scholarly journals Glutathione S-transferases in elasmobranch liver. Molecular heterogeneity, catalytic and binding properties, and purification

1981 ◽  
Vol 199 (3) ◽  
pp. 749-756 ◽  
Author(s):  
Yuichi Sugiyama ◽  
Tadataka Yamada ◽  
Neil Kaplowitz
Keyword(s):  
Enzyme Activity ◽  
Rose Bengal ◽  
Organic Anion ◽  
Gel Filtration ◽  
Anion Binding ◽  
Molecular Heterogeneity ◽  
Glutathione S Transferase ◽  
Binding Studies ◽  
Binding Properties ◽  
Glutathione S Transferases

In order to gain insight into the phylogeny and physiological significance of organic-anion-binding proteins in the liver, the hepatic glutathione S-transferases of rat and a typical elasmobranch, the thorny-back shark (Platyrhinoides triseriata), were compared with respect to both glutathione S-transferase activites and organic-anion-binding properties. On gel filtration (Sephadex G-75, Superfine grade) of rat cytosol, the elution volumes of enzyme activities with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates were identical (rat Y-fractions; Mr 45000). In contrast, two peaks of enzyme activity for 1-chloro-2,4-dinitrobenzene with elution volumes corresponding to Mr 52000 (PLAT Y1) and Mr 45000 (PLAT Y2) were detected on gel filtration of P. triseriata cytosol. Only fraction PLAT Y2 had enzyme activity with p-nitrobenzyl chloride. Enzyme kinetic studies showed that rat Y-fraction had higher affinities for both 1-chloro-2,4-dinitrobenzene and glutathione than PLAT Y1- and PLAT Y2-fractions. The two forms of P. triseriata glutathione S-transferases differed greatly in affinity for glutathione. At a glutathione concentration that we found to be physiological in P. triseriata, PLAT Y2 accounted for approx. 70% of the total glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene. Binding studies revealed that PLAT Y1 and PLAT Y2 fractions had much lower affinities for sulphobromophthalein and bilirubin than rat Y-fraction. In contrast, binding affinities of PLAT Y1 and PLAT Y2 for Rose Bengal and 1-anilino-8-naphthalenesulphonate were comparable with that of rat Y-fraction. Inhibitory kinetics suggested that sulphobromophthalein and Rose Bengal were non-competitive inhibitors of glutathione S-transferase activities when 1-chloro-2,4-dinitrobenzene was used as substrate for both PLAT Y1 and PLAT Y2. The major glutathione S-transferase from the PLAT Y2 fraction was purified 81-fold by sequential chromatography on Sephadex G-75, DEAE-Sephadex and hydroxyapatite, and consisted of two identical subunits with pI7.7. The highly enriched Y2-fraction retained high affinity binding of Rose Bengal and 1-anilino-8-naphthalenesulphonate.

scholarly journals Comparison of renal and hepatic glutathione S-transferases in the rat

1976 ◽  
Vol 158 (2) ◽  
pp. 243-248 ◽  
Author(s):  
N Kaplowitz ◽  
G Clifton ◽  
J Kuhlenkamp ◽  
J D Wallin
Keyword(s):  
Organic Anion ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Transferase Activity ◽  
Close Correspondence ◽  
Glutathione S Transferase ◽  
Competitive Inhibitors ◽  
Liver And Kidney ◽  
Glutathione S Transferases

Renal and hepatic GSH (reduced glutathione) S-transferase were compared with respect to substrate and inhibitory kinetics and hormonal influences in vivo. An example of each of five classes of substrates (aryl, aralkyl, epoxide, alkyl and alkene) was used. In the gel filtration of renal or hepatic cytosol, an identical elution volume was found for all the transferase activities. Close correspondence in Km values was found for aryl, epoxide- and alkyl-transferase activities, with only the aralkyl activity significantly lower in kidney. Probenecid and p-aminohippurate were competitive inhibitors of renal aryl-, aralkyl-, epoxide- and alkyl-transferase activities and inhibited renal alkene activity. Close correspondence in Ki values for inhibition by probenecid of these activities in kidney and liver was found. In addition, furosemide was a potent competitive inhibitor of renal alkyl-transferase activity. Hypophysectomy resulted in significant increases in aryl-, araklyl-, and expoxide-transferase activities in liver and kidney. The hypophysectomy-induced increases in renal aryl- and aralkyl-transferase activities (approx. 100%) were more than twofold greater than increases in hepatic activities (approx. 40%). Administration of thyroxine prevented the hypophysectomy-induced increase in aryltransferase activity in both kidney and liver. The renal GSH S-transferases, in view of similarities to the hepatic activities, may play a role as cytoplasmic organic-anion receptors, as previously proposed for the hepatic enzymes.

scholarly journals Identification of hepatic Z-protein in a marine elasmobranch, Platyrhinoides triseriata

1982 ◽  
Vol 203 (2) ◽  
pp. 377-381 ◽  
Author(s):  
Y Sugiyama ◽  
T Yamada ◽  
N Kaplowitz
Keyword(s):  
Oleic Acid ◽  
Protein Binding ◽  
Rose Bengal ◽  
Organic Anion ◽  
Gel Filtration ◽  
Binding Activity ◽  
Anion Binding ◽  
Lower Affinity ◽  
Binding Characteristics ◽  
Z Protein

Previous studies were unable to identify Z-protein in elasmobranch liver with bromosulphophthalein as ligand. By using 8-anilinonaphthalene-1-sulphonate and Rose Bengal as ligands, however, we demonstrated in hepatic cytosol from Platyrhinoides triseriata an organic-anion-binding protein with gel-filtration characteristics identical with those of rat Z-protein. By comparison with pooled rat Z-protein, Pl. triseriata Z-protein had slightly lower affinity for 8-anilinonaphthalene-1-sulphonate and Rose Bengal, greatly decreased binding affinity for bromosulphophthalein and no binding activity for oleic acid or squalene. The Pl. triseriata Z-protein binding site was less hydrophobic than that of rat Z-protein. This observation may explain the differences in binding characteristics between the Z-proteins of these species.

scholarly journals Glutathione S-transferases from the white-rot fungus, Phanerochaete chrysosporium

1997 ◽  
Vol 324 (1) ◽  
pp. 243-248 ◽  
Author(s):  
Caitriona A. DOWD ◽  
Catherine M. BUCKLEY ◽  
David SHEEHAN
Keyword(s):  
Molecular Mass ◽  
Phanerochaete Chrysosporium ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Preliminary Evidence ◽  
White Rot ◽  
White Rot Fungus ◽  
Glutathione S Transferase ◽  
Glutathione S Transferases ◽  
A Subunit

A glutathione S-transferase (GST) was purified to homogeneity from the white-rot fungus, Phanerochaete chrysosporium, by affinity chromatography on glutathione–agarose followed by Mono-Q ion-exchange FPLC. This protein immunoblotted with antisera to rat Theta class GST 5-5 and also showed N-terminal sequence similarity to the Theta class, including the presence of a conserved serine residue that has been specifically implicated in catalysis in this class [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133–2143] and other residues conserved in plant sequences. Catalytic activity was found to be highly labile in the purified protein, although preliminary evidence for activity (approx. 120 m-units/mg) with 1,2-epoxy-3-(p-nitrophenoxy)propane was obtained in some preparations. The enzyme seems to be a dimer with a subunit molecular mass of 25 kDa by SDS/PAGE. The native molecular masses estimated by non-denaturing electrophoresis and by Superose-12 gel filtration were 58 and 45 kDa respectively. A second protein purified in this study also gave low level of activity with 1,2-epoxy-3-(p-nitrophenoxy)propane and had a subunit molecular mass of 28 kDa (native size 62–63 kDa), but did not immunoblot with any GST class and seemed to be N-terminally blocked.

scholarly journals Partial purification of two lithocholic acid-binding proteins from rat liver 100000g supernatants

1977 ◽  
Vol 165 (3) ◽  
pp. 425-429 ◽  
Author(s):  
R C Strange ◽  
R Cramb ◽  
J D Hayes ◽  
I W Percy-Robb
Keyword(s):  
Ion Exchange ◽  
Binding Proteins ◽  
Lithocholic Acid ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Anion Binding ◽  
Partial Purification ◽  
Transferase Activity ◽  
Glutathione S Transferase

