Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “CYSTAMINE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 703 records · Page 39Linked to original sources
PubMed2000

A method for measuring disulfide reduction by cultured mammalian cells: relative contributions of glutathione-dependent and glutathione-independent mechanisms.

A method is described for measuring bioreduction of hydroxyethyl disulfide (HEDS) or alpha-lipoate by human A549 lung, MCF7 mammary, and DU145 prostate carcinomas as well as rodent tumor cells in vitro. Reduction of HEDS or alpha-lipoate was measured by removing aliquots of the glucose-containing media and measuring the reduced thiol with DTNB (Ellman's reagent). Addition of DTNB to cells followed by disulfide addition directly measures the formation of newly reduced thiol. A549 cells exhibit the highest capacity to reduce alpha-lipoate, while Q7 rat hepatoma cells show the highest rate of HEDS reduction. Millimolar quantities of reduced thiol are produced for both substrates. Oxidized dithiothreitol and cystamine were reduced to a lesser degree. DTNB, glutathione disulfide, and cystine were only marginally reduced by the cell cultures. Glucose-6-phosphate deficient CHO cells (E89) do not reduce alpha-lipoate and reduce HEDS at a much slower rate compared to wild-type CHO-K1 cells. Depletion of glutathione prevents the reduction of HEDS. The depletion of glutathione inhibited reduction of alpha-lipoate by 25% and HEDS by 50% in A549 cells, while GSH depletion did not inhibit alpha-lipoate reduction in Q7 cells but completely blocked HEDS reduction. These data suggest that the relative participation of the thioltransferase (glutaredoxin) and thioredoxin systems in overall cellular disulfide reduction is cell line specific. The effects of various inhibitors of the thiol-disulfide oxidoreductase enzymes (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), arsenite, and phenylarsine oxide) support this conclusion.

Animals↗
PubMed1993

Conformational states of human placental glutathione transferase as probed by limited proteolysis.

Limited proteolysis experiments have been carried out on human placental glutathione transferase in its different forms. The reduced enzyme, as well as the oxidized form and that inactivated with cystamine were all sensitive to 10% (w/w) trypsin, under nondenaturing conditions. The proteolytic cleavage was accompanied by a concomitant loss of enzymatic activity. On the contrary, the presence of glutathione or glutathione conjugates strongly protected the reduced enzyme against inactivation and from the proteolytic attack. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and peptide sequence analysis showed that only the peptide bond between Lys 44 and Ala 45 was cleaved. Since Lys 44 has been demonstrated to be involved in the glutathione binding, it is suggested that the region surrounding this amino acid residue (alpha B helix) could be more exposed to the solvent, in the absence of glutathione. Crystallographic data also indicated that this region is flexible, supporting the idea that it may be involved in the observed conformational change upon glutathione binding.

Alanine↗
PubMed1998

A novel calcium-independent enzyme capable of incorporating putrescine into proteins.

A Ca(++)-independent enzyme capable of incorporating [3H]-putrescine into proteins was detected in the rat intestine mucosa. The Ca(++)-independent incorporation of [3H]-putrescine into proteins was temperature-, pH-, time-, and dose-dependent. However, this enzyme was absent in the gastric mucosa. Similar to testicular Ca(++)-dependent transglutaminase, the optimal pH of intestinal Ca(++)-independent enzyme was 9.0. At 10(-5) M or less putrescine concentrations, the Ca(++)-independent enzyme in an intestinal cytosol preparation showed a greater activity than did the Ca(++)-dependent transglutaminase. However, at higher putrescine concentrations, the latter showed a greater activity than did the former. Both the intestinal Ca(++)-dependent and independent enzymes were inhibited by cystamine, thermal labile at 50 degrees C and precipitated by 30 to 50% saturation of ammonium sulfate. The fact that these two enzymes shared many similar characteristics, with the exceptions of Ca(++)-requirement, suggests that they may have similar active site and intrinsic molecular function(s).

Ammonium Sulfate↗
PubMed1994

Flow cytometric analysis of fluorescein-labeled nerve growth factor binding to A875 human melanoma cells.

The interaction of fluorescein-labeled nerve growth factor (NGF) with human melanoma cells (A875) has been studied in order to assess better methodology for rigorous NGF binding studies. The NGF was modified at a single carboxyl group with iodoacetamidofluorescein after reaction with carbodiimide and cystamine. The modified NGF showed full binding competence in competition with radiolabeled NGF and full biological activity in neurite outgrowth assays compared to native NGF. Binding to unfixed, viable cells was assayed using flow cytometry. This method offers the advantage that unbound ligand need not be separated from that which is cell-associated, thus avoiding perturbation of the binding equilibrium, and accurate, extensive statistical analysis is possible. Binding of fluorescein-NGF was mainly specific and saturable, with analysis by three methods of data treatment indicating a Kd of 0.8 to 3 nM at 4 degrees C. Time-based data acquisition allowed a continuous time course for binding to be generated. Binding reached a steady-state level within 5 min of exposure of the cells to the ligand. Kinetic and steady-state results obtained using fluorescein-NGF agree well with previous data produced by 125I-NGF binding studies. The main limitation of the flow cytometric method in the NGF system is the relative lack of sensitivity compared to the binding of radiolabeled NGF, partially due to unusual quenching of the fluorophore bound to NGF.

Animals↗
PubMed2000

Modulation of lens glycolytic pathway by thioltransferase.

The observation that the level of S-thiolated proteins (protein-thiol mixed disulfides) was transiently increased in the lens epithelial cells correlation with the transient inactivation of glyceraldehyde-3-phosphate dehydrogenase (G-3PD), a key glycolytic enzyme, when the cells were treated with a bolus of hydrogen peroxide, prompted our speculation that G-3PD may have been transiently thiolated at the SH sensitive active center. In the meantime, thioltransferase (TTase), a thiol regulating enzyme, whose activity remained constant under the same condition, may be regulating G-3PD and other sulfhydryl-sensitive glycolytic enzymes through thiol-disulfide exchange reactions ( Lou et al., 1998 ). To prove this hypothesis, several purified glycolytic enzymes from a commercial source, including hexokinase (HK), G-3PD, pyruvate kinase (PK) and fructose 1,6-bisphosphatase (FBPase), an enzyme in gluconeogenesis, were made into protein-thiol mixed disulfide and used for this study. Glycolytic enzymes in cultured rabbit lens epithelial cells pre-exposed to H(2)O(2)(0.5 m M for 15 min) were also studied for this purpose. Recombinant human lens thioltransferase (RHLT), which was isolated and purified previously in this laboratory, reactivated these pure glycolytic enzymes inactivated by forming protein-S-S-gluthathione (PSSG), protein-S-S-cysteine (PSSC) or, protein-S-S-cysteamine after thiolating with oxidized glutathione, cystine or cystamine respectively. RHLT also reactivated these enzymes in the cell extract of cultured rabbit lens epithelial cells after being briefly exposed to 0.5 m M H(2)O(2). The S-thiolation and dethiolation of FBPase however, showed an opposite effect to that of glycolytic enzymes. These results suggest that TTase may participate in the repair process of glycolytic enzymes during oxidative stress and restore their activities in situ.

Adenosine Triphosphate↗
PubMed2002

Molecular recognition of avidin on biotin-functionalized gold surfaces detected by FT-IRRAS and use of metal carbonyl probes.

Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS) was successively used to monitor the covalent immobilization of biotin molecules onto a planar gold substrate covered with a self-assembled monolayer of cystamine and to transduce the molecular recognition of avidin and biotin. This detection was greatly facilitated and made selective by the labeling of avidin and of biotin with various transition metal carbonyl probes. The binding of avidin to the surface was optimized by blocking the nonspecific binding sites by adsorption of an unrelated protein, bovine serum albumin. This work exemplifies the feasibility of detecting biomolecular associations involving molecules of any size at a liquid/solid interface by using a simple and accessible surface analysis technique.

Avidin↗
PubMed1998

Expression and purification of human gamma-glutamylcysteine synthetase.

gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the ATP-dependent ligation of L-glutamate and L-cysteine to form L-gamma-glutamyl-L-cysteine; this is the first and rate-limiting step in glutathione biosynthesis. Inhibitors of gamma-GCS such as buthionine sulfoximine are widely used as tools for elucidating glutathione metabolism in vivo and as pharmacological agents for reversing glutathione-based resistance to chemotherapy and radiation therapy in certain cancers. Although gamma-GCS is readily isolated from rat kidneys, future drug design efforts are better based on structure-activity relationships established with the human enzyme. We report here the coexpression in Escherichia coli BL21(DE3) of the human gamma-GCS catalytic (heavy) subunit and regulatory (light) subunit using pET-3d and pET-9d vectors, respectively. Intracellular assembly of the holoenzyme occurred without difficulty, and levels of expression were acceptable (approximately 32 mg holoenzyme/100 g cells). Recombinant human gamma-GCS was purified to homogeneity in an overall yield of 45% by ammonium sulfate fractionation followed by sequential chromatography on Q-Sepharose ion-exchange, Superdex 200 gel filtration and ATP-affinity resins. Trace amounts of E. coli gamma-GCS were removed by immunoaffinity chromatography. The specific activity of the isolated enzyme was >1500 units/mg, comparable to the best preparations from rat kidney. The Km values for L-glutamate, L-cysteine, L-gamma-aminobutyrate (an L-cysteine surrogate), and ATP are 1.8, 0.1, 1.3, and 0.4 mM, respectively. Recombinant human gamma-GCS, like native rat gamma-GCS, is feedback inhibited by glutathione and is potently inhibited by buthionine sulfoximine and cystamine.

Animals↗
PubMed1980

Synergistic action of cysteamine and BrdU-substituted DNA in the induction of sister chromatid exchanges.

The effect of different BrdU-concentrations on the cysteamine-induced SCE-rate was investigated in V-79 Chinese hamster cell monolayer cultures. Both cysteamine and its auto-oxidation product cystamine act synergistically with BrdU in the induction of SCEs. A given concentration of these substances produces a low SCE-frequency at low BrdU-concentrations -- but the incidence of SCEs is significantly increased at increased BrdU-concentrations. -- Using one-cycle substitution experiments and the determination of the relative level of substitution by means of 3HBrdU-incorporation, this synergism was shown to depend on the BrdU incorporated in the DNA and on the extent to which this incorporation takes place.

Animals↗
PubMed1992

Transport of putrescine in the isolated rabbit intestine.

The transepithelial fluxes of putrescine were studied in sections of the three segments of rabbit intestine mounted in an Ussing chamber. The ileum exhibited the highest mucosal-to-serosal (Jms) and serosal-to-mucosal (Jsm) unidirectional fluxes of 1 mumol/l [3H]putrescine. Putrescine net flux (Jnet = Jms - Jsm) was deduced to be positive through the duodenum (Jnet = 53.40 +/- 14.30 pmol h-1 cm-2), not significantly different from zero through the jejunum (Jnet = 8.90 +/- 19.20 pmol h-1 cm-2) and negative through the ileum (Jnet = -34.30 +/- 13.80 pmol h-1 cm-2). Increasing putrescine concentration up to 10 mmol/l led to an increase in Jms, Jsm and Jnet without affecting the transport polarity in the ileum. The tissue retention of putrescine after 100 min was higher by the serosal side than by the mucosal side of the ileum. In parallel experiments, isolated pieces of ileum accumulated putrescine to a five- to sixfold concentration gradient by a ouabain-inhibitable mechanism. In contrast with arginine and in order of decreasing potency, putrescine, cystamine (a transglutaminase inhibitor), spermidine and spermine (1 mmol/l) reduced both unidirectional fluxes of putrescine across the ileum in the Ussing chamber. The latter effectors, except spermine, and N,N-dimethylcasein (1 mg/ml) led to an important, if not complete, suppression of putrescine secretion by the ileum, while the calmodulin antagonist melittin (0.3 micrograms/ml) reversed the polarity of polyamine transport, suggesting the involvement of transglutaminase in putrescine transport. We conclude that the heterogeneous pattern of putrescine transport along the small-intestinal epithelium constitutes an important feature of the regulation of polyamine concentrations in this tissue.

Animals↗
PubMed1986

Isolation and characterization of cathepsin B from bovine brain.

Cathepsin B (EC 3.4.22.1) was purified 746-fold with a 21% recovery from bovine brain by autolysis, fractional precipitation with acetone, carboxy-methyl-Sephadex chromatography, affinity chromatography on a cystamine containing column and gel filtration chromatography. The purified cathepsin B eluted on gel filtration with an apparent molecular weight of 27,000 but was resolved into three bands of 30,000, 25,000 and 5,000 molecular weight by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Antibodies to cathepsin B, raised against the 30,000 dalton band, were shown by immunoblots to react with both the 30,000 and 25,000 dalton proteins with results suggesting that the former predominated as the immunoreactive form in bovine brain homogenates. Isoelectric focusing demonstrated multiple bands, ranging from pH 4.75-5.2 with the major band at pH 5.1-5.2, all of which were capable of degrading N alpha-carbobenzoxy-L-arginyl-L-arginine 4-methoxy-beta-naphthylamide. The cathepsin B activity against N alpha-benzoyl-DL-arginine beta-naphthylamide (BANA) and bovine myelin basic protein (MBP) had a pH optimum of pH 6.0. The Km for the degradation of BANA was 1.0 mM and 5.1 mM when assayed in the presence of 1% and 2.5% dimethylsulfoxide, respectively. Cathepsin B from bovine brain has many properties similar to cathepsin B isolated from other organs. The degradative effect of cathepsin B on MBP suggests a role for this proteinase in inflammatory demyelination.

Animals↗
PubMed1981

Hypotaurine transport in brain slices: comparison with taurine and GABA.

Hypotaurine uptake was compared to taurine and GABA uptakes in brain slices under identical experimental conditions. The slices effectively concentrated both hypotaurine and GABA from the medium, whereas taurine was taken up more slowly. The uptakes of these three structurally related amino acids were all saturable, consisting of one low- and one high-affinity transport component. The kinetic parameters of hypotaurine uptake were of the same order of magnitude as those of GABA uptake. All uptake systems were sensitive to temperature, metabolic poisons, and sodium omission. Hypotaurine uptake was inhibited by GABA, L-2,4-diaminobutyric acid (L-DABA), cysteic acid, and beta-alanine, but not by taurine. Taurine uptake was strongly reduced by hypotaurine, beta-alanine, and L-DABA, as well as by GABA, whereas GABA uptake was affected only by cystamine, L-DABA, and nipecotic acid. The uptake processes of hypotaurine, taurine, and GABA were thus fairly similar and showed properties characteristic for neurotransmitter uptake. Hypotaurine uptake resembled more GABA than taurine uptake. The present inhibition studies suggest that there may exist only one common two-component transport system for these three amino acids.

Animals↗
PubMed1988

Local interactions favor the native 8-residue disulfide loop in the oxidation of a fragment corresponding to the sequence Ser-50-Met-79 derived from bovine pancreatic ribonuclease A.

A 30-residue peptide was obtained from ribonuclease A by chemical cleavage with cyanogen bromide, subsequent sulfitolysis with concomitant S-sulfonation, and finally enzymatic cleavage with Staphylococcus aureus protease. The peptide was converted to the free thiol form by reductive cleavage of the S-sulfo-protecting groups with D,L-dithiothreitol. This peptide consisted of residues 50-79 of the native sequence of ribonuclease A, with the exception that methionine-79 had been converted to homoserine. Included in this sequence are residues cysteine-65 and cysteine-72, which form a disulfide bond in the native enzyme, as well as cysteine-58. This molecule may form one of three possible intramolecular disulfide bonds upon thiol oxidation, viz. one loop of 15 and 2 of 8 residues each. These isomeric peptides were prepared by oxidation with cystamine, 2-aminoethanethiolation of residual thiols, and fractionation by reverse-phase high-performance liquid chromatography. Disulfide pairings were established by mapping the tryptic fragments and confirming their composition by amino acid analysis. After protracted incubation under oxidizing conditions at 25.0 degrees C and pH 8.0, the 26-member ring incorporating the native disulfide bond between residues 65 and 72 is the dominant product. Assuming that equilibrium is established, we infer that local interactions in the sequence of ribonuclease A significantly stabilize the native 8-residue disulfide loop with respect to the non-native 8-residue loop (delta G degree = -1.1 +/- 0.1 kcal mole-1). The implications of this observation for the oxidative folding of the intact protein are discussed.

