scholarly journals Purification, molecular cloning and heterologous expression of a glutathione S-transferase from the Australian sheep blowfly (Lucilia cuprina)

1994 ◽  
Vol 299 (2) ◽  
pp. 425-430 ◽  
Author(s):  
P Board ◽  
R J Russell ◽  
R J Marano ◽  
J G Oakeshott
Keyword(s):  
Genomic Dna ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Lucilia Cuprina ◽  
Glutathione S Transferase ◽  
Significant Similarity ◽  
Glutathione S Transferases ◽  
Australian Sheep ◽  
Blowfly Pupae ◽  
Low Activity

Three glutathione S-transferases from Lucilia cuprina (Australian sheep blowfly) pupae were purified by affinity chromatography and anion-exchange chromatography. One isoenzyme was composed of M(r)-24,800 subunits, and two isoenzymes had subunits of M(r) 23,900. The M(r)-23,900 subunits showed immunological identity and were immunologically distinct from the M(r)-24,800 subunits. All three enzymes were active with the substrate 1-chloro-2,4-dinitrobenzene and had low activity with 1,2-dichloro-4-nitrobenzene. A cDNA clone encoding a M(r)-23,900 subunit (LuGST1) was isolated and sequenced. The sequence has close similarities (> 81%) to that of GSTs from the fruitfly Drosophila melanogaster and Musca domestica (housefly). The deduced amino acid sequence of the Lu GST1 subunit showed no significant similarity to that of the mammalian GSTs to the Alpha, Mu and Pi classes, but shows some similarity (33%) over the first 100 residues with the rat subunit 12 Theta-class GST. Southern blots of genomic DNA hybridized with the LuGST1 cDNA identified many hybridizing fragments. Taken together, these data indicated that the L. cuprina genome contains multiple glutathione S-transferase genes.

scholarly journals Variation in the expression of Mu-class glutathione S-transferase isoenzymes from human skeletal muscle. Evidence for the existence of heterodimers

1991 ◽  
Vol 273 (2) ◽  
pp. 323-332 ◽  
Author(s):  
A J Hussey ◽  
L A Kerr ◽  
A D Cronshaw ◽  
D J Harrison ◽  
J D Hayes
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
High Activity ◽  
Human Skeletal Muscle ◽  
Neutral Type ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glutathione S Transferase ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases

The cytosolic glutathione S-transferases (GST) from human skeletal muscle were purified by a combination of affinity chromatography and anion-exchange chromatography followed by either chromatofocusing or hydroxyapatite chromatography. Pi-class and Mu-class GST, but not Alpha-class GST, were isolated from muscle. In addition to a Pi-class GST subunit, which exists as a homodimer, this tissue also contains a total of three distinct neutral-type Mu-class GST subunits, which hybridize to form homodimers or heterodimers. The neutral-type subunits are referred to as N1-N3 and are defined by the decreasing isoelectric points of the homodimers; GST N1N1, N2N2 and N3N3 have estimated pI values of 6.1, 5.3 and less than 5.0 respectively. SDS/PAGE showed that N1, N2 and N3 have Mr values of 26,700, 26,000 and 26,300 respectively. The N1, N2 and N3 subunits are catalytically distinct, with N1 possessing a high activity for trans-4-phenylbut-3-en-2-one and N2 having high activity with 1,2-dichloro-4-nitrobenzene. In skeletal muscle the expression of the N1 subunit, but not of N2 and N3 subunits, was found to differ from specimen to specimen. The N1 subunit was absent from about 50% of samples examined, and the purification results from two different specimens are presented to illustrate this inter-individual variation. Skeletal muscle from one individual (M1), which did not express N1, contained only GST N2N2, N2N3 and pi, whereas the second sample examined (M2) contained GST N1N2, N2N2 and N2N3 as well as GST pi. N-Terminal amino acid sequence analysis supported the electrophoretic evidence that the N2 subunit in GST N1N2, N2N2 and N2N3 represents the same polypeptide. The peptides obtained from CNBr digests of N2 were subjected separately to automated amino acid sequencing, and the results indicate that N2 is distinct but closely related to the protein encoded by the human Mu-class cDNA clone GTH4 [DeJong, Chang, Whang-Peng, Knutsen & Tu (1988) Nucleic Acids Res. 16, 8541-8554]. GST N2N2 is probably identical with GST 4 [Board, Suzuki & Shaw (1988) Biochim. Biophys. Acta 953, 214-217], as over the 24 N-terminal residues of GST 4 there is complete identity between the two enzymes. Our data suggest that the GST 1 and GST 4 loci are part of the same multi-gene family.

scholarly journals Tissue distribution of enzymic methylation of glutathione S-transferase and its effects on catalytic activity. Methylation of glutathione S-transferase 11-11 inhibits conjugating activity towards 1-chloro-2,4-dinitrobenzene

1992 ◽  
Vol 282 (1) ◽  
pp. 279-289 ◽  
Author(s):  
J A Johnson ◽  
K A Finn ◽  
F L Siegel
Keyword(s):  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Multiple Forms ◽  
Glutathione S Transferase ◽  
Reverse Phase ◽  
Methyl Group ◽  
Catalysed Reaction ◽  
Liver And Kidney ◽  
Glutathione S Transferases

Glutathione S-transferases (GSTs) were isolated from rat liver, lung, heart, kidney, testis and brain by coupled affinity chromatography and subunits were resolved by reverse-phase h.p.l.c. The reverse-phase h.p.l.c. technique was improved from our previously published work [Johnson, Neal, Collins & Siegel (1990) Biochem. J. 270, 483-489] by changing from a C4 to a C18 wide-pore reverse-phase column; this resulted in baseline or near-baseline resolution of all GST subunits. There were significant tissue-dependent differences in the expression of GST subunits and the level of GST subunits present was quantitatively determined for each of the tissues. The extent of methylation of GSTs in vitro and distribution of GST methyltransferase (GST-MT) was determined in cytosolic fractions from each of these tissues. Purified GST isoenzymes were methylated with partially purified liver GST-MT. Methylation of Mu class subunits 3 and 4, the preferred substrates of methylation in liver, was substoichiometric in all tissues. The extent of methylation of subunit 3 ranged from 0.13% to 0.94% and subunit 4 from 0.03% to 0.60%. Methylation of Alpha class subunits was either not detectable or 5-10-fold less than that of Mu class subunits 3 and 4. Pi class subunit 7 was methylated to a greater extent than the Alpha class subunits but less than Mu class isoenzymes. A notable exception to this low level of methylation was GST 11-11, found mainly in testis and brain. Methylation of subunit 11 reached 21.9% (219 pmol of methyl group/nmol of subunit 11) when this isoenzyme was incubated with partially purified liver GST methyltransferase. Methylation of GST 11-11 was found to inhibit the conjugating activity of this isoenzyme towards 1-chloro-2,4-dinitrobenzene; the degree of inhibition of conjugating activity correlated with the extent of methylation of GST 11-11. GST-MT activity toward GST subunits 3, 4 and 11 was present in kidney and liver, detectable in lung and heart, but absent from brain and testis. Anion-exchange chromatography of GST-MTs from liver and kidney suggested the presence of four different forms of GST-MT (I-IV) and indicated that GST-MT isoenzymes III and IV were present at significantly lower concentrations in kidney than liver. The present paper shows that methylation is an enzyme-catalysed reaction that differs in substrate-specificity with respect to different GST isoenzymes, that expression of GST-MT is tissue-dependent and multiple forms of the enzyme are present in liver and kidney, and that methylation inhibits GST activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of glutathione S-transferase genes polymorphisms and the risk of schizophrenia in a sample of Iranian population

2011 ◽  
Vol 7 (2-4) ◽  
pp. 199-203 ◽  
Author(s):  
Farah Lotfi Kashani ◽  
Dor Mohammad Kordi-Tamandani ◽  
Roya Sahranavard ◽  
Mohammad Hashemi ◽  
Farzaneh Kordi-Tamandani ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Case Control Study ◽  
Gene Interaction ◽  
Case Control ◽  
Glutathione S Transferase ◽  
Chain Reaction ◽  
Healthy Control ◽  
Glutathione S Transferases ◽  
Polymerase Chain ◽  
Control Study

Glutathione S-transferases (GSTs) are major intracellular antioxidants, which, impaired in their function, are involved in the progress of schizophrenia (SCZ). The aim of this case-control study was to investigate the association between the polymorphism of glutathione S-transferases M1 (GSTM1), T1 (GSTT1), the glutathione S-transferase P1 gene (GSTP1) and SCZ. We isolated genomic DNA from peripheral blood of 93 individuals with SCZ and 99 healthy control subjects' genotypes analyzing them for GSTM1, GSTT1 and GSTP1 using polymerase chain reaction. The analysis of the gene–gene interaction between GSTs indicated that the magnitude of the association was greater for the combined AG/GSTT1 & GSTM1 genotypes (OR = 2.51; 95% CI: 1.13–5.63, P = 0.02). The AG and combined AG + GG genotypes of GSTP1 increased the risk of SCZ (OR = 1.83; 95% CI: 0.94–3.75 and OR = 1.71; 95% CI: 0.92–3.19, respectively). The genotypes of GSTT/NULL, NULL/GSTM and NULL/NULL increased the risk of SCZ (OR = 2.05; 95% CI: 0.9–4.74; OR = 2.0; 95% CI: 1.68–2.31; and OR = 1.8; 95% CI: 0.57–2.46, respectively). The present study supports previous data that suggest that impairment in the function of GSTs genes may increase the risk of SCZ.

