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At least 19 recordsLinked to original sources
PubMed1978

Effects of dithiothreitol on end-plate currents.

1. End-plate currents have been studied in frog cutaneus pectoris nerve-muscle preparations mounted in continuously flowing solution, using the voltage clamp technique. 2. Exposure of the muscle to 1 mM-dithiothreitol reduced the amplitude of end-plate currents by a factor of 2.7 (mean; range 1.6-3.4; twelve fibres). 3. 1 mM-dithiothreitol also caused a 2.7-fold (2.3-3.1) increase in the rate of decay, and a 1.4-fold (1.3-1.6) decrease in the time to peak of end-plate currents. During the onset of action of dithiothreitol, there was little or no indication of departure of end-plate current decay from a simple exponential. 4. Dithiothreitol actions on amplitude and decay of end-plate currents developed with similar time courses and both effects were slower in onset at pH 7.2 than at pH 8.5. 5. The actions of dithiothreitol were reversed by exposure of the muscle to 1 mM-5,5'-dithio-bis-(2-nitrobenzoic acid). 6. Following dithiothreitol treatment, the rates of decay of end-plate currents continued to depend on membrane potential; there was little or no change in the slope of the relation between in (rate of decay) and membrane potential, consistent with little or no change in the dipole moment of a gating molecule for ion channels. 7. Dithiothreitol changed the relation between peak end-plate current and membrane potential, so that peak conductance increased at more negative membrane potentials; this finding could be accounted for in terms of the closure of ion-channel gates becoming faster though remaining voltage-sensitive after exposure to dithiothreitol. 8. It is concluded that dithiothreitol causes changes in the kinetics of gating of ion channels associated with receptors and that these changes accompany changes in the binding of ACh to receptors.

Animals↗
PubMed1978

Regulation of protein synthesis by hemin: effect of dithiothreitol on the formation and activity of the hemin-controlled translational repressor.

Previous studies have demonstrated that the hemin-controlled translational repressor (HCR), a high molecular weight protein inhibitor of polypeptide chain initiation in rabbit reticulocyte lysate, is formed from a presynthesized prorepressor over a period of 12--18 h in three stages denoted reversible, intermediate, and irreversible. The prorepressor can, however, be completely converted to irreversible HCR within 2 min by incubation with such sulfhydryl reagents as N-ethylmaleimide. The results in this report demonstrate that dithiothreitol, which stabilizes thiol groups, will, like hemin, prevent the conversion of the prorepressor to HCR and will inactivate reversible HCR. Unlike hemin, dithiothreitol also inactives the intermediate form of HCR. Neither dithiothreitol nor hemin has any effect on the activity of irreversible HCR. Since the prorepressor used in these experiments had been separated from the supernatant factor (a soluble protein that reverses the inhibition of protein synthesis due to HCR), the effect of dithiothreitol and of hemin is independent of this factor and may be mediated by direct interaction with the prorepressor and HCR. Dithioerythritol, the erythro isomer of dithiothreitol, is as effective as dithiothreitol in preventing the formation of HCR, whereas glutathione and beta-mercaptoethanol have little or no effect.

Animals↗
PubMed1975

Inhibition of RNA-dependent DNA polymerase reaction by 6-(p-hydroxyphenylazo)-uracil: A result of drug induced dithiothreitol oxidation.

6-(P-hydroxyphenylazo)-uracil (HPUra) reduced by dithiothreitol inhibited AMV or RLV virion associated exogenous RNA-dependent DNA polymerase reactions. However, the inhibition was variable from experiment to experiment and was not consistent with the base specificity of HPUra seen for inhibition of gram positive DNA-dependent DNA polymerases. Increasing the concentration of dithiothreitol reversed the inhibition. Furthermore, at non-toxic concentrations, HPUra did not influence the plating efficiency of RLV in tissue culture, as measured by the ability to induce foci on sarcoma virus positive-leukemia virus negative cells. Oxidation of dithiothreitol in the presence or absence of HPUra was followed spectrophotometrically under enzyme conditions. HPUra catalyzed the oxidation of dithiothreitol under these conditions. Since dithiothreitol is required for optimum reaction rates, as well as complete disruption of virus in some polymerase assay systems, the oxidation of dithiothreitol in the presence of HPUra is sufficient to explain the inhibition seen.

