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At least 19 recordsLinked to original sources
PubMed1986

Alteration of intramolecular disulfides in insulin receptor/kinase by insulin and dithiothreitol: insulin potentiates the apparent dithiothreitol-dependent subunit reduction of insulin receptor.

Dithiothreitol (DTT) was observed to increase both beta-subunit autophosphorylation and exogenous substrate phosphorylation of the insulin receptor in the absence of insulin. The natural protein reducing agent thioredoxin was also observed to increase the insulin receptor beta-subunit autophosphorylation. The activation of the insulin receptor/kinase by both DTT and thioredoxin was found to be additive with that of insulin. Further, the increase in the insulin receptor beta-subunit autophosphorylation in the presence of DTT and insulin was demonstrated to be due to an increase in the initial rate of autophosphorylation without alteration in the extent of phosphorylation. Similarly, the increase in the exogenous substrate phosphorylation was due to an increase in the Vmax of phosphorylation without significant effect on the apparent Km of substrate binding. In the presence of relatively low concentrations of DTT, insulin was found to potentiate the apparent insulin receptor subunit reduction of the native alpha 2 beta 2 heterotetrameric complex into alpha beta heterodimers, when observed by silver staining of sodium dodecyl sulfate-polyacrylamide gels. N-[3H]Ethylmaleimide ([3H]NEM) labeling in the absence of DTT pretreatment demonstrated that only the beta subunit had accessible sulfhydryl group(s). However, treatment of insulin receptors with DTT increased the amount of [3H]NEM labeling in the beta subunit as well as exposing sites on the alpha subunit. Further, incubation of the insulin receptors with the combination of DTT and insulin also demonstrated the apparent insulin-potentiated subunit reduction without any increase in the total amount of [3H]NEM labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Disulfides↗
PubMed1981

Insulin-like effects of dithiothreitol on isolated rat adipocytes.

The effects of dithiothreitol on basal glucose oxidation, hormone-induced lipolysis and insulin receptors in isolated rat adipocytes were studied. Dithiothreitol produced a dose-dependent stimulation of basal glucose oxidation and inhibition of adrenaline-induced lipolysis. Dithiothreitol also inhibited corticotropin-induced lipolysis, but failed to inhibit dibutyryl cyclic AMP-induced lipolysis. Dithiothreitol did not inhibit the binding of the beta-adrenergic antagonist [3H]dihydroalprenolol to adipocytes. Neither catalase (100 micrograms/ml) nor EDTA (2 mM) abolished the antilipolytic effect of dithiothreitol. Treatment of isolated adipocytes with 1 mM-dithiothreitol for 20 min at 37 degrees C also caused stimulation of basal glucose oxidation and inhibition of adrenaline-induced lipolysis. A Scatchard plot of insulin binding to control adipocytes was curvilinear. However, treatment of cells with 1 mM-dithiothreitol decreased the curvilinearity of the plot, indicating that only a low-affinity state of the insulin receptors exists in the dithiothreitol-treated adipocytes. These findings suggest that the insulin-like activities of dithiothreitol are mediated through the interaction of dithiothreitol with insulin receptors.

Adipose Tissue↗
PubMed1994

Potentiation by 1,4-dithiothreitol of histamine-induced inositol phosphate formation in rat cerebral cortex and human HeLa cells.

Dithiothreitol (1 mM) caused a 340-fold shift of the concentration-response curve for histamine-induced [3H]inositol monophosphate ([3H]IP1) accumulation in slices of rat cerebral cortex prelabelled with [3H]inositol. Dithiothreitol had no significant effect on carbachol-induced [3H]IP1 accumulation. The effect of dithiothreitol appeared to be at the level of the H1-receptor, since curves of histamine inhibition of [3H]mepyramine binding to a membrane fraction from rat cerebral cortex were shifted to lower concentrations by dithiothreitol, with a similar concentration-dependence for dithiothreitol as observed for potentiation of [3H]IP1 accumulation. The small shift of the curve of histamine inhibition of [3H]mepyramine binding by a stable GTP analogue, 5'-guanylylimidodiphosphate, was not increased in the presence of dithiothreitol. Histamine-induced [3H]IP1 accumulation in human HeLa cells was also potentiated by dithiothreitol, but the decrease in the EC50 was only 3-fold. It is suggested that differences in the magnitude of the effect of dithiothreitol on histamine H1-receptor mediated responses between tissues may reflect in part the position of the concentration-response curve in the absence of dithiothreitol.

Animals↗
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↗
PubMedDate not supplied

Dithiothreitol tetraacetate S-acetyl esterase activity in blood and in different tissues of male rats.

