Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RBC membrane polypeptide aggregates have been quantitated by PAGE SDS and by gel filtration. Aggregates were absent in fresh RBC's from normal controls, but aggregates with MW 4.4 X 10(5) and greater than 50 X 10(6) increased progressively as GSH levels fell in RBC's incubated in PBS without added glucose or calcium. Aggregates of both MW ranges were also present in fresh RBC's from a patient with compensated congenital nonspherocytic hemolysis associated with a mutant RBC G-6-PD, Long Prairie. Since the aggregates were dissociable by treatment with mercaptoethanol or dithiothreitol, they are probably cross-linked by intermolecular disulfide bonds. Membranes containing these aggregates may represent an early and sensitive indicator of oxidative damage to red cells.
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PMID:Membrane polypeptide aggregates in glucose 6-phosphate dehydrogenase-deficient and in vitro aged red blood cells. 62 30

The discocyte-echinocyte transformation and the decrease in deformability associated with red cell ATP depletion have been attributed to changes in the physical properties of spectrin and actin, membrane proteins located at the membrane-cytosol interface. We investigated the spontaneous formation of spectrin-rich complexes in human erythrocyte membranes, employing two-dimensional SDS-polyacrylamide gel electrophoresis. Membranes of red cells depleted in ATP under aerobic conditions exhibited (1) an increase in components 4.5 and 8 and globin subunits, (2) a spontaneous formation of heterodimers of spectrin 1 + 2 and spectrin 2 + component 4.9, and (3) a large molecular weight (greater than 10(6) daltons) protein complex with a high spectrin to band 3 ratio. These complexes were dissociated with dithiothreitol and were prevented by anaerobic incubation or the maintenance of red cell ATP and GSH levels with glucose, adenine, and inosine. The complexes 1 + 2 and 2 + 4.9 were also seen in acetylphenylhydrazine-treated, glucose-6-phosphate dehydrogenase-deficient fresh erythrocytes that showed marked GSH depletion but preserved greater than 70% of the original ATP level. However, membranes of these cells did not contain the greater 10(6) dalton aggregate with a high spectrin to band 3 ratio. We concluded that the formation of the latter complex results from rearrangement of spectrin and other polypeptides in membranes of ATP-depleted red cells. Under aerobic conditions, the rearranged proteins undergo spontaneous intermolecular crosslinkings through disulfide couplings.
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PMID:Metabolic dependence of protein arrangement in human erythrocyte membranes. I. Analysis of spectrin-rich complexes in ATP-depleted red cells. 62 5

The molecular structure of plasma and erythrocyte selenium-dependent glutathione peroxidase (GSH-Px) was studied in rats drinking water containing [75Se]selenious acid, 1.3 mg Se/L. Substantial differences were found using three-step fractionation, including gel filtration of crude plasma and erythrocyte lysate, gel filtration of 75Se-GSH-Px treated by mercaptoethanol, and SDS-electrophoresis. Native plasma 75Se-GSH-Px, which exhibited a molecular weight (M(r)) of approx. 700,000, could be destroyed by mercaptoethanol action, resulting in disintegration of enzyme into several different 75Se-protein fragments and release of part of low-mol-wt 75Se. Native erythrocyte 75Se-GSH-Px M(r) value was found to be 113,000; two 75Se-protein fragments arose after mercaptoethanol treatment without 75Se release from the enzyme. The 75Se-subunits of 22,500 and 21,900 were isolated from plasma and erythrocyte 75Se-GSH-Px, respectively. Another minor 75Se-GSH-Px was identified in erythrocyte lysate (M(r) 214,000, subunit 22,100), which was considered to be a dimer of the above-mentioned erythrocyte enzyme. It can be assumed, based on these data, that native plasma GSH-Px, in contrast to erythrocyte enzyme, represents a high-molecular wt complex composed of several tetramers linked with S-S bonds. A certain part of selenium present in this complex is probably not selenocysteine and may be released with the mercaptoethanol treatment.
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PMID:Comparison of molecular properties of rat plasma and erythrocyte selenium-dependent glutathione peroxidase. 138 17

1. The present study demonstrated that the Ca(2+)-ATPase activity of the plasma membrane-rich fraction from bovine parotid gland was decreased by the addition of reducing agents. 2. Ca(2+)-ATPase activity staining on SDS-PAGE gels was lost in the presence of 2-mercaptoethanol. 3. Among all the reducing agents tested, GSH was the most effective in inhibiting Ca(2+)-ATPase. 4. The Ca(2+)-ATPase activity decreased by the GSH was restored by the addition of an oxidizing reagent. However, oxidation with an oxidizing reagent subsequent to alkylation of the reduced enzyme with iodoacetamide resulted in no restoration of activity. 5. The decrease of Ca(2+)-ATPase activity by GSH is due to a decrease in the Vmax of the enzyme. 6. These results suggest that the disulfide bond in this enzyme protein is necessary to maintain the activity of this enzyme.
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PMID:Inhibitory effect of sulfhydryl group on Ca(2+)-ATPase activity in the plasma membrane-rich fraction from bovine parotid gland. 142 64

