Characterization of phosphotyrosine phosphatase activity in sheep platelets: amphiphilic and hydrophilic forms.
Using O-phosphotyrosine as a substrate, we characterized the phosphotyrosine phosphatase (PTPase; protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) activity from sheep platelets. PTPase was found to be located in three particulate subcellular fractions and in the cytosol, with K(m) values in the millimolar range. PTPase was strongly inhibited by vanadate, molybdate and HgCl2 and only weakly inhibited by Zn2+. Other divalent cations and NaF had no significant effect on the activity associated with the membrane fraction but were slightly stimulatory as regards cytosolic activity. Heparin inhibited cytosolic activity 2-fold more than membrane-bound activity and dithiothreitol only inhibited cytosolic PTPase. Polycationic compounds were seen to be weak stimulators of all the PTPase activity. Solubilization of the PTPase from membranes always required a detergent. When subjected to Triton X-114 phase partitioning, PTPase was recovered in the detergent-rich (35%) and in the detergent-poor (65%) phases. Sedimentation analysis of the cytosolic PTPase showed a peak of 3.2S that remained unmodified when Triton X-100 or Brij 97 sucrose gradients were used. Sedimentation analysis of the membrane-associated PTPase showed 6S and 3.7S peaks unchanged in Triton X-100 or Brij 97 gradients together with 7.5S and 10.3S shoulders that shifted to smaller sedimentation coefficients in Brij 97 sucrose gradients. These results support the view that sheep platelets contain amphiphilic and hydrophilic forms of PTPase.
Hernández-Hernández
A
A
Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, Edificio Departmental, Spain.
Sánchez-Yagüe
J
J
Martín-Valmaseda
E M
EM
Llanillo
M
M
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002793:Cholic Acids; D003902:Detergents; D017830:Octoxynol; D011092:Polyethylene Glycols; D017027:Protein Tyrosine Phosphatases / Q000097:blood*; D012756:Sheep / Q000097:blood*; D012995:Solubility
D002793:Cholic Acids; D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D017027:Protein Tyrosine Phosphatases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate