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PubMed2005

The activity-induced reduction of myofibrillar Ca2+ sensitivity in mouse skeletal muscle is reversed by dithiothreitol.

The aim of this study was to further characterize the reduction of myofibrillar Ca2+ sensitivity in mouse muscle which has been observed after fatigue at 37 degrees C. Muscle bundles and single fibres were isolated from mouse flexor digitorum brevis muscle and studied at 37 degrees C. The single fibres were injected with the Ca2+ indicator indo-1. Muscle fatigue was produced by 0.4 s tetani repeated at 4 s intervals until force had fallen to less than 50% of initial. Excitation-contraction coupling was assessed by measuring the cytosolic calcium concentration ([Ca2+]i) during tetani, and the maximum Ca2+-activated force and the myofibrillar Ca2+ sensitivity were estimated from a series of tetani at different stimulation frequencies. Two main results were found. (i) The reduction of Ca2+ sensitivity only occurred when the muscle was intensely stimulated leading to fatigue. When the muscle was rested for 10 min at 37 degrees C there was no significant change in Ca2+ sensitivity. (ii) If the membrane-permeant thiol-specific reducing agent dithiothreitol (0.5 mm) was applied to the muscle for 2 min following the fatigue protocol, the reduction in Ca2+ sensitivity was reversed. Dithiothreitol had no effect on Ca2+ sensitivity in unfatigued preparations. There was no effect of fatigue or dithiothreitol on tetanic [Ca2+]i or on the maximum Ca2+-activated force. These results suggest that intense activity of skeletal muscle at 37 degrees C causes the production of reactive oxygen species which oxidize a target protein. We propose that critical sulphydryl groups on the target protein(s) are converted to disulphide bonds and this reaction reduces Ca2+ sensitivity.

Animals↗
PubMed1974

Dithiothreitol-dependent antilisterial activity of lysates from normal macrophages exposed in vitro to culture fluids from spleen cells of BCG-immunized mice.

The effect of culture fluids from mouse lymphocytes on the antilisterial activity of normal macrophages in vitro was determined. Titers of lysates of macrophages incubated with or without culture fluids from lymphocytes from control mice had titers of 10 to 270 with or without a reducing agent, dithiothreitol. Lysates of macrophages after incubation with culture fluids of spleen lymphocytes from BCG-immunized mice had antilisterial titers with and without dithiothreitol of 197,000 and 810, respectively. This may indicate a dithiothreitol-dependent antilisterial system inherent in activated macrophages of cell-mediated immunity. Lysates of incubated spleen cells and their culture fluids also had slight antilisterial activity.

Animals↗
PubMed1989

Effects of GTP analogs and dithiothreitol on the binding properties of the vascular vasoactive intestinal peptide receptor.

Previous studies have demonstrated a specific vascular receptor for the neurotransmitter peptide, vasoactive intestinal peptide (VIP), and have suggested that the receptor is positively coupled to vascular adenylate cyclase. The present study addressed the questions whether the vascular VIP receptor is subject to regulation by guanine nucleotides and whether a disulfide reducing agent, dithiothreitol, would perturb the binding function of the vascular VIP receptor. Guanosine triphosphate (GTP) and its non-hydrolyzable analogs, guanylyl imidodiphosphate (Gpp(NH)p) and guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), increased the rate of dissociation of radiolabeled VIP from arterial receptors in a concentration-dependent manner. GTP-gamma-S increased the equilibrium dissociation constant (KD) of the high affinity vascular VIP binding site, a result consistent with decreased high affinity binding of VIP induced by GTP-gamma-S. These results are consistent with a regulatory role for guanine nucleotides in the function of the vascular VIP receptor. The disulfide reducing agent, dithiothreitol, caused a decrease in specific binding of radiolabeled VIP. Upon Scatchard analysis the effect of dithiothreitol was characterized by an increase in the KD and a decrease in the maximum number of binding sites (Bmax) of the high affinity binding site. These results suggest that disulfide bonds are important for ligand binding to vascular VIP receptors. The sulfhydryl alkylating agents, N-ethylmaleimide and iodoacetamide, had minimal effects on radioligand binding.

Animals↗
PubMed1995

Fine structure of the epidermal basement membrane of the lip: applications of dithiothreitol separation and ultrathin frozen sectioning.

The fine structure of the epidermal basement membrane at the electron-microscopic level has already been defined. To obtain more details, two techniques, dithiothreitol separation and ultrathin frozen sectioning, were applied either alone or in combination. Negatively stained ultrathin sections showed a much thicker lamina densa than ordinary plastic-embedded sections. In the lamina lucida, bridging filaments and subbasal dense plates were observed in negative images. After the treatment with dithiothreitol, the lamina densa could be peeled off mechanically from the underlying dermis, and the anchoring fibrils were pulled off the dermis, preserving the connection with the lamina densa. With this specimen, bundles of anchoring fibrils were observed clearly and their lengths could be measured. Negatively stained ultrathin sections of dithiothreitol-separated specimens showed the fine structure of the lamina lucida. Bridging filaments in the lamina lucida were resolved by negative staining.

