Affinity chromatography of hepatic glutathione S-transferases on ω-aminoalkyl sepharose derivatives of glutathione

Inoue, Masayasu; Hara, Michiaki; Nagashima, Fujio; Matsui, Sunao; Mitsuyasu, Nobuo; Morino, Yoshimasa

doi:10.1016/0005-2744(81)90062-0

Cited by 19 publications

(12 citation statements)

References 14 publications

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“…Purification of ligandins from rat liver cytosolic fractions was performed by using lysyl-glutathione affinity chromatography as described in [17]. The specific activity of purified ligandins was 12.3 unit/mg protein.…”

Section: Purification Of Microsomal Membrane Vesicles and Ligandinsmentioning

confidence: 99%

Topological aspects of microsomal N‐acetyltransferase, an enzyme responsible for the acetylation of cysteine S‐conjugates of xenobiotics

Okajima

Inoue

Morino

et al. 1984

European Journal of Biochemistry

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Acetylation of cysteine S-conjugates of xenobiotics by microsomal N-acetyltransferase is the final step of detoxicative metabolism leading to mercapturic acid biosynthesis. To elucidate the subcellular site of N-acetylation and the effective mechanism by which the final metabolites are eliminated from the organisms, topological aspects and catalytic properties of microsomal N-acetyltransferase and mercapturic acid biosynthesis in vivo were investigated. Intravenous administration of radioactive S-benzyl-L-cysteine, a model compound of cysteine Sconjugates, resulted in rapid acetylation of the conjugate in liver and kidney to a similar extent. The acetylation was followed by a rapid excretion of the metabolite, a mercapturic acid, into the urine; about 60 % of the injected dose appeared in urine within 60 min of administration.Limited proteolysis of microsomal vesicles obtained from liver and kidney by chymotrypsin or trypsin inactivated the transferase by 49 -62 % and 62 -73 %, respectively. Proteolytic inactivation of the transferase was not significantly affected by the presence of 0.04 sodium deoxycholate by which the vesicles became permeable to macromolecules due to its detergent action.To determine the sidedness of the active site of N-acetyltransferase on the microsomal membranes, two Sacetyldextran polymer derivatives ( M , 500000) of cysteine and N-acetylcysteine which represent an nonpermeant substrate and product for this enzyme, respectively, were examined for their effects on the vesicle-associated enzyme activity. Both derivatives ihhibited the transferase activity in a dose-dependent fashion ; maximum inhibition of the enzyme activity was 40 by the latter. Sulfobromophthalein strongly inhibited the enzyme activity and this inhibition was completely reversed by adding an equimolar amount of hepatic glutathione S-transferases (ligandins). In contrast to the strong inhibition by sulfobromophthalein itself, its glutathione S-conjugate did not inhibit the enzyme activity.These results indicate that the active site and the protease-sensitive domain(s) of the microsomal Nacetyltransferase are localized on the outer surface (cytoplasmic side) of endoplasmic reticulum and that the ligandin(s) might protect membranous N-acetyltransferase from inhibition by organic anions by binding them and catalyzing the conjugation with glutathione.

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Section: Purification Of Microsomal Membrane Vesicles and Ligandinsmentioning

confidence: 99%

Topological aspects of microsomal N‐acetyltransferase, an enzyme responsible for the acetylation of cysteine S‐conjugates of xenobiotics

Okajima

Inoue

Morino

et al. 1984

European Journal of Biochemistry

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“…The purification procedures for GSHtransferases have been described basically by others [11,15]. Affinity chromatography on S -glutathione -carbamidomethyl-e-aminolysyl-Sepharose CL 4B has been used to isolate prepurified GSH-transferase from rat liver [11], We chose to apply the affinity chroma tography to the crude tissue homogenates, followed by a gel filtration chromatography on Sephadex G-75.…”

Section: Resultsmentioning

confidence: 99%

“…Tissue was homogenized in 10 mM Tris-HCl, pH 7.5. Affinity chromatography of the homogenate was carried out according to the proce dure of Inoue et al [11]. The GSH-transferase-containing fraction was further purified by chromatogra phy on a Sephadex G-75 column (3 X 121 cm) in 100 mM Tris-HCl, pH 7.4.…”

Section: Enzyme Purificationmentioning

confidence: 99%

Acidic and Basic Forms of Glutathione S-Transferases from Human Placenta and Comparison with Human Kidney Glutathione S-Transferase

Steisslinger

Pfleiderer²

1988

Enzyme

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Glutathione S-transferase (GSH-transferase) was purified from human placenta and kidney by affinity chromatography on S-glutathione-carbamidomethyl-e-aminolysyl- Sepharose CL 4B and gel filtration chromatography on Sephadex G-75. Electrophoretically pure enzyme with the specific activities of 50.7 and 55.9 U/mg, respectively, were obtained. In addition to the known acidic isoenzyme from human placenta (isoelectric point, pi, 4.5), we describe here for the first time the presence of 6 basic forms with pi values between 8.0 and 9.0. The kidney GSH-transferase contained 2 acidic forms with isoelectric points at 4.6 and 4.65, and 6 basic forms with pi values between 8.7 and 9.4. The basic and acidic isoenzymes from placenta were separated by ion exchange chromatography on Sephadex DEAE A-25. The acidic form accounted for 36% of the total GSH-transferase activity from placenta. Antibodies against the kidney enzyme were raised in rabbit. Total cross-reactivity of placental GSH-transferase with antikidney-GSH-transferase antibodies was obtained, suggesting that the kidney and placental enzymes are immunologically closely related.

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“…The precipitates were dissolved in water and dialyzed against water for 16 hr at 4°C. Dialyzed samples were centrifuged at 10,000 X g for 20 min to eliminate insoluble materials and used for isoelectric focusing (pH range of 3.0-10.0) (10).…”

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confidence: 99%

“…GSH S-transferase (fraction Y) is responsible for binding of organic The ammonium sulfate fractions of liver cytosol obtained from the control rats (A) or NAR (B) were subjected to isoelectric focusing at 40C in the pHrange 3.0-10.0 as described in ref. 10. Each sample contained8 units of GSH S-transferase activity as determined with 1 mM GSH and 2,4-dinitrochlorobenzene.…”

mentioning

confidence: 99%

Plasma clearance of sulfobromophthalein and its interaction with hepatic binding proteins in normal and analbuminemic rats: is plasma albumin essential for vectorial transport of organic anions in the liver?

Inoue¹,

Okajima²,

Nagase³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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To investigate a possible function of plasma albumin in the vectorial transportof organic anions bythe liver, the plasma disappearance of sulfobromophthalein (BSP) and its interaction with plasma and liver cytosolic proteins were studied in normal rats and mutant Nagase analbuminemic rats (NAR). After intravenous administration of BSP, plasma BSP decreased rapidly in both NAR and control animals: plasma clearance values of BSP in NAR and controls were 12.45 and 7.40 ml/min per kg, respectively. Gel exclusion Sephadex G-100 chromatography of BSP with control rat serum revealed a protein peak in the void volume and another in the albumin fraction. BSP chromatographed exclusively with the albumin fraction; binding of BSP to plasma albumin occurred stoichiometrically. Similar studies with NAR serum revealed a single protein peak, in the void volume; a small amount of BSP chromatographed with this protein peak. The amount of BSP that chromatographed with NAR serum protein(s) was 8% of that with control rat serum albumin. Sephadex G-100 chromatography of BSP with control rat liver cytosol revealed four peaks of protein-bound BSP in fractions corresponding to the void volume (fraction X), albumin, glutathione S-transferases (fraction Y, Mr 45,000), and fraction Z (Mr 12,000); fraction Y was the major component of BSP binding. Gel chromatography of NAR liver cytosol with BSP revealed three BSP peaks, fractions X, Y, and Z; fraction X was the major component of BSP binding. Total BSP binding by 30 mg of hepatic cytosolic proteins was 4.5 nmol for controls and 10.4 nmol for NAR. Isoelectric focusing of liver cytosol revealed no quantitative or qualitative differences in glutathione S-transferase isozymes between control and mutant animals. Intravenously administered BSP (5 ,cmol/kg) rapidly appeared in bile as the free form and the glutathione conjugate in normal rats and NAR; 41% and 57% of injected BSP was excreted within 60 min in NAR and control rat bile, respectively. These results indicate that binding of BSP to plasma albumin is not indispensable to transhepatocyte transport of BSP in vivo.Many hydrophobic anions, such as bilirubin, bile acids, and xenobiotics, are efficiently removed from plasma during passage through the liver. Their uptake into hepatocytes involves carrier-mediated mechanisms (1). Since most of these ligands tightly bind to plasma albumin, their transport has been believed to follow dissociation from albumin with interaction of free ligands. with "translocases" in hepatocyte sinusoidal-plasma membrane (1, 2). However, it remains obscure whether or not binding by plasma albumin is essential for vectorial transport of hepatophilic organic anions.Recently, mutant Nagase analbuminemic rats (NAR) were described (3). Despite the absence of plasma albumin, the mutants show no remarkable sign of pathology except for hyperlipidemia and mild anemia (3, 4). Total serum protein concentration of NAR is similar to that of control Sprague-Dawley rats due to an increased globulin fraction (3). There is...

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Affinity chromatography of hepatic glutathione S-transferases on ω-aminoalkyl sepharose derivatives of glutathione

Cited by 19 publications

References 14 publications

Topological aspects of microsomal N‐acetyltransferase, an enzyme responsible for the acetylation of cysteine S‐conjugates of xenobiotics

Topological aspects of microsomal N‐acetyltransferase, an enzyme responsible for the acetylation of cysteine S‐conjugates of xenobiotics

Acidic and Basic Forms of Glutathione S-Transferases from Human Placenta and Comparison with Human Kidney Glutathione S-Transferase

Plasma clearance of sulfobromophthalein and its interaction with hepatic binding proteins in normal and analbuminemic rats: is plasma albumin essential for vectorial transport of organic anions in the liver?

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