though it requires oligomeric assembly for membrane insertion. We determined the accessibility of Q291 by investigating the impact of impermeant sulfhydryl reagents on cysteine residues engineered in close proximity to Q291. The effect of these reagents indicates that Q291 faces the external aqueous milieu. The introduction of a steric hindrance close to Q291 by means of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide modification of C74A/T290C altered the affinity of the mutant for cations. Taken together, these results suggest that this irreplaceable residue is involved in the interaction with sodium or in maintaining the cation accessibility to the transporter.

Amino Acid Sequence↗
PubMed2000

Appearance of a stage-specific immunodominant glycoprotein in encysting Entamoeba invadens.

The appearance of cyst-specific proteins in encysting Entamoeba invadens and their immunogenicity were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using an axenic encystation system in vitro. A rabbit antiserum against trophozoites of E. invadens reacted with a number of proteins of cysts after 1-4 days of incubation. Thus, a number of cyst proteins remained antigenically unchanged as common antigens of the two forms after transformation from trophozoites to cysts. A rabbit antiserum against cysts also reacted with the trophozoite proteins as well as the cyst proteins. The most interesting result was that the rabbit anticyst serum reacted predominantly with an 88-kDa protein of cysts after 1 day of incubation. The 88-kDa protein reacted with the anticyst serum absorbed with trophozoite proteins and was thus cyst-specific. The reactivity of the 88-kDa protein of cysts with the absorbed anticyst serum decreased as encystation proceeded. When soluble and particulate fractions prepared from cysts after 1 day of incubation were examined by electrophoresis and immunoblotting, the 88-kDa protein that had reacted with the absorbed anticyst serum was found to be present in the particulate fraction, which was rich in cell-wall fragments, and stained with periodic acid-Schiff's reagent, indicating that it is a glycoprotein. The results indicate that encystation is accompanied by appearance of the cyst-specific 88-kDa glycoprotein, which is immunodominant and most abundantly expressed in cysts after 1 day of incubation and appears to be associated with the cyst wall.

Animals↗
PubMed1984

Mouse bone collagenase inhibitor: purification and partial characterization of the inhibitor from mouse calvaria cultures.

The organ culture of neonatal mouse calvaria produced both collagenase and collagenase inhibitor. The inhibitor was purified by a series of column chromatographies: DEAE-cellulose and CM-cellulose ion-exchange chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, and finally by Sephacryl S-200 gel filtration. The purified inhibitor migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular mass of 28,000. The inhibitor was purified 140-fold to a specific activity of 163 units/mg with a yield of 18% over the first step of the purification by DEAE-cellulose chromatography. The inhibitor stained positively for carbohydrate with periodic acid-Schiff's reagent indicating, in conjunction with its affinity to concanavalin A, that the inhibitor is a glycoprotein. In addition to mouse bone collagenase, this inhibitor also inhibited chick bone, rat bone, rabbit corneal, and human gingival collagenase, but did not inhibit bacterial collagenase.

Animals↗
PubMed1984

Erythrocyte Lii-Nao countertransport system. Inhibition by N-ethylmaleimide probes for a conformational change of the transport system.

Human erythrocytes were treated by a series of SH-reagents, including maleimides, iodo compounds, mercurials and oxidizing agents. Rates of Li efflux into Na-rich medium, Li leak and Lii-Nao countertransport were then determined. Of the 13 different reagents studied, only N-ethylmaleimide, iodoacetamide and iodoacetate inhibited selectively the countertransport activity. The effect of the various reagents indicates that the sensitive SH-groups of the countertransport system are not externally exposed. N-Ethylmaleimide was used to probe for changes elicited by substrate cations in Lii-Nao countertransport. In Na- and Li-free medium, inhibition of Lii-Nao countertransport by N-ethylmaleimide of 35% was reached within 2 s. In Na or Li medium, maximal inhibition was twice as great, but was attained much more slowly, within 10 min. Kinetic data and Hill plot analysis indicate the involvement of two classes of SH-groups: one expressed in the various media with and without substrate cations, and an additional one, which becomes specifically available to N-ethylmaleimide in the presence of external Na or Li. The affinity of Na to the site promoting inhibition by N-ethylmaleimide (apparent Km = 12 mM) is higher than the affinity of Na to its external countertransport site (apparent Km = 25 mM, as reported by Sarakadi, B., Alifimoff, J.K., Gunn, R.B. and Tosteson, D.C. (1978) J. Gen. Physiol. 72, 249-265). Reactivity of N-ethyl[14C]maleimide was not modified by the media tested. It is concluded that external Na and Li cause a conformational change in the protein(s) of the countertransport system in human erythrocytes.

Biological Transport↗