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PubMed1989

Dithiothreitol-induced triphasic response of dog coronary arteries.

Exposure of dog coronary arterial strips to dithiothreitol resulted in a triphasic response which was characterized by an initial rapid, weak contraction (phase A), an intervening slow relaxation (phase B) and finally, a slowly developing intense contraction (phase C). No responses were observed when either oxidized dithiothreitol or threitol were used. No contractions were observed if the extracellular Ca2+ was omitted. Ca2+ channel blockers (nicardipine and diltiazem), a protein kinase inhibitor (1-(5-isoquinolinesulfonyl)-2-methylpiperazine, H-7), and a myosin light chain kinase inhibitor (1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine, ML-9), all suppressed the dithiothreitol-induced responses. The calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), however, augmented phase A, and W-7 and chlorpromazine depressed phase C. Prolonged exposure of the arteries to dithiothreitol was essential to elicit phase C. The implications of these data are discussed with respect to the possible mechanisms of dithiothreitol-induced vascular responses.

Animals↗

PubMed1982

Effects of dithiothreitol, 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide on synaptic transmission at sympathetic ganglion cells of frog.

The effects of disulfide bond and sulfhydryl group reagents on synaptic transmission were studied in the bullfrog sympathetic ganglion. Ten millimoles of dithiothreitol (DTT), a disulfide bond reducing agent, decreased the amplitude of the transmitted postganglionic compound action potential (CAP) to 41% of control. More than one hour wash-out of DTT with normal Ringer's solution did not reverse the block, but 1 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), an oxidizing agent, rapidly restored the compound action potential amplitude. N-Ethylmalemide (NEM), an alkylating agent, added before DTNB, prevented the restoration of transmission by DTNB. Block of transmission induced by dithiothreitol was also reversed rapidly by 50 microns 3,4-diaminopyridine (3,4-DAP), to 87% of control compound action potential amplitude, and subsequent addition of DTNB restored transmission completely and this was accompanied by the stimulus-bound repetitive firing (SBR) which diaminopyridine produced in otherwise untreated ganglia. One to 3 mM dithiothreitol decreased the amplitude of excitatory post-synaptic potentials (EPSP), and greater concentrations slightly depolarized the cell membrane and decreased membrane resistance. Washout with Ringer's solution reversed the depolarization, but not the block of excitatory postsynaptic potentials. The amplitude of excitatory post-synaptic potentials was restored to threshold by 1 mM DTNB, which also slightly increased membrane resistance and the resting potential. It is concluded that the actions of dithiothreitol and DTNB in the bullfrog sympathetic ganglion are generally similar to those previously observed in other junctional tissues. Interactions between these drugs and 3,4-diaminopyridine in the present study also raise the question whether dithiothreitol and DTNB have any presynaptic effects.

4-Aminopyridine↗

PubMed1991

Effect of dithiothreitol on the catalytic activity, quaternary structure and sulfonamide-binding properties of an extracellular carbonic anhydrase from Chlamydomonas reinhardtii.

Extracellular carbonic anhydrase from the unicellular green alga Chlamydomonas reinhardtii is an oligomeric protein containing subunits of 36 and 4 kDa which are joined by disulfide bonds to form higher molecular mass oligomers. In this study, the effect of dithiothreitol on some properties of the enzyme were examined. Dithiothreitol caused a 40% activation of the catalytic activity of the enzyme at low concentrations (0.1 mM), but an inactivation of about 85% of the catalytic activity at high (50 mM) concentrations. Chemical cross-linking of the enzyme with dimethyl suberimidate revealed the existence of oligomers containing up to three large subunits and at least two small subunits. Cross-linking analysis of dithiothreitol-treated carbonic anhydrase revealed that 0.1 mM dithiothreitol had no effect on the subunit composition of the enzyme, but 10 or 50 mM caused subunit dissociation, including the apparent complete dissociation of the small subunits from the large subunits. There was a characteristic enhancement of dansylamide fluorescence when this fluorescent sulfonamide bound carbonic anhydrase and the fluorescence enhancement was retained following the dithiothreitol-induced dissociation of the enzyme. These results indicate that disulfide bonds are essential for maintenance of the oligomeric structure of Chlamydomonas reinhardtii carbonic anhydrase, and that the small subunit may be necessary for enhancing catalysis, but not for the binding of sulfonamides to the enzyme.

Carbonic Anhydrases↗

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↗

PubMed2005

Gramicidin D and dithiothreitol effects on erythrocyte exovesiculation.

The use of either diphenylhexatriene, trimethylamino-diphenylhexatriene, or heptadecyl-hydroxycoumarin (C17-HC) allows, simultaneously and with the same molecule, the induction of erythrocyte exovesiculation and labeling of the released vesicles with the fluorescent probe. This method was used to evaluate gramicidin D (a channel-forming peptide) and dithiothreitol (a reducing agent) effects on the human erythrocytes vesiculation process. The release of cholesterol and phospholipids in exovesicles at longer incubation times was only detectable in the presence of gramicidin or dithiothreitol. When C17-HC was used to induce the vesiculation, the presence of gramicidin or dithiothreitol lead to a drastic decrease on the [phospholipids]/[cholesterol] ratio. However, in the samples with dithiothreitol, this variation did not result in the expectable decrease of membrane fluidity. These effects can be related with the presence of lipid rafts, the transbilayer lipids reorientation induced by gramicidin or dithiothreitol, and the cholesterol-dependent gramicidin channels inactivation.

Cholesterol↗

PubMed1984

Role of the gamma subunit of chloroplast coupling factor 1 in the light-dependent activation of photophosphorylation and ATPase activity by dithiothreitol.

In leaves and intact chloroplasts, oxidation and reduction have been shown previously to regulate the ATPase activity of thylakoids. Illumination of spinach chloroplast thylakoids in the presence of dithiothreitol, which activates the ability of thylakoids to catalyze sustained ATP hydrolysis in the dark, causes increased incorporation of N-ethylmaleimide into the gamma subunit of coupling factor 1 (CF1). A disulfide bond in the gamma subunit is reduced during activation. The residues involved in this disulfide bond are the same as those in the disulfide linkage reduced during dithiothreitol activation of soluble CF1. The disulfide and dithiol forms of the gamma subunit may be separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. N-Ethylmaleimide is preferentially incorporated in the dark into the reduced form of the gamma subunit of CF1 in thylakoids previously exposed to dithiothreitol. Only a subpopulation of the CF1 in thylakoids is susceptible to dithiothreitol reduction and subsequent reaction with N-ethylmaleimide in the dark. Alkylation of the thiol groups exposed by reduction of the disulfide bond protects ATPase activity from inhibition by oxidants. At a given value of the transmembrane pH differential, photophosphorylation rates in dithiothreitol-activated thylakoids can be as much as seven to eight times those of nonactivated controls. N-Ethylmaleimide treatment of activated thylakoids in the dark prevents the loss of the stimulation of ATP synthesis on storage of the thylakoids. Photophosphorylation by intact chloroplasts lysed in assay mixtures is also activated in comparison to that by washed thylakoids. At a low ADP concentration, the rate of photophosphorylation approaches saturation as delta pH increases. These results suggest that the gamma subunit of CF1 plays an important role in regulation of ATP synthesis and hydrolysis.

Dithiothreitol↗

PubMed1983

Comparison of the 2-mercaptoethanol and dithiothreitol tests for determining Brucella immunoglobulin G agglutinating antibody in bovine serum.

The dithiothreitol test was evaluated as a substitute for the 2-mercaptoethanol test for determining Brucella immunoglobulin G agglutinating antibody in bovine serum. The tests were compared on 207 card-positive sera that showed a standard tube-agglutination titer of incomplete 1:50 or higher. The tests agreed within one dilution with 182 of the 207 sera tested for an 87.9% rate of agreement. When titers were not the same, those obtained with the dithiothreitol test were more frequently lower than higher than those obtained with the 2-mercaptoethanol test. Sixteen sera that showed a titer with the 2-mercaptoethanol test were negative with the dithiothreitol test and two that showed a titer with the dithiothreitol test were negative with the 2-mercaptoethanol test. Results suggest that the dithiothreitol test is not a reliable substitute for the 2-mercaptoethanol test to detect immunoglobulin G agglutinating antibody in bovine serum.

