Determination of intracellular glutathione in human skeletal muscle by reversed-phase high-performance liquid chromatography

A chromatographic method for the specific determination of cellular low molecular mass thiols has been applied to human muscle tissue. The method is based on the derivatisation of thiols using monobromobimane, which is a specific reagent for the sulphydryl group. The glutathione and cysteine bimane adducts were separated by reversed-phase HPLC, whilst quantitation of the cysteine and glutathione adducts was achieved by fluorescence spectroscopy. The method was found to yield a quantitative recovery of glutathione (ca. 96%), to be sensitive (down to 20 pmol glutathione/per injection) and reveal a low intra-individual coefficient of variation (C.V. < 5%) of the glutathione concentrations in human skeletal muscle. The concentrations of reduced and total glutathione were 1320 ± 37 μmol/kg wet weight (mean ± S.E.M.) and 1525 ± 66 μmol/kg wet weight, respectively. The method was also applied to tissues from nine healthy volunteers to determine if fluctuations in glutathione level occurred over a 24-h period. No diurnal variation of glutathione level in human skeletal muscle was observed.     

Microsomal glutathione transferase. Purification in unactivated form and further characterization of the activation process, substrate specificity and amino acid composition

The procedure developed for purification of the N-ethylmaleimide-activated microsomal glutathione transferase was applied successfully to isolation of this same enzyme in unactivated form. The microsomal glutathione transferases, the unactivated and activated forms, were shown to be identical in terms of molecular weight, immunochemical properties, and amino acid composition. In addition the microsomal glutathione transferase purified in unactivated form could be activated 15-fold with N-ethylmaleimide to give the same specific activity with 1-chloro-2,4-dinitrobenzene as that observed for the enzyme isolated in activated form. This activation involved the binding of one molecule N-ethylmaleimide to the single cysteine residue present in each polypeptide chain of the enzyme, as shown by amino acid analysis, determination of sulfhydryl groups by 2,2'-dithiopyridyl and binding of radioactive N-ethylmaleimide. Except for the presence of only a single cysteine residue and the total absence of tryptophan, the amino acid composition of the microsomal glutathione transferase is not remarkable. The contents of aspartic acid/asparagine + glutamic acid/glutamine, of basic amino acids, and of hydrophobic amino acids are 15%, 12% and 54% respectively. The isoelectric point of the enzyme is 10.1. Microsomal glutathione transferase conjugates a wide range of substrates with glutathione and also demonstrates glutathione peroxidase activity with cumene hydroperoxide, suggesting that it may be involved in preventing lipid peroxidation. Of the nine substrates identified here, the enzymatic activity towards only two, 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide, could be increased by treatment with N-ethylmaleimide. This treatment results in increases in both the apparent Km values and V values for 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Thus, although clearly distinct from the cytosolic glutathione transferases, the microsomal enzyme shares certain properties with these soluble enzymes, including a relative abundance, a high isoelectric point and a broad substrate specificity. The exact role of the microsomal glutathione transferase in drug metabolism, as well as other possible functions, remains to be established.     

A thin-gel isoelectric focusing method for quantitation of protein S-thiolation

A thin-gel isoelectric focusing method has been developed for analysis of protein S-thiolation (formation of mixed disulfides with low molecular weight thiols). The method is rapid and it can be used with 3 to 5 micrograms of a pure protein, or 15 to 20 micrograms of tissue extract protein. It is possible to detect a modification of the protein sulfhydryl by either charged or uncharged thiols, and to determine the quantity of different S-thiolated protein species in a modified sample. The method was used to quantitate the amount of S-thiolation of phosphorylase b in a reaction with oxidized glutathione that produced four S-thiolated forms of the enzyme. The method was also used to detect S-thiolation of two proteins in a cardiac tissue extract treated with diamide. One of the protein bands was shown to be S-thiolated with both cysteine and glutathione, while the other band was S-thiolated only with glutathione.     

Purification and properties of carbonic anhydrase from salmon erythrocytes

Carbonic anhydrase (CA) from erythrocytes of the pink salmon, Onchorhyncus gorbushka, was purified using chloroform-ethanol extraction and Sephadex G-75 gel filtration. A single, high specific-activity CA isozyme having a molecular weight of 29,000 was found. The enzyme sedimented as a single boundary at a sedimentation velocity of 2.9S. Amino acid analysis revealed a composition similar to other submammalian CAs with the exception that the cysteine content was low (1 mol cysteine/mol enzyme). Like other submammalian CAs, the presence of a sulfhydryl reducing agent was required to maintain full activity and to prevent structural changes in the enzyme.     

Purification and properties of cyclostome carbonic anhydrase from erythrocytes of hagfish

1. Carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been purified from erythrocytes of hagfish (Myxine glutinosa). A single form with low specific CO2 hydration activity was isolated. The purified carbonic anhydrase appeared homogeneous judging from polyacrylamide gel electrophoresis and gel filtration experiments. The protein has a molecular weight of about 29 000, corresponding to about 260 amino acid residues. This molecular weight is in accordance with other vertebrate carbonic anhydrases with the exception of the elasmobranch enzymes, which have Mr 36 000--39 000. 2. The molecular weight obtained for hagfish carbonic anhydrase indicates that a carbonic anhydrase with Mr approx. 29 000 is the ancestral type of the vertebrate enzyme rather than, as in sharks, a heavier carbonic anhydrase molecule. 3. The circular dichroism spectrum may indicate a somewhat different structural arrangement of aromatic amino acid residues in this enzyme than in the mammalian carbonic anhydrases. 4. The enzyme is strongly inhibited by acetazolamide and also to a lesser extent by monovalent anions. 5. Zn2+, which is essential for activity, appears, contrary to other characterized carbonic anhydrases, less strongly bound in the active site of the enzyme.     

Characterization of 73 kDa thiol protease from Serratia marcescens and its effect on plasma proteins

The 73-kDa protease (73K protease) was purified from a clinical isolate of Serratia marcescens kums 3958. The purified protease appeared homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. The protease is active in a broad pH range with maximum activity at pH 7.5-8.0. The protease appeared to be a thiol protease, since it was inhibited by sulfhydryl reactive compounds such as p-chloromercuribenzoic acid, fluorescein mercuric acetate (FMA), iodoacetamide, and N-ethylmaleimide, and the protease activity was enhanced by various reducing agents such as cysteine, glutathione, 2-mercaptoethanol, and dithiothreitol. The protease contained 2 mol of free sulfhydryl residues per mol of protease. When the protease was reacted with FMA, a maximum of 2 mol of FMA per mol of enzyme was found reacted, based on fluorescence quenching in which the enzyme inactivation was paralleled linearly with the loss of both SH groups. This indicates possible equal involvement of the two thiol groups for the enzyme activity. The inactivation of the protease by FMA was partially restored by a dialysis in the presence of cysteine or dithiothreitol. The protease was not inhibited by high molecular weight kininogen but was inhibited by alpha 2-macroglobulin. The protease bound stoichiometrically to alpha 2-macroglobulin with 1:1 molar ratio and 25% activity remained constant even after the addition of 4 molar excess of alpha 2-macroglobulin. The protease extensively degraded IgG, IgA, fibronectin, fibrinogen, and alpha 1-protease inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-Adrenergic receptors in brown-adipose tissue. Characterization and alterations during acclimation of rats to cold.

Aldehyde dehydrogenase from bovine liver has been purified to homogeneity. Amino acid composition showed a high content of cysteine of 32 mol/mol enzyme. The enzyme is composed of four identical subunits as judged by sodium dodecyl sulfate gel electrophoresis and end-group analysis. The molecular weight was determined to be 220 000 +/- 10 000 by sedimentation equilibrium analysis in an analytical ultracentrifuge. The Michaelis constants for NAD+, glyceraldehyde and acetaldehyde were found to be 47 micron, 170 micron and 130 micron, respectively.     