Dithiothreitol is known to be effective in vitro to prevent or even revert effects of oxidative stress and radiation in biological systems. However, its use in in vivo conditions has been hampered by its chemical instability and toxicity. Dithiothreitol tetraacetate is stable and less toxic than dithiothreitol. In this work, GLC-MS evidence is presented that dithiothreitol tetraacetate is hydrolyzed in vivo to give dithiothreitol. The activity of a dithiothreitol tetraacetate thiol esterase in blood and in different organs was determined and found to be: small intestine >> liver > pancreas >> testes approximately kidney > or = brain approximately adrenals approximately bone marrow approximately colon approximately lung > plasma approximately heart > total blood. These results suggest that dithiothreitol tetraacetate might be a suitable source of more stable and less toxic dithiothreitol in vivo, and that its production is due to an hydrolytic process requiring a dithiothreitol tetraacetate thiol esterase present in different organs and in blood.

Acetylesterase↗
PubMed1994

Dithiothreitol effects on human sperm quality.

Human semen normally coagulates immediately after ejaculation and then undergoes liquefaction during the next 15 to 60 minutes. Incomplete seminal liquefaction can result in impaired sperm motility and make clinical evaluation and manipulation difficult. Dithiothreitol, a mucolytic agent that reduces the mucoprotein disulfide bonds in sputum, has been found to induce liquefaction of incompletely liquefied semen in vitro. We studied the effects of dithiothreitol on sperm motility, viability, acrosomal integrity and morphology. A semen sample was provided by 45 healthy, young men at the University of Arizona. Of the specimens 10 (22%) demonstrated incomplete liquefaction. Sperm motility and motion characteristics of untreated (control) semen and semen treated with dithiothreitol were objectively evaluated using computer assisted semen analysis. Sperm cell membrane integrity and mitochondrial integrity were measured by fluorescence microscopy using the deoxyribonucleic acid specific fluorochrome propidium iodide and the mitochondria specific fluorochrome rhodamine-123, respectively. Acrosomal integrity was determined using the fluorescent stain chlortetracycline. Sperm morphology was evaluated using bright field microscopy. For completely liquefied semen (35 cases) dithiothreitol reduced sperm motility (59.1 +/- 1.2% untreated versus 53.2 +/- 1.2% treated, p < 0.01) and motion characteristics. However, dithiothreitol had no statistically significant effect on motility on sperm in the group with incompletely liquefied semen (10 cases). Sperm cell membrane, mitochondrial and acrosomal integrity was unaffected by dithiothreitol regardless of liquefaction status. Dithiothreitol caused a significant increase in abnormally large sperm head morphology in the group with completely liquefied semen. The minimal effects of dithiothreitol on sperm motility traits and viability support its use as a possible aid in the evaluation and manipulation of incompletely liquefied semen.

Acrosome↗
PubMed1982

Implications for in vitro studies of the autoxidation of ferrous ion and the iron-catalyzed autoxidation of dithiothreitol.

The influences of buffers and iron chelators on the rate of autoxidation of Fe2+ were examined in the pH range 6.0-7.4. The catalysis by Fe2+ and Fe3+ of the autoxidation of dithiothreitol was also investigated. In buffers which are non- or poor chelators of iron, 0.25 mM Fe2+, and 0.3 mM dithiothreitol when present with iron, oxidize within minutes at pH 7.4 and 30 degrees C. The stability of each increases as the pH is decreased and more than 90% of each remains after 1 h at pH 6.0. In the presence of buffers or oxy-ligands which preferentially and strongly chelate Fe3+ over Fe2+, Fe2+ autoxidizes rapidly in the pH range 6.0-7.4 while dithiothreitol is protected. Ligands which preferentially bind strongly to Fe2+ stabilize both Fe2+ and dithiothreitol at pH 7.4. Dithiothreitol readily reduces Fe3+ in non-chelating buffers or in the presence of strong chelators of Fe2+, however, the ferrous ions produced are prone to reoxidation at higher pH values. These results show that Fe2+ and dithiothreitol are very susceptible to autoxidation in the neutral pH range, and that the rates are strongly influenced by the presence of chelators of Fe2+ and Fe3+. The rapid autoxidations of these species need to be taken into account when designing and interpreting experiments involving Fe2+ or both dithiothreitol and iron.

Dithiothreitol↗
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↗
PubMed1986

The effect of dithiothreitol on the kinetics of dissociation of dexamethasone from the non-transformed mammary cytosolic glucocorticoid receptor.

