ISOLATION AND PARTIAL CHARACTERIZATION OF DISTINCT FORMS OF TYROSINE PROTEIN-KINASES FROM RAT SPLEEN
ISOLATION AND PARTIAL CHARACTERIZATION OF DISTINCT FORMS OF TYROSINE PROTEIN-KINASES FROM RAT SPLEEN
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DOI：
10.1016/0014-5793(85)80395-1
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发表时间：
1985-01-01
期刊：
影响因子：
3.5
通讯作者：
PINNA, LA
中科院分区：
文献类型：
--
作者：
BRUNATI, AM;MARCHIORI, F;PINNA, LA
大鼠脾颗粒级分提取物经DEAE-纤维素梯度层析可分离出酪氨