title: Purification, identification and subcellular distribution of three predominant protein-tyrosine phosphatase enzymes in skeletal muscle tissue.
abstract: Protein-tyrosine phosphatases (PTPases) play a key role in the regulation of insulin action. In order to identify PTPases in skeletal muscle, the major site of insulin-mediated glucose disposal in vivo, we purified PTPases from rat muscle tissue fractions by a series of column chromatographic techniques. PTPase activities were assayed by measuring the dephosphorylation of a rat insulin receptor kinase domain, derivatized lysozyme and p-nitrophenylphosphate, and the enzymes were further characterized by immunoblotting. Of the total PTPase activity in muscle homogenates, 51-64% was localized to the solubilized particulate fraction, with the specific PTPase activity 3.3-fold and 5.6-fold higher in the particulate fraction towards RCM-lysozyme or the insulin receptor, respectively. The major peak (> 75%) of PTPase activity in the particulate fraction was purified further to 700-fold; 75% of this activity passed through a Blue-3GA column and revealed immunoreactivity for both LAR and SH-PTP2. PTPase activity retained on the Blue-3GA column contained PTPase1B. The major peak (> 70%) from muscle cytosol was further purified to 1500-fold. After the Blue-3GA step, immunoblotting revealed both SH-PTP2 and PTPase1B in the cytosol fraction, but LAR was absent from this fraction. LRP (RPTP-alpha) was not detected by blotting the PTPase activities from the purified particulate or cytosol fractions. Immunodepletion studies demonstrated that LAR, SH-PTP2 and PTPase1B were quantitatively major PTPase activities in the initial muscle homogenate, together accounting for over 70% of the total activity towards RCM-lysozyme. These studies provide insight into the relative abundance and subcellular distribution of specific PTPases in muscle tissue that are involved in the regulation of reversible tyrosine phosphorylation in this tissue.
authors.affiliation: Dorrance H. Hamilton Research Laboratory, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: F
authors.initials: F
authors.lastname: Ahmad
authors.forename: B J
authors.initials: BJ
authors.lastname: Goldstein
mesh_terms: D000818:Animals; D015151:Immunoblotting; D008297:Male; D009132:Muscles / Q000201:enzymology* / Q000648:ultrastructure; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000737:chemistry; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D011972:Receptor, Insulin; D017027:Protein Tyrosine Phosphatases