Use of an anaerobic environment to preserve the endogenous activity of protein-tyrosine phosphatases isolated from intact cells.

Abstract

SPECIFIC AIMSProtein-tyrosine phosphatases (PTPases) have a catalytic cysteine residue whose reduced state is required for enzymatic activity. In this study, we evaluated how a strictly anaerobic working environment with deoxygenated buffers and avoiding exposure to air permits the assessment of endogenous PTPase activity as directly isolated in subcellular fractions. Using similar techniques, we also studied the reactivity of a specific PTPase, PTP1B, by immunoprecipitation. This approach provides a new framework for characterizing the activity of PTPases as isolated from the intracellular milieu that more closely reflects their endogenous reactivity and their potential effect on signal transduction pathways involving reversible protein-tyrosine phosphorylation.PRINCIPAL FINDINGS1. Cellular PTPase activity from 3T3-L1 adipocytes is reversible to a variable extent after oxidative inhibition by exposure to airThe effect of air exposure on PTPases isolated from differentiated murine 3T3-L1 adipocytes was ev...