Purification of glutathione S-transferases from rat lung by affinity chromatography. Evidence for an enzyme form absent in rat liver.

Glutathione $\text{S}$-transferases from rat lung cytosol were purified about 200-fold in one step by chromatography on $\text{S}$-hexylglutathione bound to epoxy-activated Sepharose 6B. Further purification on hydroxyapatite resolved the lung transferases into five peaks of activity as measured with 1-chloro-2,4-dinitrobenzene as substrate. Three of the peaks were identified with transferases A, B, and C of rat liver on the basis of chromatographic properties, immunochemical reactivity, and substrate specificity. The other two activity peaks were not detectable in liver: one originated from the lung tissue and one appeared to result from blood in the lung.     

Purification of cholesterol-esterifying enzymes from rat liver cytosol by high-performance liquid chromatography

Three enzymes esterifying cholesterol with long-chain fatty acids were purified approximately 31 000-fold to apparent homogeneity from the cytosol of normal rat liver. The enzymatic activity was tested by incubation of active fractions with tritiated cholesterol and separation of newly formed esters from non-reacted cholesterol by a passage through silica gel cartridges with subsequent assay for radioactivity by liquid scintillation. For the purification of enzymes, active proteins were precipitated by (NH₄)₂SO₄ to 35% saturation. The bulk of inactive proteins was removed by size-exclusion chromatography on TSK G3000 SW. The active fraction was subsequently separated on Separon HEMA BIO 1000 DEAE in gradients of 0–500 mM KCl into three enzymatic activities differing in their retention and these proteins were finally purified by affinity HPLC on columns of cholesterol immobilized on HEMA BIO 1000 E-H. Final purified enzymes showed the same single band in polyacrylamide gel electrophoresis corresponding to 16.5 kDa. Combination of individual enzymes did not increase the overall yield of cholesteryl esters but the reaction-rate was significantly accelerated. These proteins are apparently subunits of a larger complex (M_r 65 000) that can be demonstrated by electrophoresis in the absence of 2-mercaptoethanol. Results presented in this paper indicate that because of good and rapid separation of active proteins, HPLC may be a method of choice for enzyme purifications.     

Purification of major basic glutathione transferase isoenzymes from rat liver by use of affinity chromatography and fast protein liquid chromatofocusing

Seven major isoenzymes of glutathione transferase with isoelectric points ranging from pH 6.9 to 10 were isolated from rat liver cytosol. The purification procedure included affinity chromatography on immobilized S-hexylglutathione followed by high-performance liquid chromatofocusing. Characteristics, such as physical properties, reactions with antibodies, specific activities with various substrates, kinetic constants, and sensitivities to a set of inhibitors, are given for discrimination and identification of the different isoenzymes. The multiple forms of the enzyme correspond to glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4, and 4-4 in the recently introduced nomenclature [W.B. Jakoby et al. (1984) Biochem. Pharmacol. 33, 2539-2540]. A seventh form appears to be a heterodimeric protein composed of subunit 3 and an as yet unidentified subunit.     

Purification by affinity chromatography of glutathione S-transferases A and C from rat liver cytosol

Purification of glutathione S-transferases A and C from rat liver cytosol using an affinity matrix coupled with cholic acid is described. The method provides a convenient means for the rapid and homogeneous preparation of both transferases.     

Purification and characterization of glutathione S-transferases from rat pancreas

Glutathione S-transferases (GSTs) of rat pancreas have been characterized and their interrelationship with fatty acid ethyl ester synthase (FAEES) has been studied. Seven GST isozymes with pI values of 9.2, 8.15, 7.8, 7.0, 6.3, 5.9 and 5.4 have been isolated and designated as rat pancreas GST suffixed by their pI values. Structural, immunological and kinetic properties of these isozymes indicated that GST 9.2 belonged to the alpha class, GST 7.8, 7.0, 6.3 and 5.9 belonged to the mu class, whereas GST 8.15 and 5.4 belong to pi class. The N-terminal sequences and pI values of the mu class isozymes suggested that rat GST subunits 3, 4 and 6 may be expressed in pancreas. N-Terminal sequences of both the pi class isozymes, GST 8.15 and 5.4, were similar to that of GST-P, but there were significant differences in the substrate specificities of these two enzymes. Results of peptide finger print studies also indicated minor structural differences between these two isozymes. None of the GST isozymes of rat pancreas expressed FAEES activity. Rat pancreas had a significant amount of FAEES activity, but it segregated independently during the purification of GST indicating that these two activities are expressed by different proteins and are not related as suggested previously.     

Purification and characterization of glutathione S-transferases from guinea pig liver

Four types of glutathione S-transferase were purified to homogeneity from guinea pig liver by DEAE-cellulose, Sephadex G-75, CM-cellulose, and affinity chromatography. These isozymes were named a, b, c, and d based on the reverse order of elution from a CM-cellulose column, and had specific activities of 89.6, 92.2, 99.0, and 44.0 units/mg, respectively, when assayed with 1 mM each of 1-chloro-2,4-dinitrobenzene and reduced glutathione. All four transferases of guinea pig liver were homodimers. The transferases b, c, and d had a similar molecular weight of 50,000 and their subunit sizes were 25,000, but the corresponding values for transferase a were 45,000 and 23,500, respectively. Transferase a was notably different in the activities towards organic hydroperoxides and 1,2-dichloro-4-nitrobenzene from the other isozymes. Transferases a and b, the major forms in guinea pig liver, were studied with respect to their biochemical properties, including kinetic parameters, absorption and fluorescence spectra, and bilirubin binding. Glutathione peroxidase activity of the transferase a was about 100 times higher than that of other isozymes. In guinea pig liver, it is estimated that transferase a is the major glutathione peroxidase, accounting for about 75% of the total organic hydroperoxide reduction.     

Purification of rat liver phenylalanine hydroxylase by affinity chromatography

Rat liver phenylalanine hydroxylase has been purified to homogeneity on a totally synthesized affinity matrix. The affinity matrix consisted of a succinylated diaminodipropylamine arm linked to Sepharose-4B, to which the cofactor, 6,7-dimethyl-5,6,7,8-tetrahydropterin, was covalently linked. The pure enzyme was eluted with buffered 50% ethylene glycol, 1 m KCl in one step after the 50% ammonium sulfate fraction of the rat liver homogenate was applied to the affinity column. Specific activities ranging from 1.4 to 3.0 units/mg of protein were obtained. The enzyme has been shown to be homogeneous by: (i) discontinuous gel electrophoresis, and (ii) sodium dodecyl sulfate gel electrophoresis. The subunit molecular weight was determined by the same technique and was calculated to be between 51,000 and 55,000.     

Binding of bile acids by glutathione S-transferases from rat liver

Binding of bile acids and their sulfates and glucuronides by purified GSH S-transferases from rat liver was studied by 1-anilino-8-naphthalenesulfonate fluorescence inhibition, flow dialysis, and equilibrium dialysis. In addition, corticosterone and sulfobromophthalein (BSP) binding were studied by equilibrium and flow dialysis. Transferases YaYa and YaYc had comparable affinity for lithocholic (Kd approximately 0.2 microM), glycochenodeoxycholic (Kd approximately to 60 microM), and cholic acid (Kd approximately equal 60 microM), and BSP (Kd approximately 0.09 microM). YaYc had one and YaYa had two high affinity binding sites for these ligands. Transferases containing the Yb subunit had two binding sites for these bile acids, although binding affinity for lithocholic acid (Kd approximately 4 microM) was lower than that of transferases with Ya subunit, and binding affinities for the other bile acids were comparable to the Ya family. Sulfated bile acids were bound with higher affinity and glucuronidated bile acids with lower affinity by YaYa and YaYc than the respective parent bile acids. In the presence of GSH, binding of lithocholate by YaYc was unchanged and binding by YbYb' was inhibited. Conversely, GSH inhibited the binding of cholic acid by YaYc but had less effect on binding by YbYb'. Cholic acid did not inhibit the binding of lithocholic acid by YaYa.     

Purification of L-type pyruvate kinase from human liver by affinity chromatography on Blue-Dextran-Sepharose column

L-type pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase; EC 2.7.1.40) was purified from human liver by an original method. This purification included toluene extraction, a-monium sulphate fractionation, DEAE-Sephadex bactchwise, CM-Sephadex batchwise with elective elution by ATP and affinity chromatography on a Blud Dextran-Sepharose column with specific elution by fructose 1, 6-diphosphate. This purification procedure allowed us to obtain 6 mg protein with a specific activity of 420 IU/mg protein, i.e. 2,690-fold purification with an overall yield of 34%. This preparation was homogeneous as judged by immuno-diffusion, acrylamide and sodium dodecyl sulphate acrylamide-gel electrophoresis.     

Purification of liver and hepatoma membrane proteins by high-performance liquid chromatography

This paper documents the recovery of selected proteins from hepatic plasma membranes. Initial purification, achieved by a series of stepwise extractions, facilitates the subsequent purification by HPLC. Examples are provided to illustrate the recovery of specific proteins from two Morris hepatoma lines and the liver.     

Purification of a soluble ATPase from rat liver mitochondria by AMP-Sepharose affinity chromatography.

ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity was shown in the soluble fraction of rat liver micochondria. Two molecular forms (ATPase 1 and 2) were isolated. ATPase 1 has already been studied. The present paper deals with the purification method of ATPase 2 which was achieved by the following steps: (NH4)2SO4 precipitation. DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G100 filtration and AMP-Sepharose affinity chromatography. The purified protein was characterized by bidimensional polyacrylamide gel electrophoresis. Molecular weight evaluated by SDS-polyacrylamide gel electrophoresis and Sephadex G100 gel filtration was found to be 61 500 +/- 3000.     