The objective of this study was to characterize the influence of peroxisome proliferation on the metabolism of physiological concentrations of Se. In an initial series of experiments hepatocytes in primary cultures and isolated from ordinary-fed rats, were used. The cells were exposed to 75Se-selenite (30 nM) and after 24 h the labelling of selenoproteins was analysed with SDS-PAGE. Treatments with mono(2-ethylhexyl)phthalate (MEHP; a metabolite of di(2-ethylhexyl)phthalate (DEHP)), nafenopin, decreased oxygen tension and a H2O2 generating system decreased the labelling of a 23-kDa and a 15-kDa protein. The decreased labelling of the 23- and the 15-kDa proteins was usually accompanied by an increased labelling of a 58-kDa protein. Increased oxygen tension induced uncertain effects, possibly due to toxicity. In order to further evaluate the validity of the model, the labelling was also studied in hepatocytes isolated from Se-deficient and torula yeast-fed rats. In these cells there was a decreased labelling of the 23-kDa protein as compared to cells from Se-supplemented controls when 100 nM selenite was used. In in vivo experiments it was found that a DEHP-induced decrease in glutathione peroxidase (GSH-Px) activity was potentiated by high doses of selenite. To a large extent, the labelling data are compatible with enzyme activity data and in vivo data. For example, the decreased labelling of the 23-kDa protein may reflect the decreased GSH-Px activity. It is concluded that the effects induced by MEHP on Se-labelling can be explained by an increase in the steady state level of H2O2.
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PMID:Studies on Se incorporation in selenoproteins; effects of peroxisome proliferators and hydrogen peroxide generating system. 154 Sep 96

Six forms of glutathione transferase (GST) were resolved from the cytosolic fraction of Bufo bufo embryos at developmental stage 4 by GSH-Sepharose affinity chromatography followed by f.p.l.c. chromatofocusing in the 9-6 pH range. They have apparent isoelectric points at pH 8.37 (GST I), 8.22 (GST II), 8.10 (GST III), 7.84 (GST IV), 7.37 (GST V) and 7.12 (GST VI), and each displayed an apparent subunit molecular mass of 23 kDa by SDS/PAGE. The Bufo bufo embryo enzymes showed very similar structural, catalytic and immunological properties, as indicated by their substrate-specificities, inhibition characteristics, c.d. spectra, h.p.l.c. elution profiles and immunological reactivities, as well as by their N-terminal amino acid sequences. Although Bufo bufo embryo GSTs do not correspond to any other known GSTs, the results of our experiments indicate that amphibian GSTs could be included in the Pi family of GSTs. This conclusion is supported by the analysis of c.d. spectra, and by the fact that mammalian Pi class GSTs and amphibian GSTs showed about 80% identity in their N-terminal amino acid sequences. Furthermore, antisera prepared against Bufo bufo GST III cross-reacted in immunoblotting analysis with Pi class GSTs, and vice versa.
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PMID:Glutathione transferase isoenzymes from Bufo bufo embryos at an early developmental stage. 156 69

The effects of diquat metabolism on the protein thiol (PrSH) status of bis-chloronitrosourea-pretreated hepatocytes have been studied. Using a conventional, dithionitrobenzene-based assay for free PrSH in trichloroacetic acid-precipitated protein, control levels of PrSHs (83 +/- 6 nmol/mg protein) were unaltered during the initial 60 min of incubation of the cells with 1 mM diquat. However, using a radiochemical method for the determination of glutathionylation of PrSH [Grimm et al., Biochim Biophys Acta 844: 50-54, 1985], in which the hepatocytes were prepared from diethylmaleate-pretreated animals and reloaded with reduced glutathione (GSH) in the presence of [35S]methionine and cycloheximide, oxidation of hepatocellular PrSH by stimulated S-thiolation with GSH could be demonstrated. The S-glutathionylation of the protein was maximal after 30 min of treatment of the cells and preceded the onset of membrane leakage. However, the quantity of GSH mixed disulfide formed was limited to a maximum of 1.4 +/- 0.4 nmol GSH/mg protein, indicating the oxidation of only 2% of the total hepatocellular PrSH by S-thiolation. This percentage depletion is below the working variability of the colourimetric PrSH assay utilized and indicates strongly the use of the S-thiolation assay in the study of the possible effects of other redox-cycling cytotoxins on cellular PrSH status, as these may not be evident with conventional spectrophotometric techniques. The analysis of the cellular protein from diquat-treated cells by SDS-PAGE and autoradiography indicated the S-glutathionylation of a variety of cellular proteins, including species with molecular masses 17, 24, 26, 30, 40, 43 and 46 kDa. Although the identities of these species are uncertain (the 30-kDa protein may be equivalent to carbonic anhydrase as reported by Rokutan et al., Biochemistry 179: 233-239, 1989), it may be that oxidative modification of these proteins by stimulated S-glutathionylation may be an important early event in the mechanism of the hepatotoxicity of diquat.
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PMID:The S-thiolation of hepatocellular protein thiols during diquat metabolism. 163 11

