Purification and partial characterization of the glutathione S-transferase of rat erythrocytes.

The single glutathione S-transferase (EC 2.5.1.18) present in rat erythrocytes was purified to apparent homogeneity by affinity chromatography on glutathione-Sepharose and hydroxyapatite chromatography. Approx. 1.86 mg enzyme is found in 100 ml packed erythrocytes and accounts for about 0.01% of total soluble protein. The native enzyme (Mr 48,000) displays a pI of 5.9 and appears to possess a homodimeric structure with a subunit of Mr 23,500. Enzyme activities with ethacrynic acid and cumene hydroperoxide were 24 and 3%, respectively, of that with 1-chloro-2,4-dinitrobenzene. The Km values for 1-chloro-2,4-dinitrobenzene and glutathione were 1.0 and 0.142 mM, respectively. The concentrations of certain compounds required to produce 50% inhibition (I50) were as follows: 12 microM bromosulphophthalein, 34 microM S-hexylglutathione, 339 microM oxidized glutathione and 1.5 mM cholate. Bromosulphophthalein was a noncompetitive inhibitor with respect to 1-chloro-2,4-dinitrobenzene (Ki = 8 microM) and glutathione (Kis = 4 microM; Kii = 11.5 microM) while S-hexylglutathione was competitive with glutathione (Ki = 5 microM).

Glutathione S-transferase composition of rat erythrocytes.

With 1-chloro-2,4-dinitrobenzene as the electrophilic substrate, the specific activity of glutathione S-transferase in rat haemolysates was found to range from 0.002 to 0.013 mumol/min/mg haemoglobin at 30 degrees C. To establish the glutathione S-transferase composition, chromatofocusing was used which indicated the presence of a single soluble isoenzyme with an apparent pI of 6.1. A molecular weight of 48,000 was determined for the enzyme by gel filtration. The transferase enzyme in intact erythrocytes is shown to catalyze the formation of S-(2,4-dinitrophenyl)-glutathione from 1-chloro-2,4-dinitrobenzene and endogenous glutathione. Efflux of this conjugate from erythrocytes proceeded at a rate of 13 nmol/min/ml at 37 degrees C.

Characterization of the aflatoxin B1-binding site of rat albumin.

A fluorescence-enhancement method was used to investigate the non-covalent interaction between aflatoxin B1 and rat albumin. Solvent-induced shifts in the emission spectrum of aflatoxin B1 provided evidence that the aflatoxin B1-binding site of rat albumin is a highly nonpolar environment. A dissociation constant of 20 microM was determined at 20 degrees C. The possibility that aflatoxin B1 binds one of the three major drug sites of albumin was investigated by ligand-displacement experiments. Mechanisms whereby marker ligands displace aflatoxin B1 were further investigated by comparing the experimental binding parameters with those derived theoretically, assuming competitive binding. The results indicate that: aflatoxin B1 and phenylbutazone compete for a common high-affinity site on rat albumin; high-affinity binding of aflatoxin B1 and site-II marker ligands takes place independently; aflatoxin B1 does not compete with either cholate or warfarin for the same high-affinity site, but the simultaneous binding of warfarin or cholate negatively modulates the binding of aflatoxin B1 to albumin. Fluorescence energy-transfer studies show that the lone tryptophan residue, Trp-214, is not associated with the aflatoxin B1-binding site.

Intracellular aflatoxin B1-binding proteins in rat liver.

Intracellular aflatoxin B1 binding in rat liver was studied under both in vitro and in vivo conditions. Binding in vivo appeared similar to that observed in vitro except that some covalent adduct formation was detected. Participation of previously described carcinogen-binding proteins such as the Ah receptor, h2-5S protein, 4-5S receptor for 3-methylcholanthrene and the Z-protein fraction was discounted on the grounds of competition binding studies and gel-permeation chromatography. The molecular weight of 45,000 was estimated for the major aflatoxin B1-binding component. Aflatoxin B1 co-eluted with the glutathione S-transferases during gel-permeation and separation of the various isozymes by cation-exchange chromatography indicated interactions with the YaYa and YaYc-forms. These proteins, however, account for less than 20% of the total intracellular aflatoxin binding. A protein of apparent monomeric structure appears to form the major in vitro/in vivo complex with aflatoxin B1.

