Identification of a rat liver protein-tyrosine phosphatase similar to human placental PTPase-1B using quantitatively phosphorylated protein substrates.
Erythrocyte Band 3 protein (Band 3), brain microtubule associated protein 2 (MAP2), and tubulin were phosphorylated to high stoichiometries (1-6 mol Pi/mol protein) on tyrosine residues using a rat spleen protein-tyrosine kinase in the presence of polylysine. After total removal of polylysine, the quantitatively phosphorylated proteins as well as tyrosine-glutamate copolymer [Poly(Glu4, Tyr1)], which was also phosphorylated (1.5 mol/mol) by the kinase, were employed to assay rat liver protein-tyrosine phosphatases (PTPases). Of the four partially purified PTPases termed L1, L2, L3, and L4, PTPase L1 was previously purified to homogeneity and demonstrated to be a novel enzyme with sequence similarity to src-homology region 2 [Hiraga, A. et al. (1992) Eur. J. Biochem. 209, 195-206]. In the present work PTPase L2 was purified to near homogeneity by a procedure involving chromatography on DEAE-cellulose, Blue Sepharose CL-6B, hydroxylapatite, Phenyl Sepharose CL-4B, and TSKgel Heparin-5PW. PTPase L2 was purified 20,000-fold with a recovery of 0.9% from the extract and 0.005 mg was isolated from 300 g of liver. The highly purified PTPase L2 showed a major protein band of 36 kDa on SDS/polyacrylamide gel electrophoresis. PTPase L2 had a specific activity of about 6,000 nmol of P1 released min-1.mg-1 toward either Band 3 or poly(Glu4, Tyr1), the values being within the range of those obtained for PTPases purified thus far. PTPase L2 dephosphorylated Band-3 9-fold and 5-fold faster than tubulin and MAP2, respectively, under the assay conditions employed.(ABSTRACT TRUNCATED AT 250 WORDS)
Hiraga
A
A
Biochemistry Laboratory, Tohoku University, Sendai.
Hata
K
K
Suzuki
Y
Y
Tsuiki
S
S
D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010766:Phosphorylation; D010920:Placenta / Q000201:enzymology*; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D014404:Tubulin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D001457:Anion Exchange Protein 1, Erythrocyte; D005971:Glutamates; D008869:Microtubule-Associated Proteins; D014404:Tubulin; D018698:Glutamic Acid; D014443:Tyrosine; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases