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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↗

PubMed1977

Effect of dithiothreitol on mercuric chloride- and uranyl nitrate-induced acute renal failure in the rat.

The current study was undertaken to examine the effects of dithiothreitol (DDT), a sulfhydryl-reducing agent and heavy metal chelator, on the course of heavy metal-induced acute renal failure in the rat. Groups of rats in metabolic cages received uranyl nitrate (UN) alone, UN plus DTT, mercuric chloride (HgCl2) alone, and HgCl2 plus DTT. UN injected alone produced azotemia, decreased creatinine clearance, and rising fractional sodium excretion over the 48 hr of study. These effects of UN on renal function were not observed when DTT was administered 30 min after UN injection. Qualitatively similar results were obtained with HgCl2-induced acute renal failure. Groups of rats were killed at 6 hr after UN plus DTT, HgCl2 alone, or HgCl2 plus DTT; and determinations of plasma renin activity (PRA) and renin activities of the superficial and deep juxtaglomerular apparatus (JGA) were performed. PRA's and JGA renins were increased in animals receiving either UN or HgCl2 alone, but not in the rats receiving both DTT and UN or HgCl2. The effect of DTT on distribution of 203Hg was also examined. Treatment with DTT did not alter the renal accumulation of 203Hg, suggesting that this agent does not act by limiting renal exposure to the heavy metals. Thus, DTT ameliorates the course of heavy metal-induced ARF, and this effect is associated with prevention of heavy metal-induced alterations in sodium excretion and renin-angiotensin system activity.

Acute Kidney Injury↗

PubMed1979

Dithiothreitol separation of newborn rodent dermis and epidermis.

Incubation of newborn mouse skin in media containing 0.01 M dithiothreitol at 4 degrees C for 2 hr reduces dermal-epidermal cohesion, and the 2 layers may be separated readily. Unlike other methods of separation, this technique does not require heating, nonphysiologic pH or ionic strength, or addition of proteolytic enzymes. The split occurs beneath the basal lamina. The separated epidermal sheet retains the periodic acid Schiff-positive "basement membrane," and the basal cells can incorporate 3H-thymidine.

Animals↗

PubMed1976

A simple technique for the inactivation of IgM antibodies using dithiothreitol.

This report describes a simple method for inactivating 19S red cell antibodies using the odorless compound dithiothreitol (DTT). 30 sera which contained antiglobulin-active red cell antibodies showed virtually no alteration in activity after DTT treatment, while 30 different sera containing cold-active red cell antibodies showed almost total elimination of activity following DTT treatment. Six sera were separated into IgG and IgM fractions. All red cell activity was eliminated in the IgM fraction, but was unaffected by DTT treatment of the IgG fraction. This method of inactivating IgM antibodies should be of value in investigating cases of hemolytic disease of the newborn and in studying sera containing mixtures of warm and cold red cell antibodies.

Agglutinins↗

PubMed1975

The reversible depolymerization of spinach chloroplast glyceraldehyde-phosphate dehydrogenase. Interaction with nucleotides and dithiothreitol.

The ligand-dependent dissociation of spinach chloroplast glyceraldehyde-phosphate dehydrogenase (Mr 600000) to protomers of Mr about 145000, previously shown by us in 1973, has been further characterized by the technique of velocity sedimentation in sucrose gradients. The process exhibits cooperativity and is accompanied by an increase of the apparent NADP+-dependent activity (reactivation) from a ratio of 0.1-0.2 to a ratio of 1 to 2 with respect to the NAD+-dependent activity. In addition to NADP+ and NADPH, most nucleotide triphosphates and, to some extent, Pi, act as dissociating agents. The enzyme is depolymerized and progressively inactivated in the presence of 2'-AMP. 2. Incubation with 20 mM dithiothreitol or 8-10 mM GTP increases the apparent NADP/H)-dependent activity, although addition of a small amount of a dissociating compound, such as 0.06 mM NADP+, is required for depolymerization. 3. NAD+, NADH and, to a lesser extent, glyceraldehyde 3-phosphate, NMN and cyclic AMP act as inhibitors of the dissociation and reactivation, however induced. They also favour the reassociation of protomers to tetramers. 4. The NADP(H)-linked activity is probably a property of the protomers only. The system described here resembles in many respects the light-dependent regulation of the NADP(H)-linked activity in vivo.

Adenine Nucleotides↗

PubMedDate not supplied

Effect of dithiothreitol on histamine receptors in rabbit colon and guinea-pig ileum.

1. Dithiothreitol, an agent which reduces disulphide linkages to sulphydryl groups, potentiated the contractile responses of the rabbit colon and guinea-pig ileum to histamine, but had no effect on the resting tone or on the responses of these preparations to acetylcholine. 2. The potentiation was greater than that produced by antagonism of histamine's weak inhibitory action by metiamide, and still occurred after blockade of H2-receptors with metiamide; it is concluded that DTT potentiates responses to stimulation of H1-receptors in both preparations. 3. In this respect, these H1-receptors resemble those in vascular smooth muscle in the rabbit but not in the guinea-pig; it is concluded that there is a tissue variation rather than a species variation in the response of H1-receptors in the rabbit and guinea-pig to DTT.

Animals↗

PubMed1976

A note on the effect of dithiothreitol (DTT) on the depolarization of isolated sympathetic ganglia by carbachol and bromo-acetylcholine.

The S-S reducing agent, dithiothreitol (DTT) altered the properties of nicotinic receptors in rat superior cervical ganglia such that (i) carbachol became less active as a depolarizing agent and (ii) bromo-acetylcholine produced an irreversible depolarization. The latter was temporarily annulled by hexamethonium (which retained antagonist properties), but returned when hexamethonium was removed. It is concluded that ganglionic nicotinic receptors might be quite similar to those for monoquaternary agonists in leech dorsal muscle.

Acetylcholine↗

PubMed1978

Characterization of a DNA-protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N'-tetraacetic acid and dithiothreitol.

Treatment of polyoma virions with ethyleneglycol-bil-N,N'-tetraacetic acid (EGTA) and dithiothreitol (DTT) at pH 8.5 resulted in the dissociation of the virions into a DNA-protein complex and individual structural capsomere subunits. The sedimentation value of the DNA-protein complex in sucrose gradients was approximately 48S, and it had a density of 1.45 g/cm3 in equilibrium CsCl gradients. Alkaline sucrose analysis of the DNA within this DNA-protein complex demonstrated that approximately 75% of the DNA is component 1. The proteins associated with the DNA were dissociated by treatment with either NaCl or the anionic detergent Sarkosyl. VP1 and the histone proteins VP 4--7 were the major proteins associated with the DNA. Treatment of the DNA-protein complex with alkaline pH resulted in the specific removal of FP1. Electron microscopy of the 48S DNA-protein complex demonstrated that it is a very tightly coiled structure that is slightly larger than the intact virion. Treatment of the complex with either NaCl or with pH 10.5 buffer resulted in the loss of protein and subsequent loosening of the DNA-protein complex such that the DNA could be visualized. The capsomere subunits released as a result of the EGTA-DTT treatment sedimented as 18S, 12S, and 5S subunits in sucrose gradients. Electrophoretic analysis of the isolated capsomeres demonstrated that VP1, VP2, and VP3 were present in each species, although the ratios of the proteins varied. In addition to the structural proteins, histones VP 4--7 were found to be predominantly associated with the 5S capsomere subunit.

Capsid↗

PubMed1978

Evaluation of dithiothreitol (DTT) for inactivation of IgM antibodies.

A newly introduced sulphydryl compound, dithiothreitol (DTT), is evaluated for its optimal conditions of inactivation of IgM antibodies. The maximal effects of DTT reagent are observed when its final concentrations are between 0.0025 M and 0.005 M, pH between 7.0 and 8.0, and incubation at 37 degrees C. Concentrations over 0.01 M, pH values greater than 8.0, and an incubation temperature over 40 degrees C resulted in a gel formation of the specimen. Examination of both 'cold' and 'warm' type antibodies demonstrated that the results obtained by the DTT reagent are in close agreement with those obtained by 2-mercaptoethanol reagents and DEAE Sephadex treatment. Since the procedure is simple and rapid and lacks offensive odour, DTT is recommended for routine use in blood banking for the inactivation of IgM antibodies.

Dithiothreitol↗

PubMed1978

Measuring IgG anti-A/B titres using dithiothreitol (DTT).

In comparing the use of the sulphydryl compounds 2-mercaptoethanol (2ME) and dithiothreitol (DTT) a rapid one-stage technique for titrating IgG anti-A and anti-B using DTT in an antiglobulin test was tried. The results correlated well with those obtained by either of the two-stage methods using 2ME or DTT.

ABO Blood-Group System↗

PubMed1977

The in vivo use of dithiothreitol in cystinosis.

Two male patients with late stage (uremic) infantile nephropathic cystinosis (INC) (Table 1) were treated by mouth with the reducing agent dithiothreitol (DTT), at doses not exceeding 25 mg-kg-1 body weight three times per day. Three sequential periods of observation were obtained in both patients: on thiol (8.5 months); off thiol (8-9 months); on thiol again (7 months or longer). Other than nausea and vomiting at the maximum dose range, no apparent toxicity was observed. One subject died in uremia in the 24th month of the study. The half-cystine concentration in peripheral blood leukocytes decreased during both treatment periods in each patient from initial pretreatment levels in excess of 8 nmol-mg-1 protein (normal less than 0.1 nmol-mg-1) to 10-20% of initial values (Table 2 and Fig. 1, A and B). Reduction in total number of blood leukocytes or in the neutrophil fraction, where cystine storage occurs selectively in cystinosis, did not occur (Table 3) as a possible explanation for these findings; nor did storage of samples, a possible artifact, influence the cystine content of cystinotic cells (Fig. 2). Multiple site rectal mucosa biopsy clearly revealed cystine storage but serial biopsies did not reflect a positive DTT response when compared with the leukocyte assay (Table 4). High intersample variation in cystine content, even between samples taken at one time, prevented measurement of a treatment response. DTT had no apparent detrimental effect on the concentration of representative proteins, including hemoglobin (Table 3), serum insulin, and serum immunoglobulin during the treatment trials. Renal function (glomerular and tubular) was severely depressed and did not improve during the period of observation in either patient (Table 2; Fig. 3, A and B). Postmortem tissues from one patient revealed 10-40-fold excess cystine accumulation in kidney cortex and liver (Table 5). However, these levels of accumulation are at the lower range of or even below published values for cystine in cystinotic kidney and liver. Whereas chemical methods are not reliable for detecting and measuring DTT in biologic fluids, preliminary evidence indicates that a silylated derivative of oxidized DTT can be detected in the urine of patients receiving DTT by mouth (Fig. 4). This finding suggests that the thiol is absorbed and excreted.

Autopsy↗

PubMed1975

Microsomal O-demethylation, N-demethylation and aromatic hydroxylation in the presence of bisulfite and dithiothreitol.

Microsomal O-demethylation of p-nitroanisole, N-demethylation of aminopyrine and aromatic hydroxylation of aniline, were not altered by sodium bisulfite or dithiothreitol at concentrations effective for their use as antioxidants. Incorporation of these antioxidants in microsomal metabolism studies should have general utility in preventing decomposition of readily air oxidizable compounds.

Aminopyrine↗

PubMed1992

[Properties of skeletal muscle sarcoplasmic reticulum membranes prepared in the presence of dithiothreitol].

The authors studied the effect of dithiothreitol (DTT), an agent modulating the redox state of SH-groups, on functional properties of sarcoplasmic reticulum membranes of skeletal muscle. DTT (5 mmol.l-1) exerted a protective effect not only on (Ca2 + Mg2)-ATPase activity but also on the activity of Ca2+ uptake by vesicles, while the most pronounced effect was recorded at low levels of ionized Ca2+. The presence of DTT in the isolation medium decreased also passive permeability of membranes for Ca2+. In the light of the results obtained, modulation of the redox state is recommended for each isolation of membranes designed for the study of ion transport.

Animals↗

PubMed1979

[Detection of rubella specific IgM on gel filtration through Sephadex G 200: use of dithiothreitol and limits with MnCl2-heparine pretreatment (author's transl)].

The detection of rubella haemagglutination inhibiting antibody, in the IgM fraction of the serum, on gel filtration through Sephadex G 200, needs precautions to exclude false results. Treatment with dithiothreitol is a satisfactory method for confirming the content of rubella IgM antibody. The failure of MnCl2-heparin pretreatment to remove non specific inhibitors of rubella hemagglutinin is unfrequent (7/108) and so do be repeated. Rarely (1/108) aggregated IgG fractionates with IgM and yield false positive results.

Antibodies, Viral↗