Aflatoxin B1 transport in rat blood plasma. Binding to albumin in vivo and in vitro and spectrofluorimetric studies into the nature of the interaction.

Binding of [3H]aflatoxin B1 to rat plasma was investigated in vivo and in vitro. Column chromatographic and polyacrylamide gel electrophoretic analyses clearly demonstrated that aflatoxin B1 bound primarily plasma albumin. Very little binding activity was shown by other plasma proteins. Spectrofluorimetric studies were undertaken to gain some insight into the nature of the aflatoxin-albumin interaction. Quenching of the lone tryptophan fluorescence intensity upon aflatoxin binding was due, at least in part, to a ligand-induced conformational change in the albumin molecule. Aflatoxin B1 binds an apolar site with an association constant of 30 mM-1 at pH 7.4 and 20 degrees C. Neither charcoal treatment of rat albumin nor the presence of 0.15 M NaCl had any significant effect on the interaction. The association constant was pH-dependent, increasing about 1.7-fold as the pH increased from 6.1 to 8.4. This pH dependence is ascribed to a pH-induced conformational change in the albumin molecule. Thermodynamic studies indicated that the aflatoxin-albumin interaction was exothermic (delta H = -29.3 kJ X mol-1), with a delta S value of -13.8 J X mol-1 X K-1.

Cucurbitacin delta 23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions.

Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 X 10(-7) cm2 X s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed.

Aflatoxin B1 alters protein phosphorylation in rat livers.

A study was conducted on the effect of aflatoxin B1 on protein phosphorylation in rat livers by incubation of soluble and insoluble cell fractions with [gamma 32P] ATP. SDS-polyacrylamide gel electrophoresis indicated that a total of eight rat liver phosphoproteins were affected during a feeding period of 36 weeks on a carcinogenic aflatoxin B1-containing diet compared to the phosphoprotein patterns obtained from the livers of rats on a normal noncarcinogenic diet. The appearance of only two of these phosphoproteins were cAMP dependent. DEAE-cellulose chromatography employed to separate the different histone kinase activities in soluble rat liver cell fractions, showed that the specific activity of histone kinase I activity was increased but its total activity decreased while the histone kinase II activity stayed unchanged for rats submitted to the aflatoxin B1-containing diet compared to those on the normal noncarcinogenic diet.

Purification of human plasma catalase and monoamine oxidase and the partial characterization of monoamine oxidase.

Human plasma catalase and monoamine oxidase were purified for the first time to homogeneity. Monoamine oxidase purified 3200-fold with the aid of DEAE-Sepharose Cl-6B and Sepharose Cl-6B chromatography was devoid of catalase, aldehyde dehydrogenase and reductase activity. Homogeneous catalase was obtained during DEAE-Sepharose C1-6B chromatography of monoamine oxidase. Monoamine oxidase was characterized physico-chemically in terms of molecular weight, electrophoretic mobility, iso-electric point, amino acid composition and specific activity. The molecular weight was estimated to be 234.5 K by exclusion chromatography and with iso-electric focusing the pI was estimated to be 5.6.

Rhodanese from Cercopithecus aethiops (vervet monkey) liver. I. Purification and some physical characteristics.

Rhodanese (thiosulphate sulphurtransferase , EC 2.8.1.1.) from Cercopithecus aethiops (vervet monkey) liver has been isolated and purified by means of extraction, ammoniumsulphate and pH fractionation, anion-exchange chromatography, Sephacryl S-300 gel chromatography and cation-exchange chromatography. A yield of about 10% pure enzyme with a specific activity of 242 U/mg protein corresponding to a purification factor of 523 was obtained. The enzyme was physically characterized and its homogeneity determined by electrophoretic studies and gel chromatography. The rhodanese enzyme has a molecular weight of 37,000 daltons, a D020 ,w value of 7.6 X 10(-7) cm2 sec-1, a Stokes radius (molecular size) of 2.75 X 10(-7) cm and a frictional ratio of 1.071.

Rhodanese from Cercopithecus aethiops (vervet monkey) liver. II. Aspects of enzyme kinetics and mechanism of action.