Animals↗
PubMed1991

[Action of dithiothreitol on protein pattern and phosphorylation in nuclear matrix preparations isolated from the rat liver and Zajdela's hepatoma].

After rat liver nuclei had been treated with 10 mM dithiothreitol nuclear matrix contained 40 per cent less protein than without treatment. Protein composition did not change qualitatively. The protein with a molecular weight of 35 kD was not phosphorylated in the treated samples. After Zajdela hepatoma nuclei had been treated in the same way, nuclear-matrix contained 25% less protein. High molecular weight proteins (140, 150 and 220 kD) were solubilized by media containing dithiothreitol. After dithiothreitol treatment phosphatase and protein-phosphatase activities reduced dramatically both in rat liver and Zajdela hepatoma nuclear matrices.

Animals↗
PubMed1990

Pharmacological differentiation of angiotensin effects in the rabbit isolated vas deferens with dithiothreitol and pertussis toxin.

Angiotensin II inhibits nonadrenergic (purinergic) neurotransmission in the vas deferens and potentiates adrenergic neurotransmission and prostaglandin (PG)E synthesis. Other angiotensin responses are sensitive to either dithiothreitol or pertussis toxin. The present study tested the hypothesis that dithiothreitol or pertussis toxin selectively depress angiotensin responses in the vas deferens. The dithiothreitol (10 mM) eliminated the potentiation of both adrenergic neurotransmission and PGE synthesis but did not alter the depression of purinergic neurotransmission. In contrast, pertussis toxin (100 ng/ml for 3 hr) eliminated the depression of purinergic neurotransmission but had no effect on adrenergic neurotransmission or PGE synthetic responses to angiotensin II. The results are consistent with the existence of at least two transduction pathways for angiotensin II, one enhancing adrenergic neurotransmission and PGE synthesis and the other depressing purinergic neurotransmission. The results indicate that the vas deferens is a useful preparation in defining selective actions of angiotensin receptor agonists or antagonists.

Angiotensin II↗
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↗
PubMed1994

Reduction of disulfide bonds in sarcoplasmic reticulum Ca(2+)-ATPase by dithiothreitol causes inhibition of phosphoenzyme isomerization in catalytic cycle. This reduction requires binding of both purine nucleotide and Ca2+ to enzyme.

Sarcoplasmic reticulum vesicles were treated with 6 mM dithiothreitol in the presence of 2 mM ATP and 0.1 mM Ca2+ at 25 degrees C and pH 7.0 for various periods. The Ca(2+)-ATPase was inhibited almost completely in 100 min. The content of the sulfhydryl group, which was measured in 2.5% SDS with 5,5'-dithiobis(2-nitrobenzoic acid), increased during the treatment. Extrapolation of the plot of the Ca(2+)-ATPase activity versus the increment of the sulfhydryl group showed that reduction of two disulfide bonds per phosphorylation site leads to a complete inhibition of the enzyme. This reduction required binding of both Ca2+ (with a high affinity) and a purine nucleotide to the enzyme. Adenosine 5'-(beta,gamma-methylene) triphosphate (a nonhydrolyzable ATP analog) was also effective for the reduction, while phosphorylation of the enzyme with acetyl phosphate or P(i) did not trigger the reduction. These results indicate that formation of a substrate-enzyme-calcium complex is responsible for the dithiothreitol-induced disulfide bond reduction. The partial reactions of the Ca(2+)-ATPase were examined with the vesicles that had been treated with dithiothreitol in the presence of ATP and Ca2+ for 100-200 min. Neither isomerization of the unphosphorylated enzyme from the low Ca2+ affinity form to the high Ca2+ affinity form nor phosphorylation of the enzyme with ATP or P(i) was inhibited. In contrast, isomerization of the phosphoenzyme intermediate from the ADP-sensitive form to the ADP-insensitive form was strongly inhibited. These results show that the observed inhibition of the Ca(2+)-ATPase is due to a selective blockage of the phosphoenzyme isomerization.

Adenosine Triphosphate↗
PubMed1976

The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. II. Products formed by lysis with sperm enzymes and dithiothreitol.

