Isolation and partial characterization of distinct forms of tyrosine protein kinases from rat spleen

FEBS Letters

Brunati, Anna Maria²  Marchiori, Fernando¹  Pinna, Lorenzo A.² 

[1] Centro Studi Biopolimeri CNR, Padova, Italy;Istituto di Chimica Biologica dell' Università di Padova, via Marzolo 3, 35131 Padova, Italy

关键词: Protein kinase;    Phosphotyrosine;    Protein phosphorylation;    (Spleen);    D10G1 and D10G1-Orn;    peptides Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly and its derivative with Orn in place of Arg;    EGF;    epidermal growth factor;    SDS-PAGE;    11% polyacrylamide gel electrophoresis in 0.1% SDS;    PMSF;    phenylmethylsulfonyl fluoride;    poly(Glu;    Tyr) 4:1;    synthetic random polymer with a 4:1 ratio of glutamic acid residues and tyrosine residues;   

DOI  :  10.1016/0014-5793(85)80395-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Three peaks of tyrosine protein kinase activity (TK-I, TK-II and TK-III) can be resolved when the extract of rat spleen particulate fraction is subjected to DEAE-cellulose gradient chromatography. TK-I and TK-II, insensitive to both EGF and insulin, have been further purified by Sephacryl S200 gel filtration and characterized. TK-I has an apparent m _r of 65000, by far prefers Mn²⁺ over Mg²⁺ as activator, can use GTP besides ATP as phosphate donor and is stimulated 2-3-fold by polylysine. TK-II, whose m _r approximates 50000, is equally activated by Mg²⁺ and Mn²⁺, does not use GTP and is insensitive to polylysine. TK-I and TK-II can phosphorylate the synthetic peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly (as well as its derivative with Orn in place of Arg), angiotensin II and poly(Glu, Tyr) 4:1 which exhibits different k _m, values with TK-I and TK-II, (100 and 10 μM, respectively). When TK-I was incubated with [γ-³²P]ATP and MnCl₂ a doublet of alkali-stable radiolabeled bands with molecular masses of 55 and 60 kDa were observed. Under identical conditions TK-II gives rise to a single alkali-stable radiolabeled band of 51 kDa, which may represent the autophosphorylation product of TK-II itself.

【 授权许可】

Unknown