Characteristics of human milk glutathione peroxidase

Human milk glutathione peroxidase (GPx) was purified 4500-fold using acetone precipitation and purification by repetitive ion-exchange and gel filtration chromatography with an overall yield of 34%. Homogeneity was established by gel electrophoresis. Using gel filtration, the molecular weight (mol wt) of the enzyme was estimated to be 92 kdalton (kD). The monomeric molecular weight was estimated to b 23 kD from polyacrylamide gel electrophoresis, indicating that the native enzyme consists of four identical subunits. The molecular weight of each subunit was supported by amino acid analysis. Selenium (Se) content of the purified enzyme was 0.31%, in a stoichiometry of 3.7 g-atoms/mol. Data from these studies reveal that GPx provided approximately 22% of total milk Se, but only 0.025% of the total protein.     

Purification and some properties of carbonic anhydrase from bovine skeletal muscle

Procedures for the purification of bovine muscle carbonic anhydrase (isoenzyme III) are described. The purified enzyme has a molecular weight near 29,000 and contains one Zn2+ ion per molecule. The sedimentation coefficient, s(0)20,w, is 2.8 X 10(-13) s, the isoelectric pH is 8.5, and A280(0.1%) = 2.07 cm-1. The CO2 hydration activity, expressed as kcat/Km, is about 1.5% of that of human isoenzyme I (or B) and about 0.3% of that of human isoenzyme II (or C) at pH 8 and 25 degrees C. The activity is nearly independent of pH between pH 6.0 and 8.6. The muscle enzyme is weakly inhibited by the sulfonamide inhibitor, acetazolamide, whereas some anions, particularly sulfide and cyanate, are efficient inhibitors. Bovine carbonic anhydrase III contains five thiol groups, two of which react readily with Ellman's reagent without effect on the catalytic activity. A reinvestigation of the amino acid sequences of cysteine-containing tryptic peptides has shown that cysteine residues occur at sequence positions 66, 183, 188, 203, and 206.     

The mouse liver content of carbonic anhydrase III and glutathione S-tranferases A3 and P1 depend on dietary supply of methionine and cysteine

The contents of glutathione S-transferase (GST) subunits, carbonic anhydrase III (CAIII), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a 230 kDa protein are affected by protein deprivation in mouse liver. In order to know if particular amino acids control these contents, the effects of feeding for 5 days with diets containing different amino acids were examined. After an exploration using SDS-PAGE analysis, the action of selected diets was further examined by distinct techniques. The 230 kDa protein was identified as fatty acid synthase (FAS) by both mass spectrometry and amino acid sequence analyses. Dietary tests showed that: (1) a protein-free diet (PFD) increased the content of glutathione S-transferases P1 and M1, and glyceraldehyde-3-phosphate dehydrogenase, while the content of glutathione S-transferase A3, fatty acid synthase and carbonic anhydrase III decreased; (2) a protein-free diet having either methionine or cysteine preserved the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anydrase III; (3) a protein-free diet having threonine preserved partially the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anhydrase III; (4) a protein-free diet having methionine, threonine and cysteine prevented in part the loss of fatty acid synthase; and (5) the glyceraldehyde-3-phosphate dehydrogenase content was controlled by increased carbohydrate level and/or by lower amino acid content of diets, but not by any specific amino acid. These data indicate that methionine and cysteine exert a main role on the control of liver glutathione S-transferases A3 and P1, and carbonic anhydrase III. Thus, they emerge necessary to prevent unsafe alterations of liver metabolism caused by protein deprivation.     

Membrane-associated carbonic anhydrase purified from bovine lung

We found carbonic anhydrase activity associated with particulate fractions of homogenates of rat, rabbit, human, and bovine lungs. These membrane-associated carbonic anhydrases were remarkably stable in solutions containing sodium dodecyl sulfate (SDS). The bovine enzyme was dissolved with SDS and purified by affinity chromatography and gel filtration. The purified enzyme contains glucosamine, galactose, and sialic acid; it is at least 20% carbohydrate. The apparent molecular weight by SDS-polyacrylamide gel electrophoresis (52,000) may be higher than the actual molecular weight due to the presence of carbohydrate. The enzyme contains cystine, an amino acid that is absent in bovine erythrocyte carbonic anhydrase. Dithiothreitol greatly accelerated the rate of inactivation of the membrane-associated enzyme in SDS, so disulfide bonds appear to stabilize this enzyme. The specific CO2-hydrating activity was about half that of the erythrocyte enzyme. Acetazolamide inhibits the membrane-associated enzyme (Ki = 10 nM) nearly as well as the erythrocyte enzyme (Ki = 3 nM). Antibody to bovine erythrocyte carbonic anhydrase did not inhibit the membrane-associated enzyme. Other investigators have accumulated a good deal of evidence for carbonic anhydrase on the luminal surface of pulmonary capillaries. The enzyme described here appears to be a new isozyme whose properties are consistent with such a localization.     

Isolation from rat liver of a peroxisomal enzyme which converts molecular form 1 of biliverdin reductase into molecular form 3

Cobaltous chloride induced in rat liver an enzyme which converted biliverdin reductase molecular form 1 into the molecular form 3. This conversion involves the oxidation of two sulfhydryl groups of form 1 giving rise to a disulfide bond in form 3. The converting enzyme was isolated from the liver peroxisomal fraction (which was devoid of biliverdin reductase activity), and was absent in liver peroxisomes of control rats. The enzyme was solubilized by treatment of the peroxisomes with 0.1% sodium deoxycholate, and partially purified by DEAE-cellulose and Sephadex G-100 filtration. It is a NAD⁺ dependent enzyme which was inactivated by trypsing and heat treatments. It did not oxidize either reduced glutathione or cysteine. The converting enzyme had a molecular weight of about 54,000 daltons. The oxidation of biliverdin reductase molecular form 1 mediated by the converting enzyme did not affect the latter's molecular weight or activity.     

The low-molecular-weight acid phosphatase from bovine liver: Isolation,amino acid composition,and chemical modification studies

The smallest of the three molecular weight forms of acid phosphatase from bovine liver was purified to a specific activity of 100 μmol min^?1 mg^?1 (measured at pH 5.5 and 37 °C with p-nitrophenyl phosphate). Using several chromatographie and electrophoretic methods, no evidence of heterogeneity was detected. The enzyme was characterized with respect to its stability as a function of pH, molecular weight, amino acid composition, steady-state kinetic parameters in the pH range 4–7 and inhibition by common acid phosphatase inhibitors at pH 5.5. The amino acid composition differed somewhat from a previous literature report. The enzyme was stoichiometrically inactivated upon incubation with Hg²⁺, Ag⁺, and iodoacetate. Inactivation also occurred upon photoinactivation in the presence of Rose Bengal but no inactivation occurred with diethyl pyrocarbonate. The alkylation of one of five cysteine residues by iodoacetate was shown to cause complete inactivation of the enzyme. This alkylation was prevented by the presence of phosphate ion. A tryptic dipeptide containing this essential cysteine was isolated following inactivation with iodo[2-¹⁴C]acetate.     

Purification and characterization of glutathione reductase from corn mesophyll chloroplasts