The egg vitelline envelope of the marine invertebrate, Megathura crenulata, was lyzed either by sperm lysins A, B, C or by dithiothreitol. In each case the lysis mixture consisted of two major fractions, I and II, that could be separated by hydroxylapatite chromatography and had different electrophoretic mobilities on cellulose acetate strips. The amino acid, amino sugar, and neutral sugar compositions of fractions I and II were similar and resembled that of the intact vitelline envelope. Fractions I and II of each lysis mixture emerged in the exclusion volume of a Sepharose 6B column. A vitelline envelope fragment enzymatically formed by lysin was further degraded by dithiothreitol to form smaller fragments. A model of the vitelline envelope of the Megathura crenulata egg is suggested whereby the envelope is composed of polypeptide chains cross-linked by disulfide bonds and built to a large extent of closely spaced threonine residues. Most of the threonine residues are linked to carbohydrate units. Dithiothreitol dissolves the envelope by reducing disulfide bonds, whereas lysins most likely dissolve the envelope by degrading polypeptide chains.

Amino Acids↗
PubMed1990

Inhibition of zeaxanthin formation and of rapid changes in radiationless energy dissipation by dithiothreitol in spinach leaves and chloroplasts.

Dithiothreitol, which completely inhibits the de-epoxidation of violaxanthin to zeaxanthin, was used to obtain evidence for a causal relationship between zeaxanthin and the dissipation of excess excitation energy in the photochemical apparatus in Spinicia oleracea L. In both leaves and chloroplasts, inhibition of zeaxanthin formation by dithiothreitol was accompanied by inhibition of a component of nonphotochemical fluorescence quenching. This component was characterized by a quenching of instantaneous fluorescence (F(o)) and a linear relationship between the calculated rate constant for radiationless energy dissipation in the antenna chlorophyll and the zeaxanthin content. In leaves, this zeaxanthin-associated quenching, which relaxed within a few minutes upon darkening, was the major component of nonphotochemical fluorescence quenching determined in the light, i.e. it represented the ;high-energy-state' quenching. In isolated chloroplasts, the zeaxanthin-associated quenching was a smaller component of total nonphotochemical quenching and there was a second, rapidly reversible high-energy-state component of fluorescence quenching which occurred in the absence of zeaxanthin and was not accompanied by F(o) quenching. Leaves, but not chloroplasts, were capable of maintaining the electron acceptor, Q, of photosystem II in a low reduction state up to high degrees of excessive light and thus high degrees of nonphotochemical fluorescence quenching. When ascorbate, which serves as the reductant for violaxanthin de-epoxidation, was added to chloroplast suspensions, zeaxanthin formation at low photon flux densities was stimulated and the relationship between nonphotochemical fluorescence quenching and the reduction state in chloroplasts then became more similar to that found in leaves. We conclude that the inhibition of zeaxanthin-associated fluorescence quenching by dithiothreitol provides further evidence that there exists a close relationship between zeaxanthin and potentially photoprotective dissipation of excess excitation energy in the antenna chlorophyll.

Journal Article↗
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↗
PubMed1993

Antioxidative stress therapy with dithiothreitol tetraacetate. I. Protection against carbon tetrachloride induced liver necrosis.

Dithiothreitol (DTT) is known to prevent or even reverse several deleterious effects of radiation or of chemical agents operating via free radical and oxidative stress. However, its use has been hampered by its chemical instability and toxic properties. In this work, we synthesized and characterized dithiothreitol tetraacetate (DTT-Ac) which is less toxic and chemically stable, and we provided GLC/MS evidence that it is able to rapidly generate fully deacetylated DTT in liver after its administration to rats. Treatment with DTT-Ac simultaneously with CCl4 or 3 h after the hepatotoxin was able to significantly prevent the CCl4-induced liver necrosis at 24 h after poisoning. DTT-Ac administration was able to significantly reduce the intensity of the covalent binding of CCl4 reactive metabolites to microsomal lipids (CB), but it did not prevent the CCl4-induced initiation of a lipid peroxidation (LP) process as evidenced by diene hyperconjugation of microsomal lipids. Results suggest that DTT-Ac protective effects might be due to its in vivo conversion to DTT which in turn would decrease the intensity of CB via different potential mechanisms to be explored. Protection cannot be attributed to decreases in levels of CCl4 reaching the liver or to chain breaking effects on LP.

Animals↗
PubMed1990

Dithiothreitol liberates non-acid labile sulfide from brain tissue of H2S-poisoned animals.

Acid-labile sulfide measured by conventional gas dialysis and ion chromatography with electrochemical detection accounts for only a proportion of the total sulfide present in brain tissue after poisoning with NaHS, an H2S precursor. Dithiothreitol (DTT) displaced additional measurable sulfide not detectable by the conventional techniques from NaHS-poisoned brain tissue. Sulfide liberation by DTT was dose-dependent and maximal at higher DTT concentration (10 and 30 mM) and was thought to represent non-acid labile sulfide. Dithiothreitol was also found to be significantly protective against H2S poisoning. Furthermore, in vitro inhibition by sulfide of monoamine oxidase (MAO) was reversed by DTT, thus suggesting a molecular mechanism consistent with known persulfide chemistry. Persulfide formation may thus underlie some aspects of hydrogen sulfide neurotoxicity. The rational development of antidotes for use in H2S poisoning may thus have to be centered on strategies concentrating on known thiol, disulfide and persulfide chemistry.