Agglutination Tests↗

PubMed1980

Restoration of lead-inhibited 5-aminolevulinate dehydratase activity in whole blood by heat, zinc ion, and (or) dithiothreitol.

We examined effects of heat, zinc, ion, and dithiothreitol in restoring the activity of lead-inhibited-5-aminolevulinate dehydratase (EC 4.2.1.24). The ratio of non-activated to activated activity produced by dithiothreitol correlated well with blood lead concentration among 35 lead workers. The individual effects of heat, zinc, or dithiothreitol differ from each other in the shift of pH optimum as well as in the extent to which activity is restored. Dual or triple combinations of these agents show additive or complementary restoration of activity. The combination of heat and zinc or zinc and dithiothreitol expands the range of optimum concentration of zinc in restoring activity. Using these combinations of agents, we can expect more accurate evaluation of lead exposure than by measuring only activation of zinc. Although dithiothreitol most powerfully restores activity, it restores not only the activity inhibited by lead exposure but also the activity removed by oxidation of SH-groups in the enzyme molecule.

Dithiothreitol↗

PubMed1987

Hemin-mediated oxidation of dithiothreitol reduces oxygen to H2O.

Hemin catalyses the oxidation of dithiothreitol. One mole of oxygen is consumed for every 2 moles of dithiothreitol oxidized and the product is shown by spectral studies to be the intramolecular disulphide. The reaction shows a specificity for dithiol and for free heme moieties. Hemin molecules exhibit cooperativity in oxygen reduction. Oxygen radicals do not seem to be involved. H2O2 is not required for this oxidation of dithiothreitol and does not appear to be an intermediate in the reduction of O2 to H2O. However, an independent minor reaction involving a 2-electron transfer with the formation of H2O2 also occurs. These studies on the hemin-catalyzed oxidation of dithiothreitol provide a chemical model for a direct 4-electron reduction of O2 to H2O.

Catalase↗

PubMed1991

Increased dithiothreitol-insensitive, type 2 angiotensin II receptors in selected brain areas of young rats.

1. Angiotensin II receptors have been studied by quantitative autoradiography in selected brain areas of young (2-week-old) and adult (8-week-old) rats. 2. In young rats, angiotensin II receptors were present in brain areas which did not express receptors in the adult brain, such as thalamic nuclei, cortical areas, and the cerebellum. 3. Young rats had more angiotensin II receptors in the subfornical organ than adult rats. In the inferior olive, the number of angiotensin receptors in young animals was 10 times higher than that in adult rats. Angiotensin II binding in the inferior olive was insensitive to incubation in the presence of dithiothreitol. 4. Conversely, the number of angiotensin II receptors in the nucleus of the solitary tract was lower in young rats compared to adults. Incubation in the presence of dithiothreitol resulted in a more than 90% inhibition of angiotensin II binding in the nucleus of the solitary tract. 5. Our results indicate the presence of two types of angiotensin II receptor in brain, one sensitive (type 1) and one insensitive (type 2) to the reducing agent dithiothreitol. 6. The expression of type 2 angiotensin II receptors, insensitive to dithiothreitol, is more marked in young rats, indicating a role for this type of angiotensin receptors in brain development.

Age Factors↗

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↗

PubMed1991

Inhibition by EDTA and enhancement by divalent cations or polyamines of the dithiothreitol-induced activation of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum.

Binding of cAMP to cell surface receptors evokes the transient activation of of adenylate cyclase in Dictyostelium discoideum. Dithiothreitol is also known as an activator of this enzyme. We found that the dithiothreitol-induced activation was specifically enhanced by extracellular polyamines or divalent cations. Furthermore, EDTA, a chelating agent of divalent cations, completely inhibited the dithiothreitol-induced activation of adenylate cyclase while EDTA did not inhibit the cAMP-induced activation. The inhibition was nullified by addition of polyamines or divalent cations. These results suggest that extracellular polyamines and divalent cations play a specific role in the dithiothreitol-induced activation of adenylate cyclase.