Animals↗
PubMed1979

Effect of dithiothreitol of the reaction of renin and angiotensinogen.

Effects of several sulfhydryl reagents on the reaction of renin and angiotensinogen were investigated by measuring the production of angiotensin I. Renin and angiotensinogen were prepared from the kidneys of dogs and from plasma of nephrectomized dogs, respectively. The amount of angiotensin I generated was increased with application of dithiothreitol, dithioerythritol and 2,3-mercapto-1-propanol. The greatest enhancement of the generation of angiotensin I was observed with dithiothreitol. Dithiothreitol had no influence on renin activity, on radioimmunoassay for angiotensin I and the recovery of synthetic angiotensin I from the incubation medium. When heterologous angiotensinogen was used, a great enhancement of generation of angiotensin I was observed. Dithiothreitol did not accelerate the reaction following replacement of the natural substrate, angiotensinogen, with the synthetic tetradecapeptide as substrate. It is postulated that dithiothreitol augments angiotensin I generation by acting on the angiotensinogen and not on the renin.

Angiotensinogen↗
PubMed1977

Modification of cardiovascular responses to histamine by dithiothreitol.

1 Histamine produced dose-dependent contractile responses on both isolated perfused ear arteries and aortic strips of the rabbit. These responses were blocked by mepyramine and potentiated by both metiamide and dithiothreitol. 2 In the presence of maximum potentiation by metiamide, dithiothreitol still potentiated the contractile response to histamine of both preparations. 3 In the presence of mepyramine, histamine produced dose-dependent reductions in the contractile response to noradrenaline. This vasodilator action of histamine was abolished by metiamide but was unaffected by dithiothreitol. 4 The vasodilator action of histamine on the human isolated perfused temporal artery and the positive inotropic effect of histamine on the isolated spontaneously beating atria of the rabbit were blocked by metiamide but unaffected by dithiothreitol. 5 It is concluded that the rabbit aorta, like the ear artery, contains both H1 and H2 histamine receptors and that dithiothreitol potentiates cardiovascular responses mediated by H1-receptors but not by H2-receptors.

Animals↗
PubMed1977

Glyceraldehyde-3-phosphate dehydrogenase of Scenedesmus obliquus. Effects of dithiothreitol and nucleotide on coenzyme specificity.

NADH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.--) of the photosynthetic alga Scenedesmus obliquus is converted to an NADPH specific form by incubation with dithiothreitol. The change in nucleotide specificity is accompanied by a reduction in the molecular weight of the enzyme from 550 000 to 140 000. Prolonged incubation with dithiothreitol results in the further dissociation of the enzyme to an inactive 70 000 dalton species. The 140 000 dalton, NADPH-specific enzyme is stabilized against dissociation and inactivation by the presence of NAD(H) or NADP(H). Optimum stimulation of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase activity is achieved on incubation of the NADH-specific enzyme with dithiothreitol and NADPH, or dithiothreitol and a 1,3-diphosphoglycerate generating system. The relevance of these observations to in vivo light-induced changes in the nucleotide specificity of the enzyme is discussed.

Cyanobacteria↗
PubMed1979

Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide.

Thioredoxin from Escherichia coli was shown to catalyze the reduction of insulin disulfides by dithiothreitol. A quantitative assay was developed which measures the rate of insulin reduction spectrophotometrically at 650 nm as turbidity formation from the precipitation of the free insulin B chain. Thioredoxin, at 5 microM concentration, accelerated the reaction between 0.130 mM insulin and 1.0 mM dithiothreitol at pH 7 around 20-fold. The pH optimum of the reaction was 7.5. Thioredoxins from E. coli and calf liver showed similar specific activities. Stopped flow fluorescence measurements of the rate of reduction of thioredoxin-S2 by dithiothreitol showed a second order rate constant of 1647 M-1 s-1 at pH 7.2. This is between 10(2) to 10(3) times larger than the reaction between insulin or linear model disulfides and dithiothreitol. It is consistent with a ping-pong mechanism of thioredoxin catalysis since reduced thioredoxin is known to react very fast with insulin. Thioredoxin also catalyzed lipoamide-dependent reduction of the insulin disulfides in a coupled system with NADH, lipoamide, and lipoamide dehydrogenase. The fast spontaneous reaction between dihydrolipoamide and thioredoxin-S2 provides a mechanism for NADH or pyruvate-dependent disulfide reduction. The implication of the dithiol-disulfide oxidoreductase activity of thioredoxin for the regulation of enzyme activities by thiol oxidation-reduction control is discussed.