Sulfhydryl reducing agents such as dithiothreitol are required for maximum binding of dexamethasone to the mammary cytosolic glucocorticoid receptor, but little is known concerning the effects of dithiothreitol on the kinetics of the binding reaction. In this report we have examined the influence of dithiothreitol on the dissociation kinetics of dexamethasone from the non-transformed glucocorticoid-receptor complex at 0-4 degrees C under various experimental conditions. Without dithiothreitol, the rate of dissociation of dexamethasone remains essentially the same (t1/2 approximately 17 h) regardless of the method chosen to monitor dissociation. With dithiothreitol, however, there is a marked acceleration in the rate of dissociation of receptor-bound dexamethasone when an excess of unlabeled dexamethasone is used to study dissociation (t1/2 approximately 5 h) but not when dissociation is investigated by removal of free labeled dexamethasone by charcoal adsorption (t1/2 approximately 21 h); dithiothreitol also accelerates the observed rate of dissociation when a combination of these methods is used. An acceleration in the rate of receptor-bound dexamethasone is also observed when an excess of the synthetic progestin, R5020, is used in the dissociation assay. The possible reasons and importance underlying these findings have been discussed.

Animals↗
PubMed1983

Amelioration of mercuric chloride-induced acute renal failure by dithiothreitol.

Experiments were conducted to determine if administration of the sulfhydryl reducing agent and metal chelator dithiothreitol (31 mg/kg body wt) alters the development of renal dysfunction in the first 3 hr after injection of mercuric chloride (3 mg/kg). Mercuric chloride alone resulted in elevation of urine flow rate and fractional excretion of solutes within 30 min of injection. In animals injected with dithiothreitol 60 min after mercuric chloride, urine flow rate and fractional excretion of solutes were reduced within 30 min to values intermediate between control and mercuric chloride-treated rats. Neither the injection of mercuric chloride alone nor when followed by dithiothreitol resulted in changes in mean arterial blood pressure or glomerular filtration rate. In addition, dithiothreitol did not reduce urine flow rate or fractional excretion of solutes when these parameters were elevated during extracellular fluid volume expansion. Measurement of mercury in organs of those rats injected with mercuric chloride alone or prior to dithiothreitol revealed no alteration in organ distribution. The renal cortex contained the highest concentrations of mercury, and these concentrations were comparable in both groups of rats. These studies demonstrate that dithiothreitol can ameliorate the renal toxicity of mercury and suggest that this effect is mediated through an intrarenal site of action.

Acute Kidney Injury↗
PubMed1982

Effects of dithiothreitol on insulin-sensitive phosphodiesterase in rat fat cells.

Dithiothreitol activates the low-Km membrane-bound cyclic AMP phosphodiesterase when incubated with the enzyme in a cell-free system. To investigate the mechanism of its activation, we studied the effect of protease inhibitors. Isolated fat cells obtained from Sprague-Dawley rats were incubated in Krebs-Henseleit Hepes buffer, pH 7.4, at 37 degrees C with and without insulin (2 nM, 10 min). A crude microsomal fraction prepared by differential centrifugation was suspended in 0.25 M sucrose containing 10 mM Tes buffer, pH 7.5, with and without 2 mM dithiothreitol and protease inhibitors at 4 degrees C for 48 h. Dithiothreitol stimulated the phosphodiesterase, in a time-dependent manner. As little as 0.02 mM dithiothreitol activated the enzyme, and the maximally effective dose was 2-10 mM. Among the various protease inhibitors tested, antipain, leupeptin, chymostatin and E-64 were the most effective in preventing activation of the enzyme by dithiothreitol. Antipain also inhibited release of the enzyme from the bound fraction. These results suggest that activation of the low-Km phosphodiesterase by dithiothreitol may be provoked by stimulation of an endogenous thiol protease.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Partial disruption of naturally occurring groups of insulin receptors on adipocyte plasma membranes by dithiothreitol and N-ethylmaleimide: the role of disulfide bonds.

In this ultrastructural study, monomeric ferritin-insulin was used to further elucidate the role of disulfide bonds in maintaining the natural groups of insulin receptors on adipocyte plasma membranes. Dithiothreitol (1 mM) caused partial disruption of the occupied receptor groups with an increase in single receptors to greater than 50% of total occupied receptors. N-Ethylmaleimide (1 mM) disrupted the groups to the same extent as dithiothreitol and the effect was partly additive with the dithiothreitol effect. The magnitude of the disruption caused by dithiothreitol or N-ethylmaleimide was similar to that caused by cytochalasin B. Dithiothreitol, a reducing agent, caused a marked increase in binding of insulin to the plasma membranes while N-ethylmaleimide and cytochalasin B, both thiol reagents, had little if any effect on insulin binding. These data suggest that two different sets of disulfide bonds are involved. One set was susceptible to both reducing and thiol reagents and responsible for holding the receptor groups together, and the other set was susceptible to reducing agents only and related to the increased insulin binding caused by dithiothreitol. A proposed model is discussed.

Adipose Tissue↗
PubMed1986

Enhancement of histamine H1-receptor agonist activity by 1,4-dithiothreitol in guinea-pig cerebellum and cerebral cortex.

