Reduction Of Cystine Research Articles

Reactivity of hair cystine in microemulsion media

Synopsis Reduction of keratin cystine by thioglycolic acid incorporated in microemulsions of the water/sodium dodecilsulphate/n-pentanol/n-dodecane system has been determined. The results obtained have been interpreted in relation to the properties of the reaction media. Microemulsions with constant oil-to-surface active mixture weight ratios (R(o/s)) and different concentrations of water were chosen as reaction media. At low water concentrations a steep increase in reactivity with the increase of water was observed at all values of R(o/s). However it was more pronounced the higher the oil content. A relation between maximum cysteine formation and percolative behaviour of the microemulsion was found at high R(o/s) values.

Intracellular cystine loading inhibits transport in the rabbit proximal convoluted tubule.

Cystinosis is an autosomal recessive disorder characterized by a high intracellular cystine concentration. To establish an in vitro model of this disorder and examine the mechanism of the proximal tubule transport defect seen with elevated intracellular cystine concentrations, rabbit proximal convoluted tubules (PCT) were perfused in vitro. PCTs were loaded with cystine using cystine dimethyl ester, a permeative methyl ester derivative. Bath cystine dimethyl ester (0.5 mM) reduced volume absorption (Jv) (0.67 +/- 0.07 to 0.15 +/- 0.09 nl/mm.min, P less than 0.01), bicarbonate transport (JTCO2) (47.2 +/- 4.9 to 11.1 +/- 2.8 pmol/mm.min, P less than 0.001) and glucose transport (JGLU) (34.1 +/- 1.5 to 19.7 +/- 1.5 pmol/mm.min, P less than 0.001). The methyl esters of leucine (0.5 mM), and tryptophan (0.5 and 2.0 mM) had no effect on these parameters. To examine if intracellular reduction of cystine to cysteine could contribute to the inhibition in transport, the effect of bath cysteine methyl ester on proximal tubular transport was examined. Bath cysteine methyl ester (2 but not 0.5 mM) resulted in an inhibition in Jv, JGLU, and JTCO2. Cystine dimethyl ester had no effect on mannitol or bicarbonate permeability. These data are consistent with intracellular proximal tubular cystine accumulation resulting in an inhibition of active transport.

Effect of Captopril on Urinary Cystine Excretion in Homozygous Cystinuria

We determined if captopril could reduce urinary cystine excretion in homozygous cystinuric patients. Seven patients were treated with 150mg. captopril daily after determination of baseline 24-hour urine cystine excretion. All patients had a history of multiple cystine stones, and were on chronic fluid and alkalization therapy. Five patients had previously been on D-penicillamine. Cystine excretion studies were repeated 1 to 5 months after institution of captopril.Baseline 24-hour urinary cystine excretion ranged from 580 to 970mg. per gm. creatinine (mean 744). After institution of captopril these levels decreased to a range of 113 to 581mg. per gm. creatinine (mean 371). These values on treatment represented a statistically significant decrease to between 18 and 89% of baseline levels (p equals 0.0045).We conclude that captopril can significantly, and at times profoundly, decrease urinary cystine excretion in patients with homozygous cystinuria. Further studies are required to elucidate fully the mechanism of urinary cystine reduction and to help define a possible role for this drug in routine maintenance or dissolution therapy.

ChemInform Abstract: A Mechanistic Study of the Reduction of Cystine by Vanadium(II) in the pH Range from 7.5 to 12

The reduction of cystine by aqueous vanadium(II) was investigated in the p..H range from 7.5 to 12. The product ratio [VIV] /[VIII] reaches a maximum at pH ca. 9 and depends linearly on the excess concentration of cystine. It is also affected by cysteine, but not by initially added vanadium(III). The rate of the oxidation is first order in total vanadium(II) and also depends on cystine and on added cysteine or mercaptoacetic acid. The data are consistent with a mechanism involving two parallel paths leading to vanadium(III) and vanadium(IV), with precursors differing by one cystine ligand. In either case, the net result is scission of the SS bond.

A mechanistic study of the reduction of cystine by vanadium(II) in the pH range from 7.5 to 12

The reduction of cystine by aqueous vanadium(II) was investigated in the p..H range from 7.5 to 12. The product ratio [V IV] /[V III] reaches a maximum at pH ca. 9 and depends linearly on the excess concentration of cystine. It is also affected by cysteine, but not by initially added vanadium(III). The rate of the oxidation is first order in total vanadium(II) and also depends on cystine and on added cysteine or mercaptoacetic acid. The data are consistent with a mechanism involving two parallel paths leading to vanadium(III) and vanadium(IV), with precursors differing by one cystine ligand. In either case, the net result is scission of the SS bond.