Bacterial Proteins↗
PubMed1976

Effect of dithiothreitol on meiotic maturation of mouse oocytes in vitro: dependence of the effect on N6,O2'-dibutyryl Adenosine 3',5'-cyclic monophosphate.

The inhibitory effect of dithiothreitol on meiotic maturation of mouse oocytes in vitro is a function of the intracellular dibutyryl cyclic AMP concentration. Inhibition of nuclear (germinal vesicle) breakdown by dibutyryl cyclic AMP is reversed upon transfer of oocytes to plain culture medium, whereas, transfer to medium containing dithiothreitol results in continued inhibition. Dithiothreitol significantly enhances the effectiveness of dibutyryl cyclic AMP as an inhibitor of meiotic maturation. These results suggest that the reported effect of sulfhydryl reducing agents on membrane dissolution and reconstitution, in both meiotic and mitotic cells, may be attributed to their influence on intracelular levels of cyclic AMP.

Animals↗
PubMed1978

Dithiothreitol-induced alterations of blood pressure, vascular reactivity and aortic microsomal calcium uptake in spontaneously hypertensive rats.

Dithiothreitol, a potent sulfhydryl reducing agent, depressed systolic blood pressure to a greater extent in genetically hypertensive rats (SHR) than in normotensive Wistar-Kyoto rats (WKY). Dithiothreitol depressed the contractile response to norepinephrine and potassium chloride of isolated aortic strips from both SHR and WKY. Dithio-bis-2-nitrobenzoid acid, a sulfhydryl oxidizing agent, restored the responsiveness of rat aortic strips to these contractile agents. Microsomes isolated from rat aortae sequester calcium in the presence of ATP. This activity, generally referred to as calcium-pump activity, has been postulated to function in smooth muscle relaxation, and is significantly depressed in aortic microsomes of the SHR. Dithiothreitol (10 mM) greatly increased and dithio-bis-2-nitrobenzoic acid (100 micrometer) decreased the ATP-dependent calcium pump activity of microsomes isolated from both SHR and WKY aortae. These observations indicate that sulfhydryl groups may influence systems involved in vascular reactivity and blood pressure regulation.

Animals↗
PubMed1978

Characteristics of the activation by dithiothreitol and Fe(2+) of tryptophan hydroxylase from the rat brain.

The preincubation of tryptophan hydroxylase extracted from various areas of the central nervous system of the rat with 30 mM dithiothreitol and 50 muM ferrous ammonium sulfate under nitrogen atmosphere resulted in a persistent increase of its activity. Studies on the enzyme characteristics indicated that this activation was associated with a doubling in its Vmax and a shift (from 7.6 to 7.2) of the optimal pH for its activity. In contrast, the molecular weight and the apparent affinities of tryptophan hydroxylase for its pterin cofactor and for tryptophan were not significantly altered by the preincubation with dithiothreitol and ferrous ammonium sulfate. Since this treatment did not prevent the stimulatory effects of various compounds (phosphatidylserine, ATP and MG(2+), Ca(2+)) on tryptophan hydroxylase activity, this might be a good procedure to activate this enzyme with only minor changes in its regulatory properties.

Animals↗
PubMed1978

Stabilization of placental aromatase by dithiothreitol in the presence of oxidizing agents.

Cumene hydroperoxide, sodium periodate and iodosobenzene were not able to support aromatization by placental microsomes in the absence of NADPH or molecular oxygen. In the presence of these oxidizing agents and NADPH, aromatase was slowly inactivated. Dithiothreitol (10mM) prevented the loss of aromatizing activity in the presence of these compounds. One function of dithiothreitol may be to protect aromatase by scavenging harmful oxidizing agents.

Aerobiosis↗
PubMed1978

Effects of superoxide dismutase, dithiothreitol and formate ion on the inactivation of papain by hydroxyl and superoxide radicals in aerated solutions.