Adenylyl Cyclases↗

PubMed2003

Transmission and scanning electron microscopic observations of epithelial-mesenchymal interface of rat tooth germs using dithiothreitol separation.

Aperiodic fibrils (AF) project from the interstitial side of the lamina densa of the basement membrane (BM) of the inner enamel epithelium (IE), and show remarkable changes in their morphology during development. The three-dimensional morphology of aperiodic fibrils during development has not been observed, because of the difficulty of exposing the interstitial surface of the BM of the inner enamel epithelium. In the present study, the dithiothreitol separation method was applied to expose the interstitial side of the inner enamel epithelial BM of rat tooth germs for the purpose of observing the exposed aperiodic fibrils by transmission and scanning electron microscopy (TEM and SEM, respectively). After dithiothreitol treatment, the enamel organ (EO) was mechanically separated from the dental papilla (DP). In the region with poorly-developed aperiodic fibrils, the separation occurred at the junction between the inner enamel epithelial BM and the dental papilla, and the aperiodic fibrils were exposed, showing the typical picture of dithiothreitol separation. SEM observation of this region revealed that the aperiodic fibrils were connected to each other and they formed networks. These networks resembled those formed by the anchoring fibrils of epidermal and mucosal epithelial BMs. TEM and SEM observations revealed that there were sidechain-like structures on the surface of the aperiodic fibrils. In the region with well-developed aperiodic fibrils, dithiothreitol treatment was not entirely effective, and some mesenchymal tissues remained on the BM. In this region, TEM observation revealed that the aperiodic fibrils were arranged in parallel with each other, and were connected by the sidechains. Several thin collagen fibrils, which were thought to be immature collagen fibrils (CF) of the predentine, were also connected to the aperiodic fibrils with these sidechains and arranged in parallel with them. Based on SEM and TEM observations, the aperiodic fibrils may be regarded as a kind of anchoring fibrils and they may play a role in connecting the BM with the mesenchymal tissue below. They are also thought to guide the arrangement of collagen fibrils in the surface layer of the predentin.

Animals↗

PubMed1992

Inactivation of purified phenylalanine hydroxylase by dithiothreitol.

Purified rat liver phenylalanine hydroxylase is inactivated in vitro by ascorbate and thiol compounds, dithiothreitol being the most effective inhibitor, with a second order rate constant for the inactivation of 0.066 +/- 0.002 mM-1.min-1 at 20 degrees C and pH 7.2. Anaerobic conditions and catalase protected the enzyme from inactivation by dithiothreitol. This suggests that hydrogen peroxide, produced by oxidation of the thiol, is involved in the inactivation. The substrate, L-phenylalanine, also partially protected the enzyme from this inactivation. It is shown that incubation of the enzyme with dithiothreitol at aerobic conditions, followed by gel filtration, causes the release of iron from the active site. The inactivation by dithiothreitol was reversed by incubation of the iron-depleted enzyme with Fe(II).

Anaerobiosis↗

PubMed2001

NMDA receptors are involved in dithiothreitol-induced hypothermia.

Sulfhydryl-reducing agents, such as dithiothreitol, modulate glutamate N-methyl-D-aspartate (NMDA) receptors. Since these receptors are involved in thermoregulatory processes, we studied the effects of their positive modulation, through a dithiothreitol-induced reduction of the receptor redox site, on thermoregulation in rats maintained at an ambient temperature of 20-22 degrees C. Given intraperitoneally at the dose of 25 and 50 mg x kg(-1), dithiothreitol induced dose-dependent hypothermia. The prior administration of 0.5 mg x kg(-1) of (+/-)-dizocilpine maleate (MK801), a non-competitive glutamate NMDA receptor antagonist, blocked most of the dithiothreitol-induced hypothermia. MK801 given alone was followed by slight transient hyperthermia. This confirms the involvement of NMDA receptors in thermoregulation and suggests that they might be under redox modulation.

Animals↗

PubMed2002

Electrocyte (Na(+),K(+))ATPase inhibition induced by zinc is reverted by dithiothreitol.

The Mg(2+)-dependent (Na(+),K(+))ATPase maintains several cellular processes and is essential for cell excitability. In view of the importance of the enzyme activity, the interaction and binding affinities to substrates and metal ions have been studied. We determined the effect of Zinc ion (Zn(2+)) on the (Na(+),K(+))ATPase activity present in both conducting (non-innervated) and post-synaptic (innervated) membranes of electrocyte from Electrophorus electricus (L.). Zn(2+) is involved in many biological functions and is present in pre-synaptic nerve terminals. This metal, which has affinity for thiol groups, acted as a potent competitive inhibitor of (Na(+),K(+))ATPase of both membrane fractions, which were obtained by differential centrifugation of the E. electricus main electric organ homogenate. We tried to recover the enzyme activity using dithiothreitol, a reducing agent. Kinetic analysis showed that dithiothreitol acted as a non-essential non-competitive activator of (Na(+),K(+))ATPase from both membrane fractions and was able to revert the Zn(2+) inhibition at mM concentrations. In the presence of dithiothreitol, this metal behaved as a competitive inhibitor of (Na(+),K(+))ATPase in the non-innervated membrane fractions and presented a non-competitive inhibition of (Na(+),K(+))ATPase in innervated membrane fractions. This difference may be attributed to formation of a Zn-dithiothreitol complex, as well as the involvement of other binding sites for both agents. The consequences of the enzyme inhibition by Zn(2+) may be considered in regard to its neurotoxic effects.

Animals↗

PubMed1971

The inactivation of phenylalanine hydroxylase by 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine and the aerobic oxidation of the latter. The effects of catalase, dithiothreitol and reduced nicotinamide-adenine dinucleotide.

1. Phenylalanine hydroxylase is inhibited by its cofactor, 6,7-dimethyltetrahydropterin. The rate of inactivation, which is irreversible, increases with the concentration of cofactor. 2. Catalase, in sufficient amount relative to cofactor, prevents this inactivation. More tyrosine is formed in the presence of added catalase. 3. Dithiothreitol in the presence of liver extract also prevents inactivation of the enzyme by the cofactor and stimulates hydroxylation of phenylalanine, probably by protecting the cofactor from oxidation and regenerating it from a dihydropterin reaction product. Dithiothreitol restores linearity of rate at very low enzyme concentrations. 4. Dimethyltetrahydropterin is unstable when the solution is exposed to air but is stabilized by dithiothreitol the aerobic oxidation of which is greatly accelerated by dimethyltetrahydropterin. 5. NADH together with liver extract stabilizes the cofactor but not phenylalanine hydroxylase. 6. It is suggested that either hydrogen peroxide or an organic peroxide formed by oxidation in air of the cofactor is the substance attacking phenylalanine hydroxylase, dithiothreitol and cofactor.

Air↗

PubMed1995

Identification of spinach farnesyl protein transferase. Dithiothreitol as an acceptor in vitro.

Spinach seedlings were found to contain farnesyl protein transferase. The enzyme is activated by Zn2+, but not by Mg2+. The pH optimum is approximately 7.0 and maximal activity is obtained at 40-45 degrees C. The apparent Km for the farnesyl diphosphate substrate is 7 microM. Western blotting of soluble proteins with an antiserum raised against mammalian farnesyl protein transferase demonstrated a specific cross-reactivity with the spinach enzyme. The antiserum preferentially recognises the beta-subunit of the heterodimeric farnesyl protein transferase, and the corresponding spinach polypeptide has a molecular mass of 42 kDa on SDS/PAGE. The enzyme can employ dithiothreitol as an acceptor for the farnesyl moiety and catalyses the formation of a thioether linkage between these substrates. On the basis of this discovery, a new method was developed utilising the hydrophobicity of the reaction product, and its interaction with poly(propylene). During in vivo labelling, the plants took up dithiothreitol, which inhibited the incorporation of [3H]mevalonate metabolites into proteins, indicating that dithiothreitol might be isoprenylated in vivo as well as in vitro. However, isoprenylation of some proteins remains unaffected by dithiothreitol suggesting the existence of different isoprenylation mechanisms. Thus, it is demonstrated that plants possess farnesyl protein transferase, which resembles its mammalian and yeast homologues.

Alkyl and Aryl Transferases↗