Ian A. Nimmo

Current trends in the estimation of Michaelis-Menten parameters

Abstract This review is about two aspects of the design and analysis of steady state kinetic experiments: (i) how one decides whether an enzymic reaction conforms to the Michaelis-Menten equation; and (ii) given that it does, how one then estimates the characteristic parameters (Km, Ki, V etc.). 1 The answers to these problems depend on several factors, of which perhaps the most important is the reason for doing the experiment in the first place. There are many examples in the biochemical literature of investigations in which kinetic data are used simply to give an indication of an enzymes properties. In these instances graphical analysis with little or no computational backup is the usual choice, and is perfectly satisfactory. On the other hand, the subjectivity of graphical methods precludes their use when the experiment has more precise objectives, such as (i) the determination of the mechanism of action of the enzyme; (ii) the derivation of rate constants; and (iii) the detection of kinetic variants. In these cases some form of statistical analysis is required. Fortunately, the advent of computers means that the tedious but necessary computations are easily done; it has also stimulated a search for reliable statistical techniques and (to a lesser extent) for efficient experimental designs. As a result, one can now calculate better estimates of kinetic parameters than was possible 20 years ago. (A good estimate in this context is one that is both accurate, i.e., free from bias, and precise, i.e., having a small coefficient of variation.)

The major glutathione S-transferase in salmonid fish livers is homologous to the mammalian pi-class GST

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 substrate specificities and subunit compositions of the hepatic glutathione S-transferases of rainbow trout (Salmo gairdneri)

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 (Mrs 22,400 and 24,500), and C5 predominantly an Mr 22,400 homodimer.

Distribution and some properties of the glutathione s-transferase and γ-glutamyl transpeptidase activities of rainbow trout

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.

The glutathione S-transferase activity in the kidney of rainbow trout (Salmo gairdneri)

Abstract 1. 1. Trout gills contain 1.6 mmol GSH/kg. 2. 2. The cytosolic glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate is 0.5 μmol/min/mg protein. There is no detectable activity with 1,2-epoxy-3-( p -nitrophenoxy)propane, ethacrynic acid or p -nitrobenzyl chloride. 3. 3. The activity does not bind to cholate-, glutathione- or S-hexylglutathione-affinity matrices. It was partially purified by ammonium sulphate fractionation and chromatography on Sephadex G-75. 4. 4. The half-saturation concentrations for GSH and 1-chloro-2,4-dinitrobenzene are 1.9 mM and greater than 1.0 mM respectively. 5. 5. The activity is inhibited by cholate, 1.0 mM giving 50% inhibition. 6. 6. About 70% of the activity is inhibited progressively by 1-chloro-2,4-dinitrobenzene with a rate-constant of 0.09/min.

Partitioning of bile acids into subcellular organelles and the in vivo distribution of bile acids in rat liver.

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.

A comparison of the glutathione S-transferases of trout and rat liver.

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.

Fish and mammalian liver cytosolic glutathione S-transferases: Substrate specificities and immunological comparison

Abstract The substrate specificities of cytosolic glutathione S-transferase (GST) activity were compared in several common marine fish, anadromous and freshwater salmonids. Polyclonal antisera raised against purified rat and plaice GST subunits were used to investigate phylogenetic relationships between the mammalian and fish enzymes. Flatfish contained two major isoforms, one of which is related to the mammalian group 1 family. The major GST of salmonids and the cod was related to the mammalian group 3 transferase used as a preneoplastic marker.

GSH biosynthesis in glutathione deficient erythrocytes from Finnish landrace and Tasmanian merino sheep.

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.

The nature of the random experimental error encountered when acetylcholine hydrolase and alcohol dehydrogenase are assayed.

Abstract Analysis of replicate initial velocities showed that (1) the random errors in the velocities were usually not normally distributed, and (2) their magnitude increased with that of the velocity. Consequently equations should be fitted to data by weighted least-squares or by nonparametric methods. It is also demonstrated that initial velocities derived graphically from curvilinear time courses may be underestimates, and that a numerical method may be used instead.