The role of sulfur-containing amino acids in superoxide production and modification of low density lipoprotein by arterial smooth muscle cells.

Extracellular superoxide (O2-.) was detected in cultures of monkey arterial smooth muscle cells as measured by the superoxide dismutase-inhibitable reduction of cytochrome c and acetylated cytochrome c. Reduction of cytochrome c by these cells required L-cystine in the incubation medium. A variety of other sulfur-containing amino acids, including D-cystine, L-cystathionine, L-methionine, and djenkolic acid did not support O2-. generation when present at concentrations equimolar to L-cystine. At millimolar concentrations, the chelators EDTA and diethylene triamine penta-acetic acid inhibited O2-. production by smooth muscle cells. This effect was maximal when the chelator was present at the same concentration as the sum of the Ca2+ and Mg2+ in the medium, suggesting a role for these cations in O2-. generation by cells. Modification of low density lipoprotein (LDL) by arterial smooth muscle cells, as assessed by changes in lipid peroxide content, mobility on agarose gel electrophoresis, and apoprotein B fragmentation, was also L-cystine-dependent. LDL modification also required micromolar concentrations of the transition metal ion Cu(II) or Fe(III) and was inhibited by superoxide dismutase. LDL modified by smooth muscle cells in the presence of L-cystine and Cu(II) was taken up and degraded less well than native LDL by human skin fibroblasts, suggesting that recognition by the LDL receptor was lost. In contrast, LDL modified by smooth muscle cells was taken up and degraded to a greater degree than native LDL by mouse peritoneal macrophages, consistent with recognition by the scavenger receptor. These results indicate that monkey arterial smooth muscle cells produce O2-. and modify LDL by an L-cystine-dependent process. This may involve reduction of cystine to a thiol, possibly cysteine or a cysteine-containing peptide such as glutathione. Sulfur-containing amino acids may play a role in atherogenesis by supporting cell-mediated generation of reactive oxygen species and modification of lipoprotein to a form recognized by the scavenger receptor.

Cystine antagonism of the antibacterial action of lactoperoxidase-thiocyanate-hydrogen peroxide on Streptococcus agalactiae.

Cystine reduction in Streptococcus agalactiae, resulting in sulfhydryl formation, may account for antagonism of the antibacterial effect of lactoperoxidase-thiocyanate-hydrogen peroxide when cystine is present in excess of the amount needed for maximum growth. Accumulation of cystine by S. agalactiae and its reduction to form sulfhydryl compounds were demonstrated. The reduction of cystine appeared to occur by a couple reaction between glutathione reductase and glutathione-disulfide transhydrogenase activity, both of which were found in the supernatant fraction from cell homogenates. NADPH-specific glutathione reductase activity was found in the pellet and supernatant fractions from cell homogenates. Two sulfhydryls were formed for each mole of NADPH used during cystine reduction. The information presented offers a plausible explanation of how cystine, when present in excess of growth needs, may be reduced to generate sulfhydryl compounds which neutralize the antibacterial effect of lactoperoxidase-thiocyanate-hydrogen peroxide on S. agalactiae.

Mechanism of augmentation of the antibody response in vitro by 2-mercaptoethanol in murine lymphocytes : II. A major role of the mixed disulfide between 2-mercaptoethanol and cysteine

Five thiol compounds including 2-mercaptoethanol (2-ME) were examined for their augmenting effects on in vitro antibody response to sheep erythrocytes. Three compounds were effective with the following order of activity; 2-ME > dithiothreitol > cysteamine. Glutathione and thioglycollate failed to enhance the response. The same order of effectiveness was seen in the stimulation of [ 35S]cystine uptake by murine lymphocytes by these thiols. Murine lymphocytes took up cysteine five to six times more rapidly than cystine. It is, however, unlikely that 2-ME stimulation of cystine uptake is solely due to the reduction of cystine into cysteine, because 2-ME was still stimulatory after free thiol groups had disappeared in the medium containing 2-ME and [ 35S]cystine. The mixed disulfide of cysteine with 2-ME (Cys-2-ME) was found to be an only product after free thiols had been oxidized. [ 35S]Cys-2-ME was taken up by the lymphocytes with a comparable rate to cysteine via a transport system common to that of leucine and phenylalanine. Cysteine was, however, transported via a different route. It was observed that Cys-2-ME was readily metabolized to cysteine and glutathione after the uptake. Cys-2-ME added to cystine-free RPMI 1640 medium could support the antibody response as efficiently as cystine plus 2-ME. These observations strongly suggest that 2-ME stimulates cystine uptake and, therefore, enhances the antibody response through the formation of the mixed disulfide with cysteine.

