Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-05-02
1999-01-19
Spiegel, Carol A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435961, 435975, 436518, 436543, 436547, 436808, 530327, 5303879, 530402, 530403, 530806, 530810, G01N 33573
Patent
active
058612629
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for assaying human plasma glutathione peroxidase (pl.GPx) and a ready-for-use kit for the implementation of the said method; it is based on the selection of peptide sequences of pl.GPx and the in vitro synthesis of the said sequences as well as on the production and use of antibodies against the said sequences and which specifically recognize pl.GPx.
BACKGROUND OF THE INVENTION
Three types of human glutathione peroxidase have been characterized: plasma glutathione peroxidase (pl.GPx), intracellular glutathione peroxidase (c.GPx) which is essentially active on hydrophilic substrates, and glutathione peroxidase which is active on phospholipid hydroperoxides as well as on other lipid substrates (PHGPx). These enzymes catalyse the reduction of hydroperoxides (H.sub.2 O.sub.2 or ROOH) by glutathione (GSH). These enzymes are selenoproteins which contain a selenocysteine within their active site. c.GPx and pl.GPx are homotetramers whereas PHGPx is exclusively monomeric Biochem. Biophys. Acta, 839, 62-70 (1985).
Originally purified from plasma J. Biol. Chem., 262, No.36, 17398-17403 (1987); Arch. Biochem. Biophys., 256,No.2, 677-686 (1987), pl.GPx represents 0.007% of the total plasma protein mass. pl.GPx has also been isolated from human maternal milk J. Nutr., 121, No.8, 1243-1249 (1991).
Of glycoprotein nature, pl.GPx exists in the form of a homotetramer of a molecular weight of 94 kDa J. Biol. Chem., 262, No.36, 17398-17403 (1987). Each subunit, characterized by a molecular weight of 21.5 to 23 kDa J. Biol. Chem., 262, No.36, 17398-17403 (1987); J. Biochem., 108, 145-148 (1990), contains a selenocysteine in its active site.
Recently, a study performed in rats has shown that pl.GPx is predominantly synthesized by the renal cell (Yoshimura et al. J. Biochem. 109; (1991); 918-923) whereas another study has shown that a tumorous hepatic cell line (Hep G2) of human origin is capable of synthesizing this pl.GPx (AVISSAR et al. J. Biol. Chem. 264; 1989; 15850-15855).
Given the low concentrations of plasma glutathione, the principal role of pl.GPx remains uncertain. The specific activity of pl.GPx is about ten times lower than that of c.GPx Arch. Biochem. Biophys., 256, No.2, 677-686 (1987).
The primary sequence of human pl.GPx was determined from the cDNA and published by Takahashi et al. J. Biochem., 108; (1990); 145-148. It was not possible to completely sequence the pl.GPx and to identify the N-terminal end because of the glycosylation affecting this end. The exact number of residues per subunit therefore remains unknown.
pl.GPx differs considerably from c.GPx from the structural point of view:. absence of glycosylation and intramolecular disulphide bridges for the cellular form Arch. Biochem. Biophys., 256, 677-686 (1987); Blood, 73, 318-323 (1989), weak sequence homology between the two enzymes (of the order of 44% Nucleic Acids Res., 15; (1987); 5484; Nucleic Acids Res., 15; (1987); 7178).
Measurement of the pl.GPx level in plasma is a good indicator of a possible state of selenium deficiency in the body. Within the framework of experiments designed to quantify the influence of a deficiency and then of a supply of selenium in man, it has been shown that the average glutathione peroxidase levels do not vary at the same rate depending on whether the plasma form or the cellular form is involved. Thus, the pl.GPx level in plasma begins to increase from the early days of treatment to reach a normal value after two to four weeks. The time necessary for the value of the normal level of c.GPx to be reestablished is longer: three to four months Amer. J. Clin. Nutr., 41; (1985); 735-747.
Furthermore, and following the studies previously carried out in rats, a number of renal pathologies could be detected by the measurement of pl.GPx.
None of the known methods of assaying enzymatic activity currently permit the specific assay of pl.GPx in biological fluids or in tissue extracts.
The most commonly used method of assaying glutathione peroxidase activity involves an enzymatic coup
REFERENCES:
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patent: 5187078 (1993-02-01), Ohya et al.
Harlow, E. and D. Lane, Antibodies, Cold Spring Harbor Laboratory, pp. 86-87, 1988.
Avissar, N. et al., Blood, vol. 73, No. 1, pp. 318-323, Jan. 1989.
Yoshimura, S. et al., J. Biochem., vol. 109, pp. 918-923, 1991.
Chu et al., "Expression of Plasma Glutathione Peroxidase in Human Liver . . . " Blood, vol. 79, No. 12, pp. 3233-3238, Jun. 15, 1992.
Yoshimura et al., "The human plasma glutathione peroxidase--encoding gene . . . ," Gene, vol. 145, 293-297, 1994.
Esworthy et al., "Characterization and Partial Amino Acid Sequence of Human Plasma Glutathione Peroxidase," Arch. Biochem. Biophys. 286(2):330-336, 1991.
Journal of Biochemistry., vol. 108, 1990, Tokyo JP pp. 145-148 K. Takahashi et al. Primary structure of human plasma glutathion peroxidase deduced from cDNA sequences'.
Proceedings of The National Academy of Sciences of USA., vol. 81 Jul. 1984, pp. 3998-4002; H.M. Geystn et al. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid'.
Proceedings of The National Academy of Sciences of USA., vol. 82, Jan. 1985, Washington US pp. 178-182; H.M. Geysten et al. "Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised against the native protein".
Chaudiere Jean
Lemainque Arnaud
Malette Patricia
Oxis Isle of Man, Limited
Spiegel Carol A.
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