CHARACTERIZATION OF PHOSPHOTYROSINE PHOSPHATASE-ACTIVITY IN SHEEP PLATELETS - AMPHIPHILIC AND HYDROPHILIC FORMS

Using O-phosphotyrosine as a substrate, we characterized the phosphoty rosine phosphatase (PTPase; protein-tyrosine-phosphate-phosphohydrolas e, EC 3.1.3.48) activity from sheep platelets. PTPase was found to De located in three particulate subcellular fractions and in the cytosol, with K-n values in the millimolar range. PTPase was strongly inhibite d by vanadate, molybdate and HgCl2 and only weakly inhibited by Zn2+ O ther divalent cations and NaF had no significant effect on the activit y associated with the membrane fraction bur were slightly stimulatory as regards cytosolic activity. Heparin inhibited cytosolic activity 2- fold more than membrane-bound activity and dithiothreitol only inhibit ed cytosolic PTPase. Polycationic compounds were seen to be weak stimu lators of all the PTPase activity. Solubilization of the PTPase from m embranes always required a detergent. When subjected to Triton X-114 p hase partitioning, PTPase was recovered in the detergent-rich (35%) an d in the detergent-poor (65%) phases. Sedimentation analysis of the cy tosolic PTPase showed a peak of 3.2S that remained unmodified when Tri ton X-100 or Brij 97 sucrose gradients were used. Sedimentation analys is of the membrane-associated PTPase showed 6S and 3.7S peaks unchange d in Triton X-100 or Brij 97 gradients together with 7.5S and 10.3S sh oulders 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. (C) 1997 Elsevie r Science Inc.