Characterization of a Selenocystine-Resistant Carrot Cell Line

A selenocystine-resistant carrot cell line, C-1, was isolated from a haploid carrot (Daucus carota) cell culture, HA. The C-1 variant takes up cystine, but not cysteine, more slowly than does HA. The selenocystine resistance is maintained in culture in the absence of selection and is expressed in regenerated plants. Results based on chromatographic separation of sulfur metabolites from cells fed with [(35)S]cystine suggest a block either in the uptake or reduction of cystine in the variant. Both lines can grow on cystine as sole sulfur source. Growth of the HA line on cystine suppressed the development of sulfate uptake capacity (Furner, Sung 1982 Proc Natl Acad Sci USA 79: 1149-1153), while cystine-grown C-1 cells have high levels of sulfate uptake capacity.We suggest that the C-1 line, grown on cystine, accumulates an insufficient quantity of some sulfur metabolite, which is involved in the control of sulfate uptake, to suppress the uptake. C-1 grown on cystine is more sensitive than HA to growth inhibition by the sulfate analog selenate.

Turnover of cellular glutathione in isolated rat-kidney cells. Role of cystine and methionine.

Turnover of cellular glutathione in isolated rat kidney cells was studied using cystine or methionine as sulfur donor. In the absence of any sulfur donor a continuous decrease of intracellular reduced glutathione (GSH) during incubation of the cells was observed. This decrease was abolished in the presence of cystine, and, as indicated by incorporation of 35S, there was also a rapid synthesis of GSH. In the presence of gamma-glutamyltransferase inhibitor, the synthesis of intracellular GSH was accompanied by an accumulation of extracellular cysteine-glutathione mixed disulfide whereas only minor amounts of GSH and glutathione disulfide could be detected. The intracellular levels of both the cysteine-glutathione and glutathione disulfides were at all times points very low. Even though the uptake of cystine was rapid and not rate-limiting for GSH synthesis, almost no cystine could be detected intracellularly. An increasing intracellular cysteine concentration was however observed, indicating a rapid reduction of cystine. In contrast to cystine, methionine did not protect from the loss of intracellular GSH and only a low rate of incorporation of 35S into GSH was observed. Methionine was rapidly taken up into the cells but was apparently converted to cysteine only to a very limited extent. This is most likely due to a low activity of the enzyme cystathionase since neither homocysteine nor cystathionine was very effective in supporting GSH synthesis.

Cystine uptake by rat renal brush-border vesicles.

Uptake of L-cystine by brush-border membrane vesicles isolated from rat renal-cortical tissue was time-dependent and occurred in the absence of cystine reduction. A significant capacity for vesicular binding of cystine was observed. The amount bound increased with time of incubation and could be displaced by thiol reagents. At early time points, cystine uptake measured the transport of cystine into the intravesicular space. Total cystine uptake was mediated by multiple transport systems, including a low-Km high-affinity component which was shared by lysine, arginine, ornithine and glutamine and on which hetero-exchange diffusion of lysine and cystine was demonstrated.

L-[35S]Cystine uptake by rat brain synaptosomes.

Abstract— The uptake of [35S]cystine at 37°C by synaptosomal fractions isolated from adult rat cerebrum can be divided into two components. About 60% of the uptake is due to binding to synaptosomal proteins while the remainder exists as a free amino acid pool. Chemical analysis of this soluble component indicates that considerable reduction of cystine to cysteine occurs with 75% or more of the labeled molecular species being cysteine. The process involved in the uptake into the soluble pool was composed of two saturable systems with apparent Km values of 0.14 and 1.4 mm. The low Km system was sodium and oxygen independent but inhibited by dinitrophenol. Dibasic amino acids, lysine, arginine and ornithine, did not inhibit cystine uptake. The characteristics of cystine uptake by synaptosomes from newborn brain are very similar to those of adult brain.

Derivatization of cysteine and cystine for fluorescence amino acid analysis with the o-phthaldialdehyde/2-mercaptoethanol reagent.