1. The partial purification of two lithocholic acid-binding proteins from liver 100 000g supernatants is described. 2. Gel-filtration, (NH4)2SO4 fractionation, Ca3(PO4)2 fractionation and ion-exchange chromatography were used. 3. Both proteins exhibited glutathione S-transferase activity; one may be the non-specific anion-binding protein ligandin. 4. Glutathione S-transferase activity of one of the binding proteins was inhibited by lithocholic acid.

scholarly journals Glutathione S-transferases of the bovine retina. Evidence that glutathione peroxidase activity is the result of glutathione S-transferase

1982 ◽  
Vol 205 (1) ◽  
pp. 213-217 ◽  
Author(s):  
R P Saneto ◽  
Y C Awasthi ◽  
S K Srivastava
Keyword(s):  
Glutathione Peroxidase ◽  
Peroxidase Activity ◽  
Structural Parameters ◽  
Gel Filtration ◽  
Glutathione Peroxidase Activity ◽  
Glutathione S Transferase ◽  
Ph Optimum ◽  
Bovine Retina ◽  
Apparent Homogeneity ◽  
Glutathione S Transferases

We have purified two isoenzymes of glutathione S-transferase from bovine retina to apparent homogeneity through a combination of gel-filtration chromatography, affinity chromatography and isoelectric focusing. The more anionic (pI = 6.34) and less anionic (pI = 6.87) isoenzymes were comparable with respect to kinetic and structural parameters. The Km for both substrates, reduced glutathione and 1-chloro-2,4-dinitrobenzene, bilirubin inhibition of glutathione conjugation to 1-chloro-2,4-dinitrobenzene, 1-chloro-2,4-dinitrobenzene inactivation of enzyme activity and molecular weight were similar. However, pH optimum and energy of activation were found to differ considerably. Retina was found to have no selenium-dependent glutathione peroxidase activity. The total glutathione peroxidase activity fractionated with the transferases in the gel-filtration range of mol.wt. 49000 and expressed activity with only organic hydroperoxides as substrate. Only the more anionic isoenzyme expressed both transferase and peroxidase activity.

Synthesis and anion binding properties of porphyrins and related compounds

2016 ◽  
Vol 20 (08n11) ◽  
pp. 950-965 ◽  
Author(s):  
Flávio Figueira ◽  
João M.M. Rodrigues ◽  
Andreia A.S. Farinha ◽  
José A.S. Cavaleiro ◽  
João P.C. Tomé
Keyword(s):  
Anion Recognition ◽  
Anion Binding ◽  
Anion Receptors ◽  
Binding Studies ◽  
Binding Properties ◽  
Related Research ◽  
Expanded Porphyrins ◽  
Recent Developments ◽  
Related Compounds

Over the last two decades the preparation of pyrrole-based receptors for anion recognition has attracted considerable attention. In this regard porphyrins, phthalocyanines and expanded porphyrins have been used as strong and selective receptors while the combination of those with different techniques and materials can boost their applicability in different applications as chemosensors and extracting systems. Improvements in the field, including the synthesis of this kind of compounds, can contribute to the development of efficient, cheap, and easy-to-prepare anion receptors. Extensive efforts have been made to improve the affinity and selectivity of these compounds and the continuous expansion of related research makes this chemistry even more promising. In this review, we summarize the most recent developments in anion binding studies while outlining the strategies that may be used to synthesize and functionalize these type of macrocycles.

scholarly journals The glutathione S-transferases in selenium and vitamin E deficiency

1985 ◽  
Vol 227 (3) ◽  
pp. 823-831 ◽  
Author(s):  
A Mehlert ◽  
A T Diplock
Keyword(s):  
Vitamin E ◽  
Time Course ◽  
Gel Filtration ◽  
Selenium Deficiency ◽  
Total Activity ◽  
Supernatant Fraction ◽  
Glutathione S Transferase ◽  
Vitamin E Deficiency ◽  
Glutathione S Transferases ◽  
Two Peaks