Animals↗
PubMed2003

Scanning electron microscopic observations of the basement membranes with dithiothreitol separation.

Scanning electron microscopic (SEM) studies of the interstitial surface of the lamina densa can be performed with dithiothreitol separation, which is the only method of exposing this surface. SEM observation revealed the three-dimensional structures of the meshwork in the lamina densa and anchoring fibrils in dithiothreitol-separated specimens. Detection of the components of the basement membrane can be performed by immunoscanning electron microscopy on this exposed surface by comparing the backscattered and the secondary electron images. SEM observation also revealed the fine structure of the lamina fibroreticularis using separated dermis or the lamina propria mucosae.

Animals↗
PubMed1988

Separation of ricin A- and B-chains after dithiothreitol reduction.

After complete cleavage of ricin interchain disulfide bridge by 0.05 M dithiothreitol in nondenaturing conditions at 37 degrees C during 1 h 30 min, A- and B-chains were separated on a lactosaminyl-aminoethyl Biogel P-150 column at 4 degrees C, in the presence of 0.01 M dithiothreitol and 0.5 M MgCl2. A- and B-chains have been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunology. Their specific activities have been tested by protein synthesis inhibition in a cell-free assay (rabbit reticulocyte lysate) and on whole cells (Zajdela hepatoma cells) and by hemagglutination. From these tests, the apparent cross contamination of the chains was about 0.1%.

Animals↗
PubMed1984

Ribonucleotide reductase activity in intact mammalian cells: stimulation of enzyme activity by MgCl2, dithiothreitol, and several nucleotides.

An intact cell assay system based on Tween-80 permeabilization was used to investigate ribonucleotide reductase activity in Chinese hamster ovary cells. Dithiothreitol, a reducing agent, is required for optimum activity. Analysis of dithiothreitol stimulation of CDP and ADP reductions indicated that in both cases the reducing agent served only to increase the reaction rate without altering the affinity of the enzyme for substrates. Magnesium chloride significantly stimulated the reduction of CDP but not ADP; this elevation in CDP reduction was due to an increase in both the affinity of the enzyme for substrate and the Vmax. In addition to ATP and dGTP, well-known activators of CDP and ADP reductase activities, it was found that dCTP and GTP were also able to activate CDP and ADP reductase activities, respectively. For the dCTP-activated reaction the Vmax was 0.158 nmol dCDP formed 5 X 10(6) cells-1 h-1 and the Km was 0.033 mM CDP, while for the GTP-activated reduction a Vmax of 0.667 nmol dADP formed 5 X 10(6) cells(-1) h-1 and Km of 0.20 mM ADP were observed. Kinetic analysis revealed that dCTP, dGTP, and GTP stimulate ribonucleotide reduction solely by increasing the affinity of the enzyme for substrate without affecting the Vmax of the respective reactions. ATP behaves in a different manner as it stimulates CDP reduction by altering both the affinity of the enzyme for substrate and the Vmax. Cellular concentrations of ribo- and deoxyribonucleoside di- and triphosphate pools were measured to help evaluate the relative physiological importance of the nucleotide activators. These determinations, along with the reaction kinetic studies, strongly imply that ATP is a much more important regulator of CDP reduction that dCTP, whereas GTP may serve as well or better than dGTP as the in vivo activator of ADP reduction.

Animals↗
PubMed1976

The oxidation of dithiothreitol by peroxidases and oxygen.

Horseradish peroxidase (1.11.1.7), lactoperoxidase (1.11.1.7), and the fragment of cytochrome c known as microperoxidase have been shown to catalyze the oxidation of reduced dithiothreitol in an oxygen-consuming reaction. Evidence for horseradish peroxidase intermediates compound III and compound II has been observed, although ferroperoxidase was not identified during the course of the reaction. The stoichiometry has been extablished as 1 : 1 for oxygen consumed to dithiothreitol oxidized. Cysteine and glutathione have also been shown to be substrates for horseradish peroxidase oxidase reaction.

Catalase↗
PubMed1985

Urea treatment allows dithiothreitol to release the binding subunit of the insulin receptor from the cell membrane: implications for the structural organization of the insulin receptor.

The sequence of the human insulin receptor has only one identifiable transmembrane region which is located in the beta subunit. The structure predicts that the alpha subunit, which binds insulin, is attached to the cell only by disulfide bonds to the beta subunit. However, treatment of membranes with dithiothreitol is ineffective at releasing the alpha subunit. If the receptor structure is unfolded with urea, dithiothreitol is able to release the alpha subunit. These data provided confirmatory evidence that the alpha subunit is not a transmembrane protein.

Cell Line↗