Losses in enzyme activity and sulphydryl content have been studied in aerated papain solutions containing formate, superoxide dismutase and dithiothreitol. Both formate and dithiothreitol converted .OH to .O2-, whereas superoxide dismutase completely suppressed the inactivation by .O2-. Using results from all three systems, the fraction of .O2- reactions with papain that caused inactivation of the enzyme was 0.33 +/- 0.07. The results also showed that the fraction of .OH reactions, which cause inactivation of papain, is significantly higher in aerated than in oxygen-free solutions.

Cobalt Radioisotopes↗
PubMed1978

Sulphydryl requirement for insulin release from the perfused pancreas. Studies with ethacrynic acid and dithiothreitol.

Using the isolated, perfused rat pancreas the importance of sulphydryl groups for the secretory process of insulin was investigated. It was found that ethacrynic acid (EA, 0.075-0.6 mmol/1) caused a dose-dependent, monophasic insulin release. Addition of EA to a glucose-stimulated (20 mmol/1) pancreas led to a sudden increase in hormone release, followed by a dose-dependent inhibition of release, which was not reversible after removal of EA. The same phenomenon was seen in the presence of 20 mmol/1 leucine. Dithiothreitol (DTT, 0.1 and 1 mmol/1) had no effect on basal insulin secretion. Added to a glucose-stimulated pancreas DTT (1 mmol/1) caused a reversible inhibition of insulin release. The persistent inhibitory action of EA on glucose-induced insulin release could be reversed by simultaneous perfusion of EA and DTT. Sequential exposure of a glucose-stimulated pancreas to EA and DTT led to a rapid release of insulin, due to DTT; however, the EA-induced inhibition of insulin secretion could not be prevented. Two kinds of thiol groups in the plasma membrane and in the beta cell might be responsible for the various kinetics of insulin release induced by EA and DTT.

Animals↗
PubMed1979

Azethoxyl nitroxide spin labels. ESR studies involving thiourea crystals, model membrane systems and chromatophores, and chemical reduction with ascorbate and dithiothreitol.

Trans- and cis-azethoxyl nitroxides 1, 2, 3 and 4 can be trapped in the cavities of thiourea crystals. The presence of a single gauche conformation on either side of the pyrrolidine ring within the crystals was indicated by the ESR spectra. Rotation about the long molecular axis then corresponds approximately to y-axis motion of the nitroxide moiety. Proxyl nitroxides in which the nitroxide group is located on the penultimate carbon of long chain lipids can also be trapped and were shown to adopt the azethoxyl conformation in the thiourea crystals. The measured deltaA values (A parallel to - A perpendicular) of oriented egg lecithin multilayers containing trans- and cis-azethoxyl nitroxides 1 and 2 were quite small, consistent with the unique orientation of the nitroxide principal axes with respect to the long axis of the molecule. The deltaA values for a series of lipids bearing a label near the terminus of the chain were very similar to that of 1, showing that the azethoxyl conformation is likely the predominant one in these labels in orienting systems. Computer simulations of the ESR spectra of 1 and 2 in egg lecithin vesicles provided values for molecular orientation and motion parameters consistent with those expected from a consideration of molecular models in the extended (all trans) conformation. Azethoxyl nitroxides have also proven useful in the investigation of motion restricted (boundary) lipid in a lipid-protein system. Thus, the values (69 +/- 10%) for the amount of boundary lipid in the chromatophore membranes from Rhodopseudomonas sphaeroides as determined using trans- 2 and cis- 2 are in good agreement with values using 16-doxylstearic acid (64 +/- 3%). The fact that all three labels show about the same fraction of boundary lipid in this system indicates that the lipid binding sites are relatively insensitive to the geometry of the lipid chain. Also, both 1 and 2 appear to be able to detect a third lipid environment not seen with the doxyl fatty acid. The apparent fluidity of this component lies between that of boundary and bilayer lipid. The unique orientation of the nitroxide principal axes with respect to the long molecular axis in azethoxyl nitroxides 1 and 2 allows detection of hindrance to rotation about the long molecular axis, in contrast to the analogous doxyl and proxyl fatty acids. Comparative reduction studies using ascorbate and dithiothreitol indicate that azethoxyl nitroxides are slightly more resistant toward reduction than proxyl nitroxides and much more resistant than doxyl nitroxides.

