Identification of a protein-tyrosine phosphatase from human platelet membranes by an immobilon-based solid phase assay.
The present investigations show that a 53-kDa platelet-membrane protein is a protein tyrosine phosphatase. Identification involved a novel methodology in which membrane proteins are resolved by polyacrylamide gel electrophoresis and then transblotted to a polyvinylidene difluoride membrane surface-labeled with [32P-Tyr](Glu4.Tyr1)n. Phosphatase activity appears as clear areas in the autoradiograph of the renatured 32P Western blot. Studies using this new solid-phase system indicate that a 53-kDa platelet-membrane protein dephosphorylates [32P-Tyr](Glu4.Tyr1)n. Dephosphorylation is both time- and dose-dependent. The protein-tyrosine phosphatase antagonists orthovanadate and molybdate block dephosphorylation in a concentration-dependent manner. Inhibitors of protein serine/threonine phosphatases and of acid and alkaline phosphatases do not significantly affect enzymatic activity. The enzyme is active toward phosphotyrosyl proteins but not phosphoseryl proteins. Results from the solid-phase phosphatase assays correlate with data from standard liquid-phase studies. A Lineweaver-Burk plot gives an apparent Km of 4.3 microM and an apparent Vmax = 168 nmol P(i) transferred/min/mg for the membrane enzyme towards the 32P-labeled polymer in a liquid system. Platelet lysate was shown by the solid-phase assay to possess a protein-tyrosine phosphatase of approximately equal to 50 kDa. The tyrosine phosphatase activities associated with a placental preparation, pure recombinant protein-tyrosine phosphatase 1B, and YOPR, the product of the YOP51 gene of Yersinia enterocolitica, which contains the C235R mutation, were also visualized by this technique. However, the catalytic activity of domain 1 of the transmembrane leukocyte antigen-related protein-tyrosine phosphatase was not detected using the solid-phase assay.
Dawicki
D D
DD
Division of Hematology/Oncology, Memorial Hospital of Rhode Island, Pawtucket 02860.
Steiner
M
M
D000595:Amino Acid Sequence; D001345:Autoradiography / Q000379:methods; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood / Q000302:isolation & purification; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010455:Peptides; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011145:Polyvinyls; D017027:Protein Tyrosine Phosphatases / Q000097:blood*; D005465:Fluorocarbon Polymers
D004229:Dithiothreitol; D008565:Membrane Proteins; D008567:Membranes, Artificial; D010455:Peptides; D010761:Phosphorus Radioisotopes; D011145:Polyvinyls; D017027:Protein Tyrosine Phosphatases; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers