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1. The hepatic glutathione S-transferase (GST) isoenzymes were isolated and characterized from salmon, sea trout and rainbow trout. 2. In all three species the predominant GST expressed comprised subunits of Mr 24,800. These subunits each co-migrated with the rat pi-class Yf polypeptide during SDS/polyacrylamide gel electrophoresis. 3. Western blotting experiments demonstrated immunochemical cross-reactivity between the major salmonid and the rat pi-class GSTs. 4. The salmon GST of subunit Mr 24,800 was digested with cyanogen bromide and the peptides, once purified by reverse-phase HPLC, were subjected to automated amino acid sequencing. 5. Over the region sequenced, the salmon GST possessed about 65% homology with the rat and human pi-class GST.

The Michaelis-Menten equation was fitted to simulated data containing different sorts of error by using the three linear transformations, and the methods of S. R. Cohen [Anal. Biochem. (1968) 22, 549-552], R. Eisenthal & A. Cornish-Bowden [Biochem. J. (1974) 139, 715-120], F. de M. Merino [Biochem. J. 143, 93-95] and G. N. Wilkinson [Biochem. J. (1961) 808 324-332). The best methods were those of Eisenthal & Cornish-Bowden (1974) and Wilkinson (1961).

1. Cytosol from trout liver, gills and intestinal caeca has substantial glutathione S-transferase activity. 2. Gel-exclusion and ion-exchange chromatography suggest that trout liver has several glutathione S-transferases with different molecular weights and ionic charges. 3. A component capable of binding lithocholic acid eluted together with glutathione S-transferase activity. Some of the transferase activity did not elute together with binding activity. 4. The enzymic activity from trout liver was less stable at 37 degrees C than that from rat liver. 5. The glutathione S-transferases of fish liver have a similar specific activity to those of rat liver but different molecular properties.

The clearance of an injected dose of sodium glycocholate has been studied in a group of 23 control subjects who had no evidence of liver disease. A significant correlation was found between bile salt clearance and age. No significant difference was found between the bile salt clearance of male and female subjects. Data derived from a simple compartmental model indicated that decreased clearance of bile salt with age was not specifically related to degenerative changes in hepatocellular integrity, hepatic uptake of bile salts or bile flow.

Substantial soluble glutathione S-transferase activity and millimolar reduced glutathione (GSH) are present in most tissues of both teleosts and elasmobranchs. The hepatic enzymes of fish conjugate a range of electrophilic substrates with GSH, although their specificities are less broad than those of the transferases in rodent liver. There is no good evidence that fish transferases have ligandin-like activity or a 'suicide' function. All fish livers tested have several transferase isoenzymes. They are dimers of subunits whose Mrs are about 25 kDa and which may have different catalytic properties. In some species transferase activity is induced by agents such as phenols or 3-methylcholanthrene. Glutathione S-transferases are important detoxication enzymes in fish.

1. The subcellular distribution of conjugates of cholic acid and chenodeoxycholic acid between cytosol, nuclei, mitochondria and microsomes in rat liver has been determined. 2. The partition coefficients for the distribution of these bile acids between subcellular fractions and buffer have been measured and used to construct a compartmental model of the amounts of conjugated bile acids present in the different subcellular organelles in vivo. 3. This model indicates that a large percentage of the bile acid in the rat liver is found in the nuclear fraction; 42% of the cholic acid conjugates and 27% of the chenodeoxycholic acid conjugates. Substantial amounts of bile acid are also present in microsomes and mitochondria suggesting that published estimates of the amounts of bile acids in these fractions are underestimates. 4. The model also allows the amount of bile acid which is in free solution in cytosol to be determined; 10.9% of the cholic acid conjugates and 4.1% of the chenodeoxycholic acid conjugates in rat liver were present in this fraction. Knowlege of the amount of free bile acid allows possible roles of the cytosolic bile binding proteins to be assessed.