Ascorbic Acid↗
PubMed1978

Effect of snap freezing, dithiothreitol and storage on estimations of estrogen receptor sites.

Estrogen receptor assays were performed on human myometrium in order to determine optimal conditions for certain steps of the assay. The findings indicate that: (1) tissue should be snap-frozen in liquid nitrogen prior to assay, (2) dithiothreitol does not increase measurable binding site numbers, (3) estrogen receptors in the cytosol fraction are unstable and should be stored in excess of 1 day; (4) if storage prior to receptor assay is unavoidable, tissue should be stored at -20 degrees C or -70 degrees C for no longer than 1 week.

Cytosol↗
PubMed1977

Partial reduction with dithiothreitol of disulfide bonds in human chorionic gonadotropin.

Of the eleven disulfide bonds of human chorionic gonadotropin (hCG), two were reduced with a 10-fold molar excess of dithiothreitol (DTT) relative to hormone. An S-carboxymethyl (SCM) derivative and a reoxidized product of this reduced hCG retained full biologic activity and were likely to be immunologically identical with native hCG. These two disulfide bonds appeared to be located in the alpha-subunit of the hormone. A 40-fold molar excess of DTT was required to reduce the third disulfide bond which was located in the beta-subunit. An SCM derivative ot this hexa-SH-hCG was only one fifth as biologically active as native hCG but its immunologic activity was only slightly decreased. However, its reoxidized product exhibited over 70% of the biologic activity of and it had nearly the same immunologic activity as the hormone. Reduction of a fourth disulfide bond, probably in the beta-subunit, by a 100-fold molar excess of the reagent was accompanied by considerable alterations in the hormone conformation as evidenced by electrophoresis. The resulting SCM derivatives as well as reoxidized products showed progressive decreases in biologic and immunologic activity and both reduced deca-SCM-hCG and a reoxidized product of deca-SH-hCG were virtually devoid of biologic activity. These findings suggest that the two first-reduced disulfide bonds, apparently in the alpha-subunit, are not important to the biologic activity of the hormone and that the third-reduced disulfide bond, probably in the beta-subunit, is very important in maintaining a biologically active conformation of the hormone. Further disulfide reduction appears to result in considerable alterations in the general conformation of the hormone, including disulfide interchange.

Binding Sites↗
PubMed1977

Relative susceptibilities of the interchain disulfides of an immunoglobulin G molecule to reduction by dithiothreitol.

The reduction by dithiothreitol (DTT) of the four interchain disulfides of a human IgGlkappa immunoglobulin has been studied by two methods: variation of the concentration of DTT relative to the protein concentration (incremental reduction); and variation of the time of reduction at fixed levels of DTT and protein (kinetic reduction). In both cases, the results depend on whether the reduction is carried out aerobically or anaerobically. Under aerobic conditions, the relative levels of intermediates (HL, H2, and H2L) which are generated as native molecules (H2L2) are converted to reduced heavy (H) and light (L) chains depend on the concentrations of protein and DTT as well as on the exposure time to DTT; no stable equilibrium is reached between reduced and oxidized states and conditions gradually revert from those favoring reduction to those favoring reoxidation. By contrast, anaerobic reduction is independent of protein concentration or time of exposure to DTT, beyond about 30 min, indicating that an equilibrium between partially reduced and oxidized states is achieved. The distribution of intermediates observed under anaerobic conditions has been analyzed according to theoretical models (Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (second in a series of three articles in this issue)). Within experimental error, both kinds of anaerobic experiments resemble a random reduction process wherein the four disulfides are equivalent and independent of each other with respect to rate and extent of reduction by D. It is concluded that there are no readily detected pathways in the process, as would occur if the intrinsic reactivities of the bonds were distinct, and no marked cooperatively between the four reaction sites, as would be observed if reduction of one bond materially facilitated or hampered reactivity at another site. Both of these characteristics of the reduction are in direct contrast to those of the reoxidative process, which is marked by the initial preference for formation of a bond between heavy and light chains, and by kinetic cooperativity in bond formation during the course of the reaction (Sears, D.W., et al. (1977), Biochemistry 16 (first in a series of three articles in this issue); Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (second in this series)).

Disulfides↗