Purification and characterization of T cell protein tyrosine phosphatase reveals significant functional homology to protein tyrosine phosphatase-1B.
We have developed a protocol for rapid purification of T cell protein tyrosine phosphatase (TCPTP) and the structurally related protein tyrosine phosphatase-1B (PTP-1B) from bacterial cells. The pH profile for TCPTP was bell-shaped with an optimum of 5.5. The catalytic domain and full-length versions of TCPTP bound a potent inhibitor with affinities similar to those of PTP-1B. The K(m) values for the catalytic domains of TCPTP and PTP-1B increased with increasing ionic strength, whereas the k(cat) values remained unchanged. Arrhenius plots revealed that TCPTP and PTP-1B possess similar activation energies of 25.3+/-1.2 and 18.4+/-3.0 kJ/mol, respectively. Increasing solvent microviscosity (up to 40% (w/v) sucrose) did not affect k(cat)/K(m) of either enzyme. However, high sucrose concentrations protected both enzymes from thermal inactivation. These studies show that, although they share a 72% amino acid sequence identity within their catalytic domains, TCPTP and PTP-1B are functionally very similar in vitro.
Romsicki
Yolanda
Y
Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Que., Canada H9R 4P8.
Kennedy
Brian P
BP
Asante-Appiah
Ernest
E
D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017124:Conserved Sequence; D004789:Enzyme Activation; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D054578:Protein Tyrosine Phosphatase, Non-Receptor Type 2; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000302:isolation & purification*; D011786:Quality Control; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D020539:Sequence Analysis, Protein / Q000379:methods; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014783:Viscosity
D011994:Recombinant Proteins; C516607:PTPN1 protein, human; C516649:PTPN2 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D054578:Protein Tyrosine Phosphatase, Non-Receptor Type 2; D017027:Protein Tyrosine Phosphatases