1. The binding of cholic acid to 100000g supernatants from rat livers was investigated by equilibrium dialysis and gel-exculsion chromatography. 2. Supernatants were found to contain at least two classes of binding site for cholic acid. 3. These recptor molecules are probably proteins since incubation with proteolytic enzymes resulted in complete loss of cholic acid binding. 4. Supernatants were added to columns of Sephadex G-75, and two groups of fractions were shown to bind cholic acid. One of these contained low-affinity binding sites and the other contained both low- and high-affinity binding sites. 5. Feeding cholestyramine had no effect on cholic acid binding. 6. Increased cholic acid binding occurred after injection of phenobarbitone. There was an increase in the amount of the low-affinity component but no change in the high-affinity component. 7. The dissociation constants of the binding of cholic acid suggest that the binding proteins may be involved in bile acid transport.

1. Two automated colorimetric methods have been developed for assaying the GSH and total thiol in protein-free extracts of erythrocytes. They employ as chromogens 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) and alloxan. 2. The concentrations of GSH, GSSG and total non-protein thiol have been estimated in high and low GSH erythrocytes from Finnish Landrace and Tasmanian Merino sheep. 3. In both breeds of sheep low GSH cells were found to have low concentrations of total non-protein thiol and GSSG as well as of GSH. 4. Nevertheless high and low GSH cells have similar values for the oxidation-reduction potential of the GSH : GSSG couple.

Pyloric caeca of trout contain 1.9 mmol GSH/kg tissue. Cytosolic glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate is 0.06 mmol/min/g protein. Cholate (3.3 mM) inhibits cytosolic transferase activity by 55% at pH 6.6 and by 4% at pH 7.4. The transferases do not bind 8-anilino-1-naphthalene sulphonate at pH 7.4. The cytosolic transferases are inactivated progressively by 1-chloro-2,4-dinitrobenzene, 50% of their activity being lost in 5.0 min. A minority of the activity does not bind to a glutathione-affinity matrix. At pH 6.6 its apparent Michaelis constants for GSH and 1-chloro-2,4-dinitrobenzene are 0.88 and 9.1 mM respectively. The rest of the activity is eluted from the affinity matrix as a single peak. Its apparent Michaelis constants for GSH and 1-chloro-2,4-dinitrobenzene are 0.33 and 2.9 mM respectively. Its subunit Mr is 22.4 kDa.

A simple model of zonal gel filtration is analysed numerically to establish semiquantitatively how the capacity of a protein-ligand complex to survive gel filtration intact depends on several experimental variables and on the kinetic characteristics of the complex. The design of gel-filtration experiments aimed at detecting and partially purifying binding proteins is discussed.

The methods of Atkins & Nimmo (1973) and Fernley (1974) for fitting the integrated Michaelis-Menten equation were compared by using the same sets of simulated experimental data. The method of Fernley (1974) is to be preferred because it gives precise and unbiased estimates of the Michaelis-Menten parameters over a wide range of substrate concentrations. However, the estimates may not be symmetrically distributed, especially at low substrate concentrations.

1. A compartmental model has been used to derive the in vivo subcellular distribution of lithocholic acid in rat liver. The model is based on the values of the partition coefficients for the distribution of lithocholic acid between subcellular fractions and buffer. It also permits calculation of the amount of lithocholic acid which is in free solution in cytosol. 2. The hypothesis that the rate of biliary excretion of a bile acid depends on the proportion in free solution was investigated by comparing the rates of biliary excretion of lithocholic acid and glycocholic acid. The rate for lithocholic acid was substantially less than for glycocholic acid while the percentages of each bile acid in free solution were 0.8% and 10%, respectively. 3. The validity of the model was supported by the observation that the amounts of lithocholic acid predicted to be present in the nuclear and cytosolic fractions were similar to the amounts found after intravenous injection of the bile acid.

Chromatofocusing separated the glutathione S-transferases of trout liver cytosol into species termed cationic (eluted from pH 8-5) and anionic (eluted by 1.0 M NaCl at pH 5). The cationic enzymes were separated from cytosol by S-hexylglutathione affinity chromatography, ultrafiltration and chromatofocusing (pH 9-7) into 4 major (C1, C2, C4 and C5) and 3 minor fractions. The anionic material was not purified in this way because only 50% of the activity bound to the S-hexylglutathione column. The major cationic enzymes had similar half-saturation concentrations for GSH (0.2 mM) and 1-chloro-2,4-dinitrobenzene (0.4 mM); those of the anionic material were higher (0.7 mM, 1.9 mM respectively). The substrate specificities of the cationic enzymes C1 and C2 were similar (e.g., conjugation of bromosulphophthalein) as were those of C4 and C5 (e.g., conjugation of 1,2-epoxy-3-(p-nitrophenoxy) propane). The anionic material had a different specificity (e.g., rapid conjugation of p-nitrobenzyl chloride). SDS-polyacrylamide gel electrophoresis showed C1 and C2 to be homodimers of subunit Mr 22,400, C4 to be a heterodimer (Mr's 22,400 and 24,500), and C5 predominantly an Mr 22,400 homodimer.