Previous reports (Drescher, D.G., and Lee, K.S. (1978) Anal. Biochem. 84, 559-569; Lee, K.S., and Drescher, D.G. (1978) Int. J. Biochem. 9, 457-467) have shown that high performance liquid chromatographic analysis of amino acids with the o-phthaldialdehyde/2-mercaptoethanol reagent (OPA/2-ME) is one of the most sensitive procedures currently available for micro amino acid analysis. In the present paper, methods are presented for the modification of cysteine and cystine in proteins for micro amino acid analysis using OPA/2-ME. Cysteine and cystine, which both show low fluorescence with OPA/2-ME, are converted to cysteic acid with performic acid directly, or to S-3-sulfopropylcysteine with 1,3-propane sultone after reduction of cystine with tri-n-butylphosphine. Cysteic acid and S-3-sulfopropylcysteine form highly fluorescent adducts with OPA/2-ME. The formation of S-3-sulfopropylcysteine in proteins and the subsequent hydrolysis of the proteins with methanesulfonic acid are particularly useful for complete amino acid analysis at the picomole level using a single sample.

525 TREATMENT OF CYSTINOSIC WITH CYSTEAMINE

Attempts at reduction of intracellular cystine in infantile cystinosis, has been reported previously in 3 patients using dithiothreitol (2 patients) or cysteamine (mercaptoethylamine) (1 patient). We evaluated the benefit of a combination of ascorbic acid and cysteamine in a 14-month-old boy with Fanconi's syndrome and cystinosis. The patient's granulocytes contained 5.2 - 13 nmol of cystine/mg of protein (normal < 0.1 nmol/mg protein) and cystine crystals were present in the bone marrow. Creatinine clearance (CCr) was 13 ml/min/1.73 m2 pre-treatment. Ascorbic acid was given as a calcium salt in a dose of 2 mg/kg/day. Cysteamine was given at 99 mg/kg/day. Within 30 days, the buffy coat cystine content had fallen to normal. The dose of cysteamine was then decreased to 30 mg/kg/day. Concurrent with the fall in leukocyte cystine content, the CCr rose to 32.8 and 50 ml/min/1.73 m2 serially and growth improved, though these changes probably reflected other elements of treatment. No side effects were noted. This is the first report of early treatment of cystinosis with cysteamine. Although the long-term benefit of this treatment will not be known for several years, the early satisfactory biochemical response emphasizes the need to evaluate cysteamine in other patients with cystinosis, before the disease is far advanced.

Patterns of cystine reduction by fibroblasts from normal and cystinotic children.

The possibility that the enzymatic reduction of cystine involves a multi-enzyme system led to re-evaluation of cystine reduction by fibroblasts from normal and cystinotic patients. Lineweaver-Burk plots of data with extracts of a normal cell line, representative of seven normal cell lines, under conditions of increasing cystine with variable levels of reduced glutathione (GSH) resulted in a two-limbed curve above 100 micronM cystine. Two cell lines from children with nephropathic cystinosis containing 1-6 micronmol half-cystine/g protein gave a family of curves similar to those of the normal. With six fibroblast lines containing more than 6 micronmol half-cystine/g protein, increasing cystine resulted in a family of lines without two-limbed curves. Plots of the data as activity against increasing cystine concentrations at ratios of cystine to GSH of 1:2 and 1:1 showed that two of the three lines from cystinotic subjects reduced cystine at a faster rate than the normal line. The third line from a cystinotic patient reduced cystine at a slightly slower rate when the substrate concentration in the assay was less than 80 micronM cystine. When the cystine to GSH ratio was maintained at 2:1, normal cells showed a linear increase in the rate of cystine reduction up to 100 micronM cystine, no increase in the rate between 100 micronM and 200 micronM cystine, and an increase again when the concentration of cystine was raised above 200 micronM. Such a stepwise phenomenon was absent with six cell lines containing more than 6 micronmol half-cystine/g cellular protein. A possible mechanism of control of cystine reduction is discussed.

ISOLATION OF LYSOSOMES FROM SKIN FIBROBLASTS OF NORMAL AND CYSTINOTIC INVIDUALS

The biochemical lesion in cystinotic cells resulting in lysosomal storage of cystine is not yet known. The lysosomal metabolism of cystine should be investigated in isolated lysosomes. In whole unfractionated cells, the cystine metabolism was found to be unchanged /Patrick 1962, Schneider et al. 1967/.Human skin fibroblasts were cultured in large scale /2 × 108 to 1 × 109 cells in one batch/. After differential centrifugation free-flow electrophoresis according to the method of Hannig et al. was used for isolating native lysosomes from these fibroblasts. The purity of lysosomal fractions was controlled by electron microscopy and by measuring enzyme markers for lysosomes, mitochondria and peroxisomes. The activity of enzymes involved in cystine reduction in cystinotic lysosomes was compared to that in normal lysosomes /cystine-glutathione-transhydrogenase, glutathione-reductase/.

