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Glutathione S-Transferases and the Disposition of Organic Anions in Rat Liver

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Date

1977

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Te Herenga Waka—Victoria University of Wellington

Abstract

1. The role of hepatic anion-binding proteins, in particular that of ligandin, in the biliary excretion of model compounds in the rat was investigated. Seven model compounds were used - bromothymol blue, bromocresol green, bromosulfophthalein, amaranth, succinylsulphathiazole, tartrazine, and succinylsulphanilamide, These compounds cover a wide range in the extent of biliary excretion in rats after intravenous dosing and they are also non-metabolizable except bromosulfophthalein. The affinity of these compounds for the purified hepatic proteins and albumin was quantitated by equilibrium dialysis. 2. Rat serum albumin has been purified by published methods, and an affinity chromatography system has been devised. It was found that CNBr-activated Sepharose to which had been attached glutathione-bromosulfophthalein conjugate offered a simple and rapid method for the isolation of albumin from rat serum. This affinity preparation bound 7.5 mg of albumin per ml of Sepharose. Elution of the albumin was effected with buffered 2M NaCl solution. The albumin eluted by salt solution was electrophoretically pure as shown by disc gel electrophoresis 3. Three dye binding proteins from rat liver have been isolated by a simple ion-exchange method. The use of affinity chromatography in their isolation has been investigated. It was found that this affinity chromatographic gel retained three proteins which were catalytically active and had bromosulfophthalein-binding activity. Other inactive proteins from rat liver 100,000 g supernatant were also adsorbed. Elution of bound proteins could be effected with glutathione, bromosulfophthalein or salt. 4. Three hepatic proteins which have high affinity for bromosulfophthalein were purified from rat liver 100,000 g supernatant by a three-step purification scheme using conventional ion-exchange chromatography. These proteins were designated as PI, PII and PIII on the basis of the order of elution from cellulose phosphate, the purification step in which these proteins were separated from each other. Evidence is presented that each of the purified proteins was homogeneous with respect to gel and column electrofocusing (pH 3 to 10). 5. The isoelectric points of PI, PII and PIII were 9.0, 8.3 and 9.0 respectively as determined by gel electrofocusing. An isoelectric point of 9.60 for PIII was obtained by column electrofocusing. Using Sephadex chromatography, it was found that PII has a molecular weight of 54,950 and that each of the other two proteins was about 40,000. PII was enzymatically inactive whereas PI and PIII were catalytically active to differing degrees in the conjugation of glutathione with the substrates tested. It was found that the iodomethane-conjugating activity of PIII was about 500 times greater than that of PI. 6. It was found that enzyme from PIII was functionally identical to glutathione S-transferase B. The enzyme from PI was functionally similar to glutathione S-transferase C. 7. With protein from PII, it was found that the binding constant for bromosulfophthalein (1.45 x 106 M¯) was the highest among the ligands studied. However, the association constant of this dye for albumin was about three times greater than that of PII. The affinity constants of the other compounds tested for PII was of the order of 5- to 20-fold less than those for albumin except in the case of amaranth. Similarly, PI was found. to be a relatively poor binder for all compounds except bromocresol green when compared to rat serum albumin. The binding constants of bromothymol blue and bromosulfophthalein for PI were respectively 10- and 20-fold less than those of albumin. These results suggested that the transfer of these compounds from the plasma to the liver could not be facilitated by the competitive binding for such ligands between PI or PII and albumin. 8. PIII had the highest affinity for the various ligands of the hepatic proteins. This protein (PIII) displayed binding affinities for all ligands except tartrazine comparable with the albumin-ligand system. It was, however, found that the number of ligand binding sites on albumin was greater than those on PIII for all compounds. At equimolar protein concentrations, the amount of any of the model compounds bound to PIII was found to be 50% or less than the amount that bound to rat serum albumin. 9. It was found that there was little correlation between the binding of the current model compounds by any of the purified proteins and the extent of biliary excretion or of hepatic uptake of such compounds in the rat.

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Keywords

Anions, Glutathione Transferase, Liver, Protein binding, Rats

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