1. The maximum activities of the enzymes for the biosynthesis of GSH (gamma-glutamyl-cysteine synthetase and GSH synthetase) have been assayed in high GSH and low GSH erythrocytes from Tasmanian Merino and Finnish Landrace sheep. 2. For the Merinos, the activities (mumol product/g haemoglobin per min +/- S.E.M. (n)) in the high and low GSH erythrocytes respectively were: gamma-glutamyl-cysteine synthetase: 0.776 +/- 0.065 (11) and 0.375 +/- 0.063 (13); and GSH synthetase: 0.069 +/- 0.003 (11) and 0.066 +/- 0.002 (13). 3. For the Finnish Landrace sheep the activities in the high and low GSH erythrocytes respectively were: gamma-glutamyl-cysteine synthetase: 0.595 +/- 0.063 (12) and 0.555 +/- 0.033 (10) and gamma-glutamyl-cysteine synthetase: 0.073 +/- 0.002 (12) and 0.070 +/- 0.002 (10). 4. gamma-Glutamyl-cysteine synthetase was markedly inhibited by physiological GSH concentrations. No evidence was found for the presence of an inhibitor of GSH biosynthesis (other than GSH) in low GSH erythrocytes from Finnish Landrace sheep. 5. Although for the Merinos the low GSH trait can be explained in terms of a diminished activity of gamma-glutamyl-cysteine synthetase, no such explanation is tenable for the Finnish Landrace sheep.

Two methods are described for fitting the Michaelis-Menten equation to sets of data with a common michaelis constant but different maximum velocities. One of them uses the method of least squares, and the other is based on the direct plot of Eisenthal & Cornish-Bowden [Biochem. J. (1974) 139, 715-720].

1. Descriptions are given of two ways for fitting non-linear equations by least-squares criteria to experimental data. One depends on solving a set of non-linear simultaneous equations, and the other on Taylor's theorem. 2. It is shown that better parameter estimates result when an equation with two or more non-linear parameters is fitted to all the sets of data simultaneously than when it is fitted to each set in turn.

It is shown that, if the asymmetrical model for glucose transport is correct, the published estimates of this half-saturation concentration must be low. As a result, the model is even less able than before to satisfy the tests of its validity.

1. The binding of glycocholic acid, chenodeoxycholic acid and lithocholic acid to rat liver 1000 000g supernatants was studied by equilibrium dialysis. 2. The binding characteristics of the bile acids suggest that the binding components are involved in bile acid transport. 3. When mixtures of [14C]lithocholic acid and liver supernatants were eluted from columns of Sephadex G-75, a prominent peak of [14C]lithocholic acid appeared with proteins of mol.wt. approx. 40000. A second, smaller, peak of [14C]lithocholic acid was eluted with proteins of mol.wt. approx. 100000. 4. The inclusion of cholic acid, glycocholic acid or chenodeoxycholic acid in the eluting buffer decreased the amount of [14C]lithocholic acid that was eluted with the higher-molecular-weight component.

1. The turnover of plasma glucose and free fatty acids was measured in ten patients within 24 h of the onset of symptoms of acute myocardial infarction and in two with symptoms of acute myocardial ischaemia. The measurements were repeated in seven of the patients 12-40 weeks after the acute episode. 2. Both for the patients with acute myocardial infarction alone and for all the individuals studied the turnover of glucose increased with plasma glucose concentration but was not related to the turnover of free fatty acids or the plasma concentrations of free fatty acids, insulin or total catecholamines. There was no obvious difference in the nature of the glucose turnover-concentration relationship between the patients with acute myocardial infarction, with acute myocardial ischaemia and on re-examination. 3. For all the individuals studied the turnover of free fatty acids increased with the concentration of these but was not related to the turnover of glucose or the plasma concentrations of glucose, insulin or total catecholamines. There was no obvious difference in the nature of the free fatty acids turnover-concentration relaionship between the patients with acute myocardial infarction, with acute myocardial ischaemia and on re-examination.