Thioltransferase, an enzyme which catalyzes the thiol/disulfide exchange reaction in the presence of GSH, was purified to homogeneity on 15% SDS-PAGE from human (36,000-fold purification) and bovine (23,000-fold) erythrocyte hemolysates. These enzymes had similar properties in their monomeric structures (M(r) = 11,000) and broad specificities for substrates ranging from low-molecular disulfides (S-sulfocysteine, cystamine, and cystine) to protein disulfides (trypsin and insulin). They were highly sensitive to SH-reagents (monoiodoacetic acid and mercuric chloride), but were protected from inactivation by the presence of disulfides (GSSG, cystamine, and cystine). Phosphofructokinase and pyruvate kinase that had been inactivated by disulfides were reactivated effectively by the addition of thioltransferase with GSH. In addition, disulfides in membrane proteins of human erythrocytes that have been oxidatively damaged by diamide treatment were reduced to the SH-free form more effectively by incubation with thioltransferase.
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PMID:Study on human erythrocyte thioltransferase: comparative characterization with bovine enzyme and its physiological role under oxidative stress. 163 68

Bovine adrenal cortex tissue expresses high levels of glutathione S-transferase (GST) from each of the alpha, mu and pi gene families. We describe the purification and characterization of an abundant alpha-class GST from this tissue that has not been identified previously because of its failure to bind to S-hexylglutathione-Sepharose 6B (S-hexG-Ag). This enzyme has been affinity purified on glutathione-Sepharose 6B (GSH-Ag) and was obtained in a highly purified form by employing S-hexG-Ag to remove the bulk of GST before chromatography on GSH-Ag. The purified GST eluted from GSH-Ag was found to exhibit marked peroxidase and delta 5-ketosteroid isomerase activities (19.2 and 1.67 U/mg respectively). The bovine enzyme also showed high GST activity towards 4-hydroxynonenal (5.09 U/mg). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the bovine GST contains two distinct polypeptides, one with an Mr of 25,900 and the other with an Mr of 26,500. An abundant alpha-class GST was also purified from human adrenal cortex that possessed properties which were similar to the bovine alpha-class GST described above; however, unlike the bovine enzyme, the corresponding human alpha-class GST bound to S-hexG-Ag. As with the bovine enzyme, the purified human GST displayed marked peroxidase and isomerase activities (27 and 4.02 U/mg respectively). Further analysis on SDS-PAGE (Mr 25,800) and reverse-phase high-performance liquid chromatography established that this abundant alpha-class GST in human adrenal cortex is equivalent to the human liver GST B1B1 enzyme. As both human and bovine adrenal cortex contain high levels of alpha-class GST with similar catalytic properties, we discuss the possible functions of these enzymes in this tissue.
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PMID:Expression of an abundant alpha-class glutathione S-transferase in bovine and human adrenal cortex tissues. 173 63

Di(2-ethylhexyl)phthalate (DEHP), a rat liver carcinogen, induces peroxisomal proliferation and a concomitant oxidative stress, but decreases liver glutathione peroxidase (GSH-Px) activity. This enzyme is a selenoprotein and we have investigated the influence of mono(2-ethylhexyl)phthalate (MEHP), a major metabolite of DEHP, on selenium incorporation in hepatocellular proteins. [75Se]Selenious acid (6 nM) was added to primary cultures of rat hepatocytes and protein incorporation was assessed by SDS-PAGE and autoradiography. High concentrations of MEHP (1.0-3.0 mM) inhibited selenium labeling of all major selenoproteins in 3-24 h experiments, but also inhibited protein synthesis as assessed by leucine incorporation. The protein synthesis inhibition was reversible. Lower concentrations of MEHP (0.3-0.5 mM) did not decrease the 75Se-labeling in 24 h experiments and did not inhibit leucine incorporation. However, conditions that significantly induced peroxisomal proliferation also affected the 75Se-labeling. Thus in 72 h experiments, 0.05-0.25 mM MEHP increased the labeling of a 58 kd protein, decreased the labeling of a 23 kd protein (with the same mol. wt as GSH-Px), had no effect on a 20 kd protein and decreased the labeling of a 15 kd protein (as compared to MEHP-free control plates). The pattern of changes associated with peroxisomal proliferation mimicked that seen in livers from selenium-deficient animals, as reported by others. These data indicate that the bioavailability of selenium is decreased by DEHP. This effect may relate to a transient inhibition of protein synthesis, but also to the DEHP-induced peroxisomal proliferation.
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PMID:Selenium metabolism in isolated hepatocytes: inhibition of incorporation in proteins by mono(2-ethylhexyl)phthalate, a metabolite of the peroxisome proliferator di(2-ethylhexyl)phthalate. 198 84


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