Dephosphorylation of tyrosine phosphorylated synthetic peptides by rat liver phosphotyrosine protein phosphatase isoenzymes.
Five phosphotyrosine-containing peptides have been synthesized by FMOC solid-phase peptide synthesis. These peptides correspond to the 411-419 sequence of the Xenopus src oncogene, to the 1191-1220 sequence of the human EGF receptor precursor, to the 1146-1158 sequence of the human insulin receptor, to the 856-865 sequence of the human beta-PDGF receptor, and to the 5-16 sequence of the erythrocyte human band 3. The peptides were used as substrates for activity assay of two isoforms (AcP1 and AcP2) of a low molecular weight cytosolic PTPase. The assay, performed in microtiter EIA plates using Malachite green to determine the released phosphate, was rapid, reproducible, and sensitive. Both PTPase isoforms were able to hydrolyze all synthesized peptides, though with different affinity and rate. The main kinetic parameters were compared and discussed with respect to the role of the two enzymes in the cell.
Stefani
M
M
Department of Molecular Biology, University of Siena, Italy.
Caselli
A
A
Bucciantini
M
M
Pazzagli
L
L
Dolfi
F
F
Camici
G
G
Manao
G
G
Ramponi
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014981:Xenopus
D001457:Anion Exchange Protein 1, Erythrocyte; D007527:Isoenzymes; D010446:Peptide Fragments; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D066246:ErbB Receptors; D011972:Receptor, Insulin; D017479:Receptors, Platelet-Derived Growth Factor; D016392:Proto-Oncogene Proteins pp60(c-src); D017027:Protein Tyrosine Phosphatases