Regulation of thiols in the brain. 4. Glutathione--cystine transhydrogenase activity during hypoxia and its substrate specificity in different parts of the rat brain.

Abstract The glutathione-cystine transhydrogenase activity measured under optimum conditions was highest in the cerebral grey matter and lowest in the cerebellum of normal (control) animals. During hypoxia the transhydrogenase activity in the cerebellum and brain stem changed slightly, but decreased in the cerebral grey matter by up to 43% after i h and then, after increase to the control value, it was unchanged. Three parts of the brains of the normoxic rats had very low activities in respect of protein disulphides, and no reaction at all with the synthetic substrate 5,5′-dithiobis-(2-nitro-benzoic acid). Of the non-protein disulphides, glutathione-cystine transhydrogenase activity was highest with cystine and the lowest with oxidized lipoic acid in the parts of the brain studied. Differences in the substrate affinity and the maximal velocity with the disulphide compounds used were observed in the different parts of the rat brain. A very strong inhibition of even slightly acidic pH on spontaneous as well as on transhydrogenase-catalysed reduction of cystine by glutathione was found in vitro. These results are compatible with the previously suggested increase of the concentration of low-molecular-weight disulphides. The significance of these results is discussed.

Selective reduction of cystine 1-8 in alpha-lactalbumin.

Selective reduction of cystine 1-8 in alpha-lactalbumin.

2—THE COMPETITIVE ADDITION REACTION OF DEHYDROALANINE RESIDUES FORMED DURING THE ALKALINE DEGRADATION OF WOOL CYSTINE

An investigation is described in which the effect of a thiol-reducing agent, thioglycollic acid, on wool in borate and ammonia solutions was ascertained in relation to the competition of thiols and amines for combination with dehydroalanine residues. Thioglycollate was found to decrease the amounts of products formed by amino-group addition to dehydroalanine residues at pH 12 and to eliminate them completely at pH 10. It is also shown that, at certain concentrations of thioglycollate, the degradation of cystine to dehydroalanine residues is greatly increased to give maximum lanthionine formation, but above this critical concentration thioglycollic acid competes successfully with the thiol group of cysteine to give S-carboxymethylcysteine rather than lanthionine. Differences between alkaline solutions of borates and ammonia with thioglycollate present are ascribed to the differences in reduction of cystine to cysteine; the blocking of free thiol groups is used to observe any difference in the amount of sul...

Enzymic reduction of cystine by subcellular fractions of cultured and peripheral leukocytes from normal and cystinotic individuals.

Extract: Lysates of cultured and freshly drawn isolated peripheral leukocytes from normal and cystinotic individuals were examined for cystine-reducing activity, with particular emphasis on possible enzyme action in lysosome-containing fractions. Whole cell extracts of cultured leukocytes were found to possess strong glutathione-cystine trans-hydrogenase activity at both pH 7.5 and 5.5; no significant difference in this enzyme activity between extracts of normal and cystinotic cells was observable at either pH. Subcellular fractionation of these cultured leukocytes indicated that the major portion of transhydrogenase activity in the 5.5–7.5 pH range was confined to the highspeed supernatant (cytoplasmic) fraction, with little or no activity being detectable in the granular fractions. Comparative enzyme assays at both pH 4.5 and 7.5 with lysosomal and supernatant fractions of peripheral leukocytes freshly drawn from normal and cystinotic donors were consistent with the foregoing results in terms of the subcellular distribution of glutathione-cystine transhydrogenase activity; again there appeared to be no significant difference in the transhydrogenase activities of these fractions from normal and cystinotic leukocytes at either pH. The relation of these observations to the pathogenesis of cystinosis is discussed.Speculation: Analogy with other lysosomal storage diseases suggests that the intralysosomal compartmentalization of cystine in cells of cystinotic individuals may be the result of an impaired mechanism of degradation of the amino acid within this cellular organelle. To date, however, convincing evidence to support this view is lacking. Alternative explanations implicating a defective membrane system associated with the egress of cystine from the lysosome, while equally plausible, likewise have yet to be confirmed experimentally; studies of subcellular transport of cystine in cystinosis are needed, but technical problems will make such investigations quite difficult.