Prosphatase binding assay

Details Prosphatase binding assay Prosphatase binding assay

1997-11-04

2000-09-05

Ceperley, Mary E.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

435 771, 435188, G01N 33573, C12N 996

Patent

active

061141321

ABSTRACT:
Disclosed is a binding assay for proteases and phosphatases, which contain cysteine in their binding sites or as a necessary structural component for enzymatic binding. The sulfhydryl group of cysteine is the nucleophilic group in the enzyme's mechanistic proteolytic and hydrolytic properties. The assay can be used to determine the ability of new, unknown ligands and mixtures of compounds to competitively bind with the enzyme versus a known binding agent for the enzyme, e.g., a known enzyme inhibitor. By the use of a mutant form of the natural or native wild-type enzyme, in which serine, or another amino acid, e.g., alanine, replaces cysteine, the problem of interference from extraneous oxidizing and alkylating agents in the assay procedure is overcome. The interference arises because of oxidation or alkylation of the sulfhydryl, --SH (or --S.sup.-), in the cysteine, which then adversely affects the binding ability of the enzyme. Specifically disclosed is an assay for tyrosine phosphatases and cysteine proteases, including caspases and cathepsins, e.g., Cathepsin K(O2), utilizing scintillation proximity assay (SPA) technology. The assay has important applications in the discovery of compounds for the treatment and study of, for example, diabetes, immunosuppression, cancer, Alzheimer's disease and osteoporosis. The novel feature of the use of a mutant enzyme can be extended to its use in a wide variety of conventional colorimetric, photometric, spectrophotometric, radioimmunoassay and ligand-binding competitive assays.

REFERENCES:
patent: 5501969 (1996-03-01), Hastings et al.
A. Brown et al, Analytical Biochemistry, vol. 217, pp. 139-147 (1994).
R. H. Skinner et al., "Direct Measurement of the Binding of RAS to Neurofibtromin Using Scintillation Proximity Assay", Anal. Biochem., vol. 223, pp. 259-265 (1994).
J. A. Patcher et al., "Scintillation Proximity Assay to Measure Binding of Soluble Fibronectin to Antibody-Captured alpha5B1 Integrin", Anal. Biochem., vol. 230, pp. 101-107 (1995).
N. A. Tonks et al., "Characterization of the Major Protein-tyrosine-phosphatases of Human Placenta", JBC, pp. 6731-6737 (1988a).
H. Charbonneau et al., "The leukocyte common antigen (CD45): A putative receptor-linked protein tyrosine phosphatase", PNAS 85, pp. 7182-7186, (1998).
N. K. Tonks et al., "Demonstration That the Leukocyte Common Antigen CD45 is a Protein Tyrosine Phosphatase", Biochemistry 27, pp. 8695-8701, (1988b).
M. Acevedo Duncan et al., "Heterogeneity of Protein Kinase C Activity In Human U-373 and G-26 Mice Glioma Cells", Biochem. Biophys. Res Comm., 205, pp. 129-134 (1994).
M. Streuli et al., "A Family of receptor-linked protein tyrosine phosphatases in humans and Drosphila", Proc. Nat'l Acad USA, vol. 86, pp. 8698-7602, Nov. 1989.
J. Chernoff et al., "Cloning of a cDNA for a major human protein-tyrosine phosphatase", Proc. Nat'l Acad Sci. USA, vol. 87, pp. 2735-2739, Apr. 1990.
D. Bromme et al., "Human Cathepsin O2, a Novel Cysteine Protease Highly Expressed in Osteoclastomas and Ovary Molecular Cloning, Sequencing and Tissue Distribution" Biol. Chem. Hoppe-Seyler, vol. 376 pp. 379-384, Jun. 1995.
F. Drake et al., "Cathepsin K, but Not Cathespin B, L, or S, Is Abundantly Expressed in Human Ostheoclasts", J. Biol. Chem. vol. 271 , No., 21, pp. 12511-12516 , (1996).
T. Burke et al., "Potent Inhibition of Insulin Receptor Dephorylation by a Hexamer Peptide Containing the Phosphotyrosl Mimetic F2Pmp", Biochem. Biophys. Res. Comm., vol. 204 pp. 129-134, (1994).
R. Singh & G. M. Whitesides, "A Reagent for Reduction of Disulfide Bonds in Proteins That Reduces Disulfide Bonds Faster Than Does Dithiothreitol", J. Org. Chem. 56, pp. 2323-2337, (1991).
Yoshimura, T., et al.; J. of Biochem., vol. 108, No. 5, pp. 699-700, 1990.

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