scholarly journals Anomalous electrophoretic behaviour of the glutathione S-transferase Ya and Yk subunits isolated from man and rodents. A potential pitfall for nomenclature

1986 ◽  
Vol 237 (3) ◽  
pp. 731-740 ◽  
Author(s):  
J D Hayes ◽  
T J Mantle
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Cross Linking ◽  
Variable Cross ◽  
Glutathione S Transferase ◽  
Relative Electrophoretic Mobility ◽  
Low Concentrations ◽  
Cross Linker ◽  
Low Degrees

GSH S-transferases are dimeric enzymes. The subunits in the rat are resolved into six types, designated Yf, Yk, Ya, Yn, Yb and Yc, by discontinuous SDS/polyacrylamide-gel electrophoresis [Hayes (1986) Biochem. J. 233, 789-798]. The relative electrophoretic mobility of the Ya and Yk subunits is dependent on the amount of cross-linker (NN'-methylenebisacrylamide) in the resolving gel. At low degrees of cross-linking, CBis 0.6% (w/w), the Yk and Ya subunits possess a faster anodal mobility than do the Yf, Yn, Yb and Yc subunits (i.e. order of mobility Yk greater than Ya greater than Yf greater than Yn greater than Yb greater than Yc), whereas at higher degrees of cross-linking, CBis 5.0% (w/w), Yf subunits possess the fastest mobility (i.e. order of mobility Yf greater than Yk greater than or equal to Yn greater than Yb greater than or equal to Ya greater than Yc). Resolving gels that contain low concentrations of cross-linker [CBis 0.6% (w/w)] allow the resolution of a hitherto unrecognized polypeptide that is isolated by S-hexyl-GSH-Sepharose affinity chromatography. This new polypeptide, which we have designated Yb, is normally obscured by the main Yb band in resolving gels that comprise concentrations of cross-linker of at least CBis 1.6% (w/w). The Ya- and Yb-type subunits in guinea pig, mouse, hamster and man were identified by immuno-blotting and their apparent Mr values in different electrophoresis systems were determined. The Ya subunits in all species studied possess a variable cross-linker-dependent mobility during electrophoresis. Since the transferase subunits are currently classified according to their mobilities during SDS/polyacrylamide-gel electrophoresis, it is apparent that the variable electrophoretic behaviour of the Ya and Yk subunits may lead to the mis-identification of enzymes.

scholarly journals Purification and characterization of a new cytosolic glutathione S-transferase (glutathione S-transferase X) from rat liver

1983 ◽  
Vol 215 (3) ◽  
pp. 617-625 ◽  
Author(s):  
T Friedberg ◽  
U Milbert ◽  
P Bentley ◽  
T M Guenther ◽  
F Oesch
Keyword(s):  
Rat Liver ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Glutathione S Transferase ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases

A hitherto unknown cytosolic glutathione S-transferase from rat liver was discovered and a method developed for its purification to apparent homogeneity. This enzyme had several properties that distinguished it from other glutathione S-transferases, and it was named glutathione S-transferase X. The purification procedure involved DEAE-cellulose chromatography, (NH4)2SO4 precipitation, affinity chromatography on Sepharose 4B to which glutathione was coupled and CM-cellulose chromatography, and allowed the isolation of glutathione S-transferases X, A, B and C in relatively large quantities suitable for the investigation of the toxicological role of these enzymes. Like glutathione S-transferase M, but unlike glutathione S-transferases AA, A, B, C, D and E, glutathione S-transferase X was retained on DEAE-cellulose. The end product, which was purified from rat liver 20 000 g supernatant about 50-fold, as determined with 1-chloro-2,4-dinitrobenzene as substrate and about 90-fold with the 1,2-dichloro-4-nitrobenzene as substrate, was judged to be homogeneous by several criteria, including sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, isoelectric focusing and immunoelectrophoresis. Results from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration indicated that transferase X was a dimer with Mr about 45 000 composed of subunits with Mr 23 500. The isoelectric point of glutathione S-transferase X was 6.9, which is different from those of most of the other glutathione S-transferases (AA, A, B and C). The amino acid composition of transferase X was similar to that of transferase C. Immunoelectrophoresis of glutathione S-transferases A, C and X and precipitation of various combinations of these antigens by antisera raised against glutathione S-transferase X or C revealed that the glutathione S-transferases A, C and X have different electrophoretic mobilities, and indicated that transferase X is immunologically similar to transferase C, less similar to transferase A and not cross-reactive to transferases B and E. In contrast with transferases B and AA, glutathione S-transferase X did not bind cholic acid, which, together with the determination of the Mr, shows that it does not possess subunits Ya or Yc. Glutathione S-transferase X did not catalyse the reaction of menaphthyl sulphate with glutathione, and was in this respect dissimilar to glutathione S-transferase M; however, it conjugated 1,2-dichloro-4-nitrobenzene very rapidly, in contrast with transferases AA, B, D and E, which were nearly inactive towards that substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Identification of a basic hybrid glutathione S-transferase from human liver. Glutathione S-transferase δ is composed of two distinct subunits (B1 and B2)

1985 ◽  
Vol 227 (2) ◽  
pp. 457-465 ◽  
Author(s):  
P K Stockman ◽  
G J Beckett ◽  
J D Hayes
Keyword(s):  
Isoelectric Point ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Glutathione S Transferase ◽  
Liver Glutathione ◽  
Glutathione S Transferases