scholarly journals Glutathione S-transferases of the yeast Yarrowia lipolytica have unusually large molecular mass

1998 ◽  
Vol 333 (3) ◽  
pp. 839-845 ◽  
Author(s):  
Vivienne FOLEY ◽  
David SHEEHAN
Keyword(s):  
Molecular Mass ◽  
Yarrowia Lipolytica ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Post Translational Modification ◽  
Native Molecular Mass ◽  
Sds Page ◽  
Glutathione S Transferases ◽  
Yeast Yarrowia Lipolytica

Two similar glutathione S-transferases (GSTs), which do not bind to glutathione– or S-hexylglutathione–agarose affinity resins, have been purified from the yeast Yarrowia lipolytica. An approx. 400-fold purification was obtained by a combination of DEAE-Sephadex, phenyl-Sepharose, hydroxyapatite and Mono-Q anion-exchange chromatography. The native molecular mass of both proteins was estimated as approx. 110 kDa by both Superose-12 gel-filtration chromatography and non-denaturing electrophoresis. SDS/PAGE indicated a subunit mass of 50 kDa. Reverse-phase HPLC of purified proteins gave a single, well-resolved, peak, suggesting that the proteins are homodimers. Identical behaviour on HPLC, native electrophoresis and SDS/PAGE, N-terminal sequencing, sensitivity to a panel of inhibitors and identical specific activities with 1-chloro-2,4-dinitrobenzene as substrate suggest that the two isoenzymes are very similar. The enzymes do not immunoblot with antisera to any of the main GST classes, and N-terminal sequencing suggests no clear relationship with previously characterized enzymes, such as that of the fungus, Phanerochaete chrysosporium [Dowd, Buckley and Sheehan (1997) Biochem. J. 324, 243–248]. It is possible that the two isoenzymes arise as a result of post-translational modification of a single GST isoenzyme.

Separation of Heparin into Subtractions by DEAE-Cellulose Chromatography

1979 ◽  
Vol 42 (05) ◽  
pp. 1452-1459 ◽  
Author(s):  
Robert H Yue ◽  
Toby Starr ◽  
Menard M Gertler
Keyword(s):  
High Activity ◽  
Uronic Acid ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Net Charge ◽  
Uronic Acid Content ◽  
Successful Separation ◽  
Salt Gradient ◽  
Structure And Activity ◽  
Low Activity

SummaryCommercial porcine heparin can be separated into three distinct subtractions by using DEAE-cellulose chromatography and a stepped salt gradient. Gram quantities of heparin can be fractionated by this technique. All three heparin subtractions can accelerate the inhibition of thrombin by antithrombin III with different efficiency. The specific activities of the high activity heparin, intermediate activity heparin and low activity heparin are 228 units/mg, 142 units/mg and 95 units/mg, respectively. Both the uronic acid content and the quantity of N-SO4 for all three heparin subfractions have been evaluated. The high activity heparin has the lowest uronic acid and N-SO4 content. The successful separation of commercial heparin into three distinct subfractions by means of ion-exchange chromatography suggests that the net charge on these three heparin components will serve as a model system in the elucidation of the structure and activity relationship to the biological function of heparin.

Extracellular polysaccharides from suspension cultures of Nicotiana plumbaginifolia

2020 ◽  
Author(s):  
Ian Sims ◽  
A Bacic
Keyword(s):  
Uronic Acid ◽  
Cell Suspension Cultures ◽  
Anion Exchange Chromatography ◽  
Nicotiana Plumbaginifolia ◽  
Suspension Cultures ◽  
Exchange Chromatography ◽  
Arabinogalactan Protein ◽  
Extracellular Polysaccharides ◽  
Selective Precipitation ◽  
The Individual

The soluble polymers secreted by cell-suspension cultures of Nicotiana plumbaginifolia contained 78% carbohydrate, 6% protein and 4% inorganic material. The extracellular polysaccharides were separated into three fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7 and the individual polysaccharides in each fraction were then isolated by selective precipitation and enzymic treatment. Monosaccharide and linkage compositions were determined for each polysaccharide after reduction of uronic acid residues and the degree of esterification of the various uronic acid residues in each polysaccharide was determined concurrently with the linkage types. Six components were identified: an arabinoxyloglucan (comprising 34% of the total polysaccharide) and a galactoglucomannan (15%) in the unbound neutral fraction, a type II arabinogalactan (an arabinogalactan-protein, 11%) and an acidic xylan (3%) in the first bound fraction, and an arabinoglucuronomannan (11%) and a galacturonan (26%) in the second bound fraction. © 1995.

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