Preliminary experiments confirmed the work of others showing that the total glutathione peroxidase (GSH-px) activity of rat liver supernatant fraction may be resolved into two peaks of activity (peaks I and II) by gel filtration, and that peak I is the selenium-containing enzyme and peak II is another peroxidase indistinguishable from glutathione S-transferase (GST). In selenium and vitamin E deficiency, the total activity of the GSH-px became very low, and the total activity of GST with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate was enhanced. Study of the time course of these changes as deficiency progressed indicated that the stimulus for the rise in GST (CDNB) activity was the fall in GSH-px activity which preceded it. The peroxidase activity of GST was found to reside only in the GST AA, B and B2 forms of the enzyme, which were shown to be respectively a homodimer of the Yc subunit, a homodimer of the Ya subunit and a heterodimer of the YaYc subunit. As vitamin E and selenium deficiency progressed, the B2 and AA forms of the enzyme showed enhanced activity, which was interpreted as implying that the Yc subunit of the enzyme becomes enriched as a consequence of the withdrawal of selenium from the animal's diet. Densitometric measurements of the Yc and Ya subunits confirmed that the amount of the Yc subunit was nearly doubled in selenium deficiency, relative to the Ya subunit.

Binding of 2,3-diphosphoglycerate by spectrin and its effect on oxygen affinity of hemoglobin

1978 ◽  
Vol 234 (1) ◽  
pp. C36-C40 ◽  
Author(s):  
N. Shaklai ◽  
L. Benitez ◽  
H. M. Ranney
Keyword(s):  
Gel Filtration ◽  
Oxygen Affinity ◽  
Oxygen Binding ◽  
Structural Protein ◽  
Binding Studies ◽  
Binding Properties ◽  
Filtration Method ◽  
Reversible Binding ◽  
Rbc Membrane ◽  
Oxygen Affinity Of Hemoglobin

The relationships between spectrin, a structural protein of the red blood cell (RBC) membrane facing the cytoplasm, and hemoglobin were studied. The oxygen-binding properties of stripped hemoglobin were not altered by the presence of spectrin, but the interaction of hemoglobin with organic phosphates was reduced by the addition of spectrin. The presence of the enzyme glyceraldehyde 3-phosphate dehydrogenase (G3PD), another component of the RBC membrane used as a control, did not change the oxygen affinity of either stripped hemoglobin or of hemoglobin solutions containing phosphates. Binding studies using the gel filtration method at pH 7.3 indicated reversible binding of 2,3-diphosphoglycerate to spectrin. A unit of 220,000 daltons was calculated to have seven binding sites and a binding constant of 1.2 X 10(4) M-1. A mechanism is proposed in which spectrin may facilitate oxygen transport for hemoglobin molecules reaching the membrane.

Synthesis, cytotoxicity, antioxidant and antimicrobial activity of indole based novel small molecule

2020 ◽  
Vol 17 ◽  
Author(s):  
Aslıhan Kurt-Kızıldoğan ◽  
Çiğdem Otur ◽  
Can Yılmaz ◽  
Sevki Arslan ◽  
Dogukan Mutlu ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cytotoxic Effect ◽  
Antimicrobial Properties ◽  
Cancer Cell Lines ◽  
Coupling Reactions ◽  
Human Liver Cancer ◽  
Glutathione S Transferases ◽  
Antioxidant And Antimicrobial Activity

Background:: Indoles probably represent one of the most important heterocyclic structures that have been attracting the interest of many scientists in drug discovery. Methods:: Pd-catalyst Sonogashira coupling reactions, MTT Assay, Antioxidant capacity test, Antimicrobial test, GST enzyme activity test. Results and Discussion:: 1-ethyl-2-phenyl-3-(phenylethynyl)-1H-indole had antioxidant and antimicrobial properties. It displayed significant induction in glutathione S-transferases (GST) enzyme activity in human liver cancer cell lines (HepG2), but cytotoxic effect on all tested cancer cell lines could not be observed. Conclusion:: All of these results showed that 1-ethyl-2-phenyl-3-(phenylethynyl)-1H-indole had antioxidant and antimicrobial properties without cytotoxic effect, which could make it a promising active component.

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