Initial velocity kinetic studies were undertaken and certain kinetic parameters ( KmSSO2 -3 = 3.1 X 10(-3) M and Vmax = 153.85 U/ml/min.) were determined and the mechanism identified as a ping-pong (double displacement) mechanism. Competitive inhibition of rhodanese by both substrates, viz. thiosulphate and cyanide, provides additional evidence of Ping-Pong Bi-Bi mechanism for this transferase.

Lysosomal acid deoxyribonuclease from vervet monkey livers--I. Purification and physico-chemical characterization.

Acid DNase from monkey liver lysosomes was purified to homogeneity by salt extraction of lysosomal membranes at pH 3.8; (NH4)2SO4 fractionation; low salt precipitation; SP-C50 and G-150 Sephadex chromatography; and polyacrylamide gel electrophoresis. The pH for optimum activity was dual in character with a labile optimum at pH 3.8 and a less active but stable one at pH 4.2. The estimated molecular weight was 40K and the pI was 4.4. Inorganic ions such as Ca2+, Mg2+, Mn2+ and SO2-4 were more than 80% inhibitory at 10-mM levels. Fe3+ ions were 80% inhibitory at 0.1-mM levels. NaCl at 100 mM is essential for activity but becomes 100% inhibitory above 200 mM.

Lysosomal acid deoxyribonuclease from vervet monkey livers--II. Enzymic properties.

Primate liver lysosomal acid DNase is an endonucleolytic enzyme. The enzyme has both 3'- and 5'-nucleotidohydrolase activities. The oligonucleotides produced by DNase are polymers mainly about 30 mononucleotides long. The Arrhenius plot shows a discontinuity with a transition temperature at 47 degrees C, with an activation energy of 107 kJ/mol below and 67 kJ/mol above this temperature. The activation enthalpy is 104 kJ/mol and the entropy -0.498 kJ/mol/K. The enzyme is subject to substrate inhibition and the Km value is 159 X 10(-3) mM DNA-P.

The effect of aflatoxins on mammalian acid deoxyribonucleases.

1. Difference spectroscopy studies indicated that tetrahydrodeoxyaflatoxin B1 and aflatoxicol bind slightly to DNA, whereas aflatoxins B2a, G2a, G2 and aflatoxicol bind to bovine and porcine spleen DNAse II but aflatoxins B1, B2, G1 and tetrahydrodeoxyaflatoxin did not. 2. Kinetic studies showed that aflatoxins B1, G1 and B2 activated bovine and porcine spleen DNAse II while aflatoxins B2a, G2a and G2 had an inhibiting effect. 3. Dissociation constants for the enzyme: substrate-aflatoxin complexes (KAS) as well as the inhibition constants (Ki) were obtained from kinetic studies.

Deviation from Michaelis-Menten kinetics for esterase E-II from the venom of Bitis gabonica, using synthetic arginine amides as substrates.

Steady state kinetic studies on the reaction between esterase E-II from the venom of Bitis gabonica and the fluorogenic substrates, N-alpha-benzoyl-L-phenylalanyl-L-valyl-L-arginine-4-methylcoumaryl-7-amide and N-alpha-benzoyl-L-arginine-4-methylcoumaryl-7-amide were found to deviate from Michaelis-Menten kinetics. Analysis of algebraic graphs and application of non-linear regression allowed an empirical rate law to be selected. The results revealed a rate equation of at least third degree at pH values above 7.0 and of 2:2 degree in the pH range 6-7. The data were interpreted in terms of a molecular model involving an enzyme with one catalytic site and several auxiliary or regulatory sites which, through cooperative effects, may either activate or inhibit the enzyme. Substrate activation is observed at low substrate values and might follow from an obligatory order of binding involving two of the sites, the modifier substrate molecule binding before the substrate molecule undergoing transformation to products. Inhibitory sites apparently become available only at alkaline pH. The inhibition is only noted at high substrate concentrations and is of the partial type.

Isolation and characterization of three lutropin isohormones from the pituitary gland of the ostrich (Struthio camelus).

Avian lutropin has been isolated from pituitary glands of the ostrich (Struthio camelus) in three homogeneous forms (designated isohormones). The different homogeneous forms of ostrich lutropin were characterized physically and chemically in terms of molecular weight, electrophoretic mobility, isoelectric points, amino acid and carbohydrate composition. From these characteristics it was evident that the isohormones are very similar. The differences between these isohormones can be attributed to differences in carbohydrate composition, especially sialic acid.