The disulphide bond-reducing agent 1,4-dithiothreitol (1 mM) produced a marked potentiation of histamine-stimulated accumulation of [3H]inositol phosphates in lithium-treated slices of guinea-pig cerebellum and cerebral cortex. This was seen as a parallel shift of the concentration-response curve for histamine to lower agonist concentrations, with no significant effect on the maximal response or Hill coefficient. Dithiothreitol similarly potentiated the augmentation of adenosine-stimulated cyclic AMP accumulation elicited by histamine in guinea-pig cerebral cortex. Studies with partial agonists suggested that this potentiating effect was associated with an increase in agonist efficacy rather than a change in agonist binding affinity. Thus, dithiothreitol increased the maximal accumulation of [3H]inositol phosphates produced by both 2-pyridylethylamine and 2-methylhistamine, which appeared to act as partial agonists in guinea-pig cerebral cortex. Dithiothreitol similarly increased the maximal extent of the augmentation of adenosine-stimulated accumulation of cyclic AMP produced by 2-methylhistamine. The site of action of dithiothreitol is not known; however, a comparison of the effect of dithiothreitol on muscarinic and histamine H1-receptor-mediated phosphoinositide responses in guinea-pig cerebral cortex suggests that it is before the stage at which the receptor-effector pathways are shared by these two receptor systems.

Animals↗
PubMed1975

New manual and automated method for determining activity of creatine kinase isoenzyme MB, by use of dithiothreitol: clinical applications.

The method is based on the selective activating capacity of dithiothreitol on creatine kinase isoenzyme MB, after isoenzyme MM is activated by glutathione. Isolated isoenzymes MM and MB of human and canine origin were assayed individually and in mixtures of known activities. When glutathione was present in the assay medium the activity of each isoenzyme could be measured individually, but glutathione did not activate isoenzyme MB if it was present in a mixture with MM. Dithiothreitol, added to the serum before assay, activated the isoenzyme MB in the mixture. Values for MB activities obtained for isolated isoenzyme MB and for the isoenzyme mixture after dithiothreitol was added averaged 110 and 111 U/liter, respectively (r = 0.998; y = 1.007 x + 0.298; n = 10). In the serum of 40 patients with documented acute transmural myocardial infarction, the mean proportion of isoenzyme MB activity measured in this way was 5.5% (coefficient of variation, 7.7%). Isoenzyme MB activities measured by use of dithiothreitol compared well with those obtained by conventional electrophoresis/spectrophotometry (r = 0.998; y = 1.09x -0.65) and spectrofluorometry (r = 0.996; y = 1.10 x + 0.80). The assay of MB activity by the dithiothreitol method was automated, by use of an Abbott Bichromatic Analyser and a Calbiochem Super-Stat Pack Kit. In 60 isoenzyme MB determinations the manual and automated method correlated well (r = 0.990; y = 1.0x -1.36). The simplicity of isoenzyme MB determination by use of dithiothreitol and its ease of automation allow routine monitoring of the isoenzyme activity in patients with ischemic heart disease.

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↗
PubMed1986

Participation of three distinct active states of chloroplast ATPase complex CF0 X CF1 in the activation by light and dithiothreitol.

Chloroplast ATPase complex (CF0 X CF1) in thylakoids is activated by illumination in the presence or absence of dithiothreitol or by incubation with both dithiothreitol and Pi in the post-illumination dark. The activation by dithiothreitol and Pi is inhibited by ADP and decreases with increasing time interval between the end of illumination and the addition of dithiothreitol and Pi. The dithiothreitol/Pi-activated ATP hydrolysis is highly sensitive to pH. The ATP hydrolysis activated by illumination in the presence of dithiothreitol decreases its sensitivity to stimulation by NH4Cl and increases its sensitivity to pH, with increasing time interval between the end of illumination and the addition of ATP. Its pH dependence approaches that of the dithiothreitol/Pi-activated ATP hydrolysis. These results suggest that in the post-illumination dark, the light/dithiothreitol-activated CFo X CF1 converts its state from the one (E2) which is sensitive to an uncoupler and relatively insensitive to pH to the one (E2i) which is insensitive to an uncoupler and highly sensitive to pH. In the post-illumination dark, the presence of both dithiothreitol and Pi brings about the activation of CFo X CF1 to E2i.

Adenosine Diphosphate↗
PubMed1976

Reaction of rhodanese with dithiothreitol.

The reaction between bovine rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) and reduced dithiothreitol has been studied. This reagent, in the absence of thiosulfate, reduces the amount of sulfur carried by rhodanese with formation of sulfide and oxidized dithiothreitol: E-S-SH + reduced dithiothreitol replaced by E-SH + HS- + oxidized dithiothreitol, (E = enzyme). An inactivation was observed at high dithiothreitol/enzyme ratios or at very low enzyme concentrations. The inactivation was not observed in the presence of thiosulfate and can be reversed by cyanide or thiosulfate. A thiosulfate reduction activity of rhodanese was also found using dithiothreitol as reductant.

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↗