Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Oxidoreductases
Patent
1992-05-13
1994-11-22
Wax, Robert A.
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Oxidoreductases
435189, 4352402, 4353201, 536 232, A61K 3750, C12N 510, C12N 902, C12N 1553
Patent
active
053667298
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a polypeptide having the superoxide dismutating property of native extracellular superoxide dismutase and a modified, i.e. reduced or increased, affinity for heparin compared to extracellular superoxide dismutase of type C, methods of producing the polypeptide and the use thereof for the purpose of therapeutic treatment.
Organisms living in the presence of oxygen have been forced to develop a number of protective mechanisms against toxic oxygen reduction metabolites, such as superoxide radicals, which are formed in connection with a variety of biological oxidations. The protective factors include superoxide dismutases (SOD) (superoxide:superoxide oxidoreductase, EC 1.15.1.1) which dismutate the superoxide radical and are found in relatively constant amounts in mammalian cells and tissue. The best known of these enzymes is CuZn SOD which is a dimer with a molecular weight of 33,000 containing two copper and two zinc atoms. CuZn SOD is found in the cytosol and in the intermembrane space of the mitochondria. Mn SOD is a tetramer with a molecular weight of 85,000 containing 4 Mn atoms, and is mainly located in the mitochondrial matrix. Recently, a superoxide dismutase was found in the extracellular fluids of mammals, birds and fish. This superoxide dismutase has been denoted extracellular superoxide dismutase which in the following will be termed EC-SOD.
EC-SOD is a tetrameric Cu and Zn containing glycoprotein (S. L. Marklund, Proc. Natl. Acad. Sci. USA. 79, 1982, pp. 7634-7638; L. Tibell et al., Proc. Natl. Acad. Sci. USA. 84, 1987, pp. 6634-6638). A cDNA sequence encoding EC-SOD has been elucidated and useful applications of the cDNA and the EC-SOD encoded thereby are described in WO 87/01387, the contents of which are hereby incorporated by reference. The cDNA sequence is the sequence shown in FIG. 1A and FIG. 1B corresponding to amino acid residues 1-222 (without any mutations). EC-SOD is a secretory protein and the cDNA encodes an 18 amino acids long signal sequence which is absent in mature and recombinant EC-SOD (L. Tibell et al., Proc. Natl. Acad. Sci. USA. 84, 1987, pp. 6634-6638). As deduced from the cDNA sequence, the subunit molecular weight of the mature enzyme is 24,200. The exact size of the carbohydrate substituent is not known, but the apparent molecular weight on gel chromatography of the tetramer is 140-150 kDa. On SDS-PAGE electrophoresis the subunits display a molecular weight of 30-32 kDa (L. Tibell et al., Proc. Natl. Acad. Sci. USA. 84, 1987, pp. 6634-6638). The sequence contains one glycosylation site Asn-89 (K. Hjalmarsson et al., Proc. Natl. Acad. Sci. USA. 84, 1987, pp. 6340-6344), and the mature enzyme binds to the lectins concanavalin A, wheat germ lectin and lentil lectin. The tetramers contain 4 Cu and 4 Zn atoms. The active site, which contains the metal atoms, is homologous to the active site of the intracellular CuZn SODs (K. Hjalmarsson et al., Proc. Natl. Acad. Sci. USA. 84, 1987, pp. 6340-6344).
EC-SOD isolated from tissues and plasma is heterologous with regard to heparin affinity, and can upon chromatography on Heparin-Sepharose be divided into three subclasses;
in vivo the correlate of the heparin affinity is binding to heparan sulfate proteoglycan (K. Karlsson and S. L. Marklund, Biochem. J. 242, 1987, pp. 55-59 , K. Karlsson et al., Biochem. J. 256, 1988, pp. 29-33; S. L. Marklund and K. Karlsson, Lab. Invest.in press, 1989), which occurs on cell surfaces and in the interstitial connective tissue.
In the vasculature, EC-SOD of subclass C, which in the following is termed EC-SOD C, exists in equilibrium between the plasma phase and heparan sulfate proteoglycan in the glycocalyx of the vessel endothelium (K. Karlsson and S. L. Marklund, Biochem. J. 242, 1987, pp. 55-59; K. Karlsson and S. L. Marklund, J. Clin. Invest. 82, 1988, pp. 762-766; K. Karlsson and S. L. Marklund, Biochem. J. 255, 1988, pp. 223-228). Upon injection of heparin, bound enzyme is released to plasma because it binds to the heparin instead of the heparan sulfate. This
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Edlund Thomas
Marklund Stefan
Cooper Iver A.
Grimes Eric
Symbicom Aktiebolag
Wax Robert A.
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