The purification of a hybrid glutathione S-transferase (B1 B2) from human liver is described. This enzyme has an isoelectric point of 8.75 and the B1 and B2 subunits are distinguishable immunologically and are ionically distinct. Hybridization experiments demonstrated that B1 B1 and B2 B2 could be resolved by CM-cellulose chromatography and have pI values of 8.9 and 8.4 respectively. Transferase B1 B2, and the two homodimers from which it is formed, are electrophoretically and immunochemically distinct from the neutral enzyme (transferase mu) and two acidic enzymes (transferases rho and lambda). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that B1 and B2 both have an Mr of 26 000, whereas, in contrast, transferase mu comprises subunits of Mr 27 000 and transferases rho and lambda both comprise subunits of Mr 24 500. Antisera raised against B1 or B2 monomers did not cross-react with the neutral or acidic glutathione S-transferases. The identity of transferase B1 B2 with glutathione S-transferase delta prepared by the method of Kamisaka, Habig, Ketley, Arias & Jakoby [(1975) Eur. J. Biochem. 60, 153-161] has been demonstrated, as well as its relationship to other previously described transferases.

EFFECT OF DIBUTYRYL CYCLIC AMP ON THE RELEASE OF MELANOCYTE-STIMULATING HORMONE FROM RAT NEUROINTERMEDIATE LOBES IN VITRO

1974 ◽  
Vol 63 (3) ◽  
pp. 533-538 ◽  
Author(s):  
BRIDGET I. BAKER
Keyword(s):  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Incubation Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Dibutyryl Cyclic Amp ◽  
Melanocyte Stimulating Hormone ◽  
Low Concentrations ◽  
Stimulating Hormone

SUMMARY A method for measuring melanocyte-stimulating hormone (MSH) in rat neurointermediate lobe in vitro and in incubation medium, using polyacrylamide gel electrophoresis, is described. Using this technique, it was shown that dibutyryl cyclic AMP increased the release of MSH in vitro, the degree of stimulation depending on the concentration of the nucleotide. The effect of low concentrations of the nucleotide was potentiated by theophylline.

scholarly journals Variant specific antigens of Trypanosoma brucei exist in solution as glycoprotein dimers

1981 ◽  
Vol 193 (2) ◽  
pp. 647-650 ◽  
Author(s):  
C A Auffret ◽  
M J Turner
Keyword(s):  
Trypanosoma Brucei ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Cross Linking ◽  
Specific Antigens

Purified variant specific antigens of Trypanosoma brucei were shown to exist in solution as dimers, and occasionally as higher oligomers, as judged by gel filtration and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after treatment with bifunctional cross-linking reagents.

Platelets Cross-Linked with Dimethyl Adipimidate Hydrochloride (DMA) Used in Ristocetin Assay For F VIII: Rcof.

1979 ◽  
Author(s):  
M. Qazzaz ◽  
B. Podolsak ◽  
E. Wenzel
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Test System ◽  
Final Concentration ◽  
Polyacrylamide Gel ◽  
Cross Linking ◽  
Membrane Glycoproteins ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Platelet Membranes

When PRP or washed platelets are incubated with dimethyl adipimidate (DMA)(final concentration 2 mg/ml, 30 - 60 min.), the aggregation of the platelets by ADP, collagen or thrombin is clearly reduced. Aggregation by ristocetin plus F VIII: Rcof is however not affected.DMA is a bifunctional imidoester which acts as a cross-linking reagent. SDS Polyacrylamide gel electrophoresis of platelet membranes shows a different pattern for membrane glycoproteins from the platelet treated with DMA when compared to untreated platelets. This different pattern is due to cross-linking of the glycoproteins.The DMA - treated platelets can be stored for at least three weeks at 4° without losing their ability to aggregate in the ristocetin F VIII: Rcof test system. Both freshly -treated and stored DMA - treated platelets could be used to assay F VIII: Rcof reproducible in normal pooled plasma and in defective plasma (von Willebrand’s disease patients; N = 8). The preparation of DMA-fixed platelets takes less than two hours.

Cross-linking of Hemoglobin, Haptoglobin, and Hemoglobin–Haptoglobin Complex with Bifunctional Imidoesters

1975 ◽  
Vol 53 (8) ◽  
pp. 861-867 ◽  
Author(s):  
W. L. Lockhart ◽  
David B. Smith
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Spatial Relation ◽  
Polyacrylamide Gel ◽  
Cross Linking ◽  
Cross Link ◽  
Complex Cross ◽  
Cross Links

Dimethyl adipimidate was used to cross-link the polypeptides within hemoglobin, haptoglobin, and hemoglobin–haptoglobin complex. Cross-linked hemoglobin retained considerable ability to bind haptoglobin, although the amounts bound were reduced and the haptoglobin reaction could be used to fractionate the modified hemoglobin. With cross-links limited to intramolecular sites, hemoglobin showed four bands on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, identified, with reference to the subunit polypeptides, as monomer, dimer, trimer, and tetramer. The dimer region consisted of at least two separable species. When hemoglobin–haptoglobin complex was cross-linked, a band of hemoglobin dimer was present, which demonstrates that at least two hemoglobin subunits have a close spatial relation when bound to haptoglobin.Some comparisons with adipimidate-reacted hemoglobin were made using malonimidate and suberimidate and some marked differences were noted.

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