1. The effect of systematic error (loss of ligand, complex or macromolecule) on three of the experimental designs by which equilibrium dialysis may be used to quantify the interaction of ligand and macromolecule is examined theoretically, and the design that is least sensitive to systematic error is identified. 2. Thirteen methods for fitting the binding isotherm to experimental data are compared by using them to analyse simulated data containing random error, and the most reliable method is identified.

1. The basic glutathione S-transferases from rainbow-trout liver were more stable than the acidic ones. 2. The apparent pI values of these enzymes were lowered when they were eluted from a glutathione affinity column by reduced glutathione at pH 8.85. 3. The pI effect was not a function of the high pH alone, was diminished under conditions less favourable to glutathione oxidation, and did not occur when S-hexylglutathione affinity chromatography was used instead.

Bromosulphophthalein and N-ethylmaleimide, inhibitors of glutathione S-transferase (EC 2.5.1.18 RX: glutathione R. transferase), have been used to identify variant forms of the erythrocyte enzyme. One 'atypical' sample was detected and was shown to have appreciably different kinetic and stability properties. These inhibitors may be useful in surveys of variation in this group of enzymes.

1. Gills, kidney, intestinal caeca and liver of trout have glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene (200 500 nmol/min/mg protein), and reduced glutathione (0.5 2.0 mmol/kg tissue). 2. Only kidney and intestinal caeca have substantial gamma-glutamyl transpeptidase activity with gamma-glutamyl-rho-nitroanilide (2-9 nmol/min/mg protein). 3. Renal gamma-glutamyl transpeptidase is membrane-bound and has similar kinetic properties to its mammalian counterparts. 4. The data are consistent with the presence of a mercapturic acid pathway in trout.

Salmon from salt water have three classes of soluble hepatic glutathione S-transferases which can be separated from cytosol by affinity chromatography and chromatofocusing. The classes have different substrate specificities and kinetic properties. All the enzymes are dimeric proteins. There are immunologically distinct subunits of Mrs 22.4, 23.0 and 24.0 kDa. Fish killed in August have enzymes with different apparent isoelectric points and subunit compositions than fish killed in February. The glutathione S-transferase activity of fresh-water salmon is similar to that of February salt-water fish except that the former binds less avidly to S-hexylglutathione-Sepharose 6B.

Glycocholate binding by plasma proteins and erythrocytes from fetal and adult blood has been studied, and the results have been used to derive a compartmental model of the distribution of glycocholate in blood. This model assumes that glycocholate is distributed between the aqueous phase, albumin binding sites, binding sites on other plasma proteins, and erythrocytes. Whereas glycocholate binding by albumin was saturable [fetal blood: dissociation constant (0.5 mM), concentration of binding sites (11.3 mumoles/g protein); adult blood: dissociation constant (0.42 mM), concentration of binding sites (20.1 mumoles/g protein)], binding by other plasma proteins was not. The association of glycocholate with erythrocytes appeared to be based on partitioning rather than binding of the bile salt to specific sites. The value of the partition coefficient was 6.0. The compartmental model indicates that, in fetal blood, 46% of the glycocholate is in free solution, and 37% is bound to albumin. In blood from adults, the corresponding values are 31% in free solution and 59% bound to albumin.

Trout kidney contains 2.3 mmol GSH/kg. The cytosolic glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate is 0.35 mumol/min/mg protein. There is no detectable activity with 1,2-epoxy-3-(p-nitrophenoxy)propane, ethacrynic acid, p-nitrobenzyl chloride or p-nitrophenyl acetate. A variable proportion of the activity does not bind to a glutathione-affinity matrix. Its Km values for GSH and 1-chloro-2,4-dinitrobenzene are 3.0 and 5.1 mM, respectively. The rest of the activity is eluted from the affinity matrix as one main and two minor peaks. The main peak has Km values for GSH and 1-chloro-2,4-dinitrobenzene of 0.4 and 4.5 mM, respectively. Its subunit Mr is 22,900. The activity in the main peak is inhibited progressively by 1-chloro-2,4-dinitrobenzene with a rate constant of 0.11/min.