Differences in the apparent molecular weights of the subunits of glutathione reductase (EC 1.6.4.2) from pea chloroplasts and corn mesophyll chloroplasts have been recently reported. In order to more fully describe the differences between the enzymes from these two sources, glutathione reductase from the mesophyll chloroplasts of corn seedlings ( Zea mays L. cv. G-4507) has been purified 200-fold by affinity chromatography using adenosine 2',5'-disphosphate agarose. The purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)^-1 min^-1. The native enzyme had a relative molecular weight of 190 ± 30 kDa and exhibited polypeptides of 65, 63, 34, and 32 kDa when separated on sodium dodecylsulfate-polyacrylamide gels. Comparisons of the results from electroblotting, native molecular weight and subunit molecular weight analyses suggest that the enzyme exists as a heterotetramer. Optimal enzyme activity was obtained at pH 8 in N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES-NaOH) buffer. The sulfhydryl reagent, n -ethylmaleimide, inhibited enzymatic activity when incubated in the presence of NADPH while no inhibition was detected with oxidized glutathione in the incubation mixture. Reduced glutathione (5 m M ) inactivated the enzyme by 50%. This inactivation followed first order kinetics with a rate constant of 0.0028 s^-1. The enzyme was also inactivated by NADPH. The inactivation reached ca 90% within 30 min and followed first order kinetics with a rate constant of 0.0015 s^-1.     

Purification and properties of soybean nodule xanthine dehydrogenase

Xanthine dehydrogenase (EC 1.2.1.37), an essential enzyme for ureide metabolism was purified from the cytosol fraction of soybean nodules. The purified xanthine dehydrogenase was shown to be homogeneous by electrophoresis and a pI of 4.7 was determined by isoelectric focusing. The enzyme had a molecular weight of 285,000 and two subunits of molecular weight 141,000 each. The holoenzyme contained 1.7 (±0.7) mol Mo and 8.1 (±2.0) mol Fe/mol enzyme and the enzyme also contained FMN and is thus a molybdoironflavoprotein. Soybean xanthine dehydrogenase is the second enzyme in plants demonstrated to contain Mo and the first xanthine-oxidizing enzyme reported to contain FMN, rather than FAD as the flavin cofactor.     

Isolation and characterization of an alkali metal ion-sensitive NAD+-dependent glyceraldehyde 3-phosphate dehydrogenase from germinating green gram (Phaseolus aurieus).

An alkali metal ion-sensitive NAD⁺-specific glyceraldehyde 3-phosphate dehydrogenase has been purified 250-fold from germinating green gram (Phaseolus aurieus). The purified enzyme shows a single protein band on gel electrophoresis. It has been shown to be a tetrameric protein (molecular weight 160,000) made up of apparently identical monomers (subunit molecular weight 40,000). It shows an $\text{A}{}_{280}\text{A}{}_{260}$ ratio equal to 1.38, which is not changed on treatment with animal charcoal or cellulosic ion exchangers. Direct estimation shows less than 0.07 mol bound NAD⁺/mol enzyme. Green gram glyceraldehyde 3-phosphate dehydrogenase is inhibited fairly strongly at physiological concentrations of Na⁺ ions. The inhibition is stronger at higher pH and lower protein concentration. Deproteinated extract, cysteine, and reduced glutathione reverse the Na⁺ ion inhibition. The effect of deproteinated extract is attributable to the presence of some SH-containing compounds. Potassium and rubidium ions have a mild activating effect at lower concentration (below 100 mm) and are inhibitory at higher, nonphysiological, concentrations. Ammonium and lithium ions have no effect. The inhibition due to Na⁺ ions is noncompetitive with respect to NAD⁺ and phosphate ions but competitive with respect to glyceraldehyde 3-phosphate, with K_i about 60 mm. Sodium ions protect the enzyme against proteolysis with trypsin. It is suggested that Na⁺ ions and the small molecular weight SH-compounds may possibly be involved in regulation of the overall rate of glycolysis via modulation of glyceraldehyde 3-phosphate dehydrogenase activity.     

Irreversible thiol oxidation in carbonic anhydrase III: protection by S-glutathiolation and detection in aging rats

Proteins with reactive sulfhydryls are central to many important metabolic reactions and also contribute to a variety of signal transduction systems. In this report, we examine the mechanisms of oxidative damage to the two reactive sulfhydryls of carbonic anhydrase III. Hydrogen peroxide (H2O2), peroxy radicals, or hypochlorous acid (HOCl) produced irreversibly oxidized forms, primarily cysteine sulfinic acid or cysteic acid, of carbonic anhydrase III if glutathione (GSH) was not present. When GSH was approximately equimolar to protein thiols, irreversible oxidation was prevented. H202 and peroxyl radicals both generated S-glutathiolated carbonic anhydrase III via partially oxidized protein sulfhydryl intermediates, while HOCl did not cause S-glutathiolation. Thus, oxidative damage from H202 or AAPH was prevented by protein S-glutathiolation, while a direct reaction between GSH and oxidant likely prevents HOCl-mediated protein damage. In cultured rat hepatocytes, carbonic anhydrase III was rapidly S-glutathiolated by menadione. When hepatocyte glutathione was depleted, menadione instead caused irreversible oxidation. We hypothesized that normal depletion of glutathione in aged animals might also lead to an increase in irreversible oxidation. Indeed, both total protein extracts and carbonic anhydrase III contained significantly more cysteine sulfinic acid in older rats compared to young animals. These experiments show that, in the absence of sufficient GSH, oxidation reactions lead to irreversible protein sulfhydryl damage in purified proteins, cellular systems, and whole animals.     

Purification and characterization of a metallo-endoproteinase from mouse kidney.

A metallo-endoproteinase was purified from mouse kidney. The enzyme was solubilized from the 100 000 g sediment of kidney homogenates with toluene and trypsin, and further purified by fractionation with (NH4)2SO4. DEAE-cellulose chromatography and gel filtration. The molecular weight of the metalloproteinase was estimated by gel filtration on Sepharose 6B to be 270 000--320 000. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of 2-mercaptoethanol, a single major protein with a mol.wt. of 81 000 was observed. Thus the active enzyme is an oligomer, probably a tetramer. It is a glycoprotein and has an apparent isoelectric point of 4.3. Kidney homogenates and purified preparations of the metalloproteinase degraded azocasein optimally at pH 9.5 and at I 0.15--0.2. The activity was not affected by inhibitors of serine proteinases (di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride), cysteine proteinases (4-hydroxymercuribenzoate, iodoacetate), aspartic proteinases (pepstatin) or several other proteinase inhibitors from actinomycetes (leupeptin, antipain and phosphoramidon). Inhibition of the enzyme was observed with metal chelators (EDTA, EGTA, 1,10-phenanthroline), and thiol compounds (cysteine, glutathione, dithioerythritol, 2-mercaptoethanol). The metalloproteinase degraded azocasein, azocoll, casein, haemoglobulin and aldolase, but showed little or no activity against the synthetic substrates benzoylarginine 2-naphthylamide, benzoylglycylarginine, benzyloxycarbonylglutamyltyrosine or acetylphenylalanyl 2-naphthyl ester. This metalloproteinase from mouse kidney appears to be distinct from previously described kidney proteinases.     

Purification and properties of a polymorphic high activity equine erythrocyte carbonic anhydrase.

A polymorphic form of the high activity or C-type of horse erythrocyte carbonic anhydrase has been isolated. It has been designated C2 and differs from the usual C1 form by having a cysteine replacement for arginine at residue 180. This second cysteine, unlike the other, is highly reactive. Isolation of the C2 isozyme by the usual methods results in most of it forming a mixed disulfide with glutathione and this product designated as C3 has an increased anodic mobility. The enzymatic activity and immunologic reactivity of both the C2 and C3 components are the same as for the usual C1 form of the enzyme. The C2 form can be stabilized by alkylation and the carboxamidomethyl derivative has been isolated in crystalline form.     

Purification of N-hydroxy-2-acetylaminofluorene reductase from rabbit liver cytosol

N-Hydroxy-2-acetylaminofluorene reductase was purified from rabbit liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose, Sephadex G-200 and hydroxylapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 34,000 by the electrophoresis and by gel filtration on Sephadex G-200. The enzyme required cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, NADPH or NADH as an electron donor. The enzyme activity was inhibited by p-chloromercuribenzoic acid, N-ethylmaleimide, cupric sulfate or disulfiram, but little by oxygen.