Development of a robust scintillation proximity assay for protein tyrosine phosphatase 1B using the catalytically inactive (C215S) mutant.
Anal Biochem
; 291(2): 269-78, 2001 Apr 15.
Article
em En
| MEDLINE
| ID: mdl-11401301
ABSTRACT
Protein tyrosine phosphatases are a class of enzymes that function to modulate tyrosine phosphorylation of cellular proteins and play an essential role in regulating cell function. PTP1B has been implicated in the negative regulation of the insulin signaling pathway by dephosphorylating the activated insulin receptor. Inhibiting this phosphatase and preventing the insulin-receptor downregulation has been suggested as a target for the treatment of Type II diabetes. A high-throughput screen for inhibitors of PTP1B was developed using a scintillation proximity assay (SPA) with GST-- or FLAG--PTP1B((1-320)) and a potent [(3)H]-tripeptide inhibitor. The problem of interference from extraneous oxidizing and alkylating agents which react with the cysteine active-site nucleophile was overcome by the use of the catalytically inactive C215S form of the native enzyme (GST--PTP1B(C215S)). The GST--PTP1B was linked to the protein A scintillation bead via GST antibody. The radiolabeled inhibitor when bound to the enzyme gave a radioactive signal that was competed away by the unknown competitive compounds. Further utility of PTP1B(C215S) was demonstrated by mixing in the same well both the catalytically inactive GST--PTP1B(C215S) and the catalytically active FLAG--CD45 with an inhibitor. Both a binding and kinetic assay was then performed in the same 96-well plate with the inhibition results determined for the PTP1B(C215S) (binding assay) and CD45 (activity assay). In this way inhibitors could be differentiated between the two phosphatases under identical assay conditions in one 96-well assay plate. The use of a mutant to reduce interference in a binding assay and compare with activity assays is also amenable for most cysteine active-site proteases.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ensaio Radioligante
/
Proteínas Tirosina Fosfatases
/
Inibidores Enzimáticos
Limite:
Animals
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2001
Tipo de documento:
Article