Hydroperoxide metabolism in mammalian organs.

Publisher Summary To determine glutathione peroxidase reliably, some factors of potential pitfall have to be considered, for example, enzymatic side reactions of substrates (especially when crude tissue samples are assayed), high and variable spontaneous reaction rates of substrates, and the peculiar kinetics of the enzyme itself. With the best documented example, the enzyme of bovine red blood cells, ping-pong kinetics with infinite limiting maximum velocities, and Michaelis constants have been established. This means that the generally recommended conditions for determination of enzyme activity––that is, “saturating” concentrations of all substrates, cannot possibly be fulfilled. In consequence, compromises are inevitable in the choice of substrate concentration for the assay and in the definition of the unit of activity. Fixed-time assay measuring H 2 O 2 consumption and continuous monitoring of Glutathione disulfide (GSSG) formation are cited here. The main differences between the assay procedure described and those proposed by others are listed in the chapter. To compare the results obtained by different procedures, appropriate empirical converting factors are also given.

Assays of glutathione peroxidase

Publisher Summary This chapter presents a procedure for the preparation of glutathione peroxidase, which is regarded as a major protective system against endogenously and exogenously induced lipid peroxidation. Two types of methods are used for determining the activity of glutathione peroxidase. One involves a direct measurement of unconsumed glutathione (GSH) at fixed time periods by polarographic GSH analysis' (Method 1), or by the dithionitrobenzoic acid method (Method 2). The second approach takes advantage of the capability of glutathione reductase, with nicotinamide adenine dinucleotide phosphate (NADPH), to regenerate GSH from oxidized GSH. The decrease in NADPH is continuously measured spectrophotometrically, while the GSH concentration in the enzymatic cycle remains essentially constant (Method 3). A convenient source for the preparation of glutathione peroxidase is bovine blood including the following steps: hemolysate; organic solvent precipitation; phosphate precipitation; absorption to phenyl-sepharose; and washing on diethylaminoethyl (DEAE)–sephadex, S-300 sephacryl, and hydroxylapatite column.

[44] Glutathione peroxidase

Publisher Summary This chapter discusses the role of plasminogen activators in various biological processes. In specific, it describes two types of plasminogen activators—namely, the urokinase-type plasminogen activator (u-PA) and the tissue-type plasminogen activator (t-PA), which are essentially different gene products. The amino acid sequences of these activators and nucleotide sequences of the corresponding cDNAs have largely been determined, and the cDNAs have been cloned using recombinant techniques. A variety of enzymatic as well as immunological assay and detection methods have also been developed that allows a precise quantification of the activators, a distinction between u-PA and t-PA, determination of whether an activator is present in its active or zymogen form, analysis of the kinetics of different steps of the cascade reaction, and immunocytochemical identification of u-PA and t-PA in tissue sections. Much of the studies on plasminogen activators and cancer has been guided by the hypothesis that proteolysis of the components of extracellular matrix, initiated by the release of plasminogen activator from the cancer cells, plays a decisive role for the degradation of normal tissue, and thereby for invasive growth and metastases.

Plasminogen activators, tissue degradation, and cancer.

Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts.

An optimized coupled test system for glutathione peroxidase in red blood cells is presented. In the improved assay, tert. butyl hydroperoxide was employed as an acceptor substrate at twenty times Km(app) concentration. By aid of a batching procedure for instant separation of hemoglobin from glutathione peroxidase, it could be proved that cyanmethemoglobin in the modified procedure does not affect the spontaneous reaction rate. Further possible interference with the determination could be excluded. Glutathione peroxidase activities in red blood cells of normal adults were measured by the improved test method. A glutathione peroxidase activity in human plasma was observed.

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An improved coupled test procedure for glutathione peroxidase (EC 1-11-1-9-) in blood.

Glutathione peroxidase, V. The kinetic mechanism.

The complete sequence of 157 amino acids of the light (A) chain of high molecular mass urokinase from human urine was determined. The fragmentation strategy included cyanogen bromide cleavage of the S-carboxymethylated A chain at the methionine and/or tryptophan residues and use of the specific endoproteinase Lys-C. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. C-terminal amino acids of the A chain were determined by consecutive treatment with carboxypeptidase A and B. The amino acid sequence obtained revealed a significant homology to peptide chains of other serine proteinases. Accordingly, the sequence of the A chain can be divided into three domains: 1) The growth factor domain with homologies to murine epidermal growth factor and a particular sequence of bovine clotting factor X, 2) The "kringle" domain with homologies to "kringle" structures, e.g. in plasminogen, and 3) the connecting peptide domain containing the A1 chain of low molecular mass urokinase. Together with the amino acid sequence of the B chain, which was presented by us in an earlier communication, the sequence data presented complete the primary structure of high molecular mass urokinase from human urine.

The primary structure of high molecular mass urokinase from human urine. The complete amino acid sequence of the A chain.

The sequence of all 253 amino acids of the heavy (B-) chain of human urinary urokinase was determined. The fragmentation strategy employed included cyanogen bromide cleavage of S-carboxymethylated B-chain at Met and/or Trp residues, cleavage of acid-labile Asp-Pro bonds, and the use of the specific endoproteinases Lys-C and Arg-C for generation of overlapping fragments. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. The amino acid sequence obtained substantiates the serine protease character of the B-chain of urokinase: a considerable homology with other serine proteinases, especially with the B-chain of human plasmin, was proved. The pertinent active site amino acids were localized: His-46, Asp-97, and Ser-198. A carbohydrate side chain, containing at least 4 glucosamine and 2 galactosamine residues, was demonstrated to be fixed at asparagine in position 144. The sequence data presented, together with the sequence of the second (A1-) chain of low molecular mass urokinase which was reported by us in an earlier communication, complete the knowledge of the whole primary structure of an active form of human urinary urokinase.

W. A. Günzler

Papers

Assays of glutathione peroxidase

An improved coupled test procedure for glutathione peroxidase (EC 1-11-1-9-) in blood.

Glutathione peroxidase, V. The kinetic mechanism.

The primary structure of high molecular mass urokinase from human urine. The complete amino acid sequence of the A chain.

The complete amino acid sequence of low molecular mass urokinase from human urine.