Amino Acid Sequence↗
PubMed1990

Transglutaminase involvement in the secretion of insulin from electropermeabilised rat islets of Langerhans.

Ca(2+)-Induced insulin release from electropermeabilised islets is inhibited by the transglutaminase inhibitors monodansylcadaverine, glycine methylester, methylamine and cystamine but not by the control compounds dimethyl monodansylcadaverine and sarcosine methylester which lack the primary amine group. Neither monodansylcadaverine nor glycine methylester inhibited insulin secretion induced by either cAMP or the phorbol ester PMA at basal levels (10 nM) of Ca2+. These data provide further evidence for the involvement of transglutaminase in Ca2+ induced insulin secretion, they also suggest that insulin secretion induced by either cAMP or PMA may act in part by a mechanism independent of that induced by Ca2+.

Animals↗
PubMed2005

Determination of total cysteamine in human plasma in the form of its 2-S-quinolinium derivative by high performance liquid chromatography.

Cysteamine (mercaptamine) can be determined in plasma by liquid chromatography with ultraviolet detection after precolumn derivatization. The plasma is reduced with sodium borohydride in order to convert disulfides to thiols, and derivatized with 2-chloro-1-methylquinolinium tetrafluoroborate. The 2-S-quinolinium derivative of cysteamine is then separated from other thiols derivatives present in the plasma, and quantitated using high-performance liquid chromatography and then detection at 355 nm. Peaks from the main plasma thiols cysteine, cysteinylglycine, glutathione and homocysteine are also observed and can be measured as needed. The cystamine standards added to the plasma before the reduction step show that the response of the detector is linear within the range studied, from 0.1 to 40 micromol/L plasma. The imprecisions at the bottom and the top of the calibration range were 11.17 and 0.8% and the inaccuracies 8.64 and 1.50%, respectively, and the lower limit of quantitation was 0.1 nmol cysteamine in 1 ml of plasma.

Calibration↗
PubMed2002

Dirofilaria immitis: further characterization of the transglutaminase enzyme and its role in larval molting.

We recently reported the cDNA cloning and functional characterization of a novel transglutaminase (TGase) from the dog filarial parasite Dirofilaria immitis. D. immitis TGase (DiTG) has no sequence similarity with any other known TGase, but has significant similarity to protein disulfide isomerase (PDI)-related endoplasmic reticulum protein ERp60. In the present study. we further characterized the recombinant DiTG (rDiTG) and studied its role in the molting process of third-stage larvae. The enzymatic activity of rDiTG requires Ca2+, and the maximum activity was observed at a calcium concentration of 4 mM. Interestingly, the rDiTG was highly thermostable, with optimal activity observed at 55 degrees C, similar to that seen with the native enzyme. Dithiothreitol (DTT) was not essential for enzyme activity. In fact, rDiTG was more active in the absence of DTT. The known inhibitors of TGase, such as monodansylcadaverine (MDC), cystamine and iodoacetamide, inhibited the TGase activity, but not the PDI activity, of rDiTG, demonstrating the dual activity of rDiTG. The TGase-specific pseudosubstrate, MDC, completely inhibited the molting of D. immitis L3 to L4 if present during the first 24-48 h of the molting process. Electron microscopic studies revealed that MDC-treated infective larvae failed to show separation between the L3 and L4 cuticles. The L4 cuticle and accompanying hypodermis were much thinner in MDC-treated worms than in controls. Using anti-rDiTG antiserum, the native DiTG antigen was localized in the hypodermis, afibrillar muscle cells and gut epithelium in adult male and female worms as well as developing embryos in the females.

Animals↗
PubMed1996

Characterization of the arylalkylamine N-acetyltransferase in Onchocerca volvulus.

The characteristics and kinetic properties of an arylalkylamine N-acetyltransferase were studied in partially purified preparations of the human filarial parasite Onchocerca volvulus. The enzyme, which had a relative molecular mass (M(r)) of 37-38 kDa, catalyzed the acetylation of arylalkylamines but did not accept arylamines or polyamines as substrates. The optimal pH for enzyme activity was found to be 8.5 in TRIS-HCI. The apparent Michaelis constant (K(m)) and maximum velocity (Vmax) determined from Lineweaver-Burk plots for tryptamine were 1.8 microM and 29 nmol min-1 mg protein-1, respectively. Except for the catecholamines, the other arylalkylamines such as 5-hydroxytryptamine (5-HT), tyramine, and octopamine similarly exhibited high affinities and reaction rates. Whereas the enzyme is inhibited by metals and p-chloro-mercuribenzoate, it is inactivated neither by amethopterin nor by cystamine and is thereby distinguished from the mammalian arylamine N-acetyltransferase. Like other N-acetyltransferases whose function is the regulation of intracellular amine levels, the enzyme may have a role in the inactivation of excess biogenic amine in this parasite.

Animals↗
PubMed1989

Chemistry on solid supports: defining events and titers by use of cleavable, assayable linking molecules.

The cleavable diamines cystamine, 5, and 1,6-diamino-3,4-dihydroxyhexane, 1, were bonded to solid supports and, with a simple, newly developed dinitrofluorobenzene-based assay, were used to define (a) titers of ligands and (b) chemistry distal to the support. Compound 1, which is cleavable with periodate, becomes a linking molecule which is stable to almost all conditions encountered in biochemistry and enjoys considerable hydrophilic character. Compound 5, which is cleavable with dithiothreitol, can be usefully applied to those systems which do not require reducing agents. These nucleophilic linking moieties were converted to cleavable electrophilic linkers by succinylation and p-nitrophenyl ester activation. The first preparation of a polysaccharide-linked support is described. The method also allows the chemical definition of ligands containing amino groups which are prepared by deblocking of protecting groups while on the support. The methodology should promote greater understanding of affinity chromatography materials and processes.

Amines↗
PubMed1988

Characteristics of taurine transport system and its developmental pattern in mouse cerebral cortical neurons in primary culture.

Developmental patterns and pharmacological and biochemical properties of taurine transport system were investigated using developing primary cultured neurons prepared from mouse cerebral cortex by trypsin treatment. [3H]Taurine was incorporated into neurons via a high-affinity transport system of which the Km value as well as the Vmax value increased during neuronal development in vitro. This transport system was also inhibited by sodium withdrawal from incubation medium and exposures for 15 h to several metabolic inhibitors such as 2,4-dinitrophenol and monoiodoacetate. In addition, [3H]taurine uptake in both neurons cultured for 3 and 14 days was competitively inhibited by beta-alanine, guanidinoethanesulfonate and hypotaurine. Cysteic acid and cysteine sulfinic acid, metabolic intermediates produced in the process of taurine biosynthesis in the brain from cysteine, induced significant reductions in [3H]taurine uptake in both types of cultured neurons, while cysteine, isethionic acid, cysteamine and cystamine exhibited no alterations in [3H]taurine transport. Moreover, non-competitive inhibition of [3H]taurine uptake by cysteic acid was observed in both neurons. These results clearly indicate that taurine uptake was mediated by the sodium- and energy-dependent transport system with high affinity in 14-day-old neurons as well as neurons cultured for 3 days and that both the Km and Vmax values of this transport system increase during neuronal development in vitro. The results described above suggest that the decrease in taurine content observed in developing brain is unlikely to be due to alteration in the capacity of the taurine transport system during neuronal development.

Amino Acids↗