Purification of a beta-D-galactosidase from bovine liver by affinity chromatography

A beta-D-galactosidase from bovine liver was purified to apparent homogeneity. The major purification step was affinity chromatography on a column of D-galactose attached to a Sepharose support activated with divinyl sulfone. Affinity media prepared by binding ligands to Sepharose activated with cyanogen bromide were unsuitable for purification of the enzyme, even though such media have been used to purify beta-D-galactosidases from other sources. The molecular weight of the denatured enzyme was 67,000. The molecular weight of the native enzyme at pH 7.0 was 68,000, and at pH 4.5 or 5.0, was 141,000. These data suggest that the enzyme has a single, fundamental subunit with a molecular weight of 67,000, and that the enzyme exists as a monomer at pH 7.0, and a dimer at pH 4.5 or 5.0. The Vmax values of the enzyme with p-nitrophenyl beta-D-galactoside, p-nitrophenyl beta-D-fucoside, lactose, and beta-Gal-(1----4)-beta-GlcNAc-1---- OC6H4NO2 -p were 10,204, 11,550, 9,479, and 8,859 nmol/min/mg of protein, respectively, and the Km values for these substrates were 0.08, 14.9, 14.2, and 1.6mM, respectively. D-Galactose, beta-D- galactosylamine , p-aminophenyl 1-thio-beta-D-galactoside, and D- galactono -1,4-lactone were competitive inhibitors of the enzyme, with Ki values of 0.9, 0.6, 0.6, and 0.8mM, respectively. The enzyme catalyzed the transfer of the D-galactosyl group from p-nitrophenyl beta-D-galactoside to D-glucose. The pH optimum of the enzyme was 4.5, and the pI was 4.7.     

Purification of nucleosidediphosphatase of rat liver by metal-chelate affinity chromatography

A procedure is presented for the purification of nucleosidediphosphatase (nucleosidediphosphate phosphohydrolase, EC 3.6.1.6) of rat liver by affinity chromatography using metal conjugated to epoxy-activated Sepharose 6B. The enzyme is eluted from the conjugate by a solution of L-histidine. The enzyme, when bound to metal-chelate gel, is active in a suspended form, suggesting that the catalytic site is different from the site that binds to the metal-chelate gels. Substrate specificity and Km value of the enzyme obtained are similar to those of the enzyme obtained from the same sources by a conventional procedure, indicating that the metal-chelate affinity chromatography does not bring about any substantial change in the catalytic properties.     

Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical Mr of 24,000 whereas a predominant subunit with Mr of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.     

Purification of aldehyde dehydrogenase from rat liver mitochondria by alpha-cyanocinnamate affinity chromatography.

1. alpha-Cyano-4-hydroxycinnamate was coupled to Sepharose CL-4B activated with 1,2:3,4-bisepoxybutane. 2. The low-Km rat liver mitochondrial aldehyde dehydrogenase was specifically bound to this affinity medium, and could subsequently be eluted with alpha-cyano-4-hydroxycinnamate. 3. The enzyme purified in this manner had a subunit molecular mass of 55 kDa and a pI of approx. 6.5. A minor component of approx. 57 kDa was also present and had a significantly higher pI value; this may be the precursor for aldehyde dehydrogenase. 4. alpha-Cyanocinnamate and some related compounds were found to be uncompetitive inhibitors of the enzyme. 5. No cytosolic aldehyde dehydrogenase was bound to the affinity column, but a protein from a rat liver post-mitochondrial supernatant with a molecular mass of approx. 25 kDa was bound, and could be eluted subsequently with alpha-cyano-4-hydroxycinnamate.     

Cholic acid binding by glutathione S-transferases from rat liver cytosol.

Cholic acid-binding activity in cytosol from rat livers appears to be mainly associated with enzymes having glutathione S-transferase activity; at least four of the enzymes in this group can bind the bile acid. Examination of the subunit compositions of different glutathione S-transferases indicated that cholic acid binding and the ability to conjugate reduced glutathione with 1,2-dichloro-4-nitrobenzene may be ascribed to different subunits.     

Purification of adenosine deaminase from chicken-egg yolk by affinity column chromatography

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity of the enzyme preparation obtained is 81.4 mU/mg.     

Determination of rat liver triglycerides by gas–liquid chromatography and reversed-phase high-performance liquid chromatography

Rats fed with a fat-free or an olive oil-rich diet were employed to compare the response of two chromatographic techniques in the determination of rat liver triglyceride (TG) molecular species composition. Gas–liquid chromatography (GLC) on polarizable liquid phase and reversed-phase high-performance liquid chromatography (RP-HPLC) have been commonly employed for TG analysis, obtaining a similar number of chromatographic peaks when used for animal tissue TG determination. In the present study similar results were achieved with regard to most relevant chromatographic peaks, however, important differences were found in the content of minor TGs. Indeed, RP-HPLC permitted separation of long chain polyunsaturated fatty acids, which were not detected by GLC, while the latter technique reported a higher number of myristoyl-containing TG species. RP-HPLC analysis reported a greater number of TGs, with more similarity to a random composition, made up from the liver fatty acid composition. Therefore, it was concluded that utilization of both techniques would be helpful for liver TG analysis as the use of only one of them does not provide a complete profile of liver TGs. Nevertheless RP-HPLC seems to be more useful for this purpose since revealed a more extensive profile.     

Isoelectric focusing of glutathione S-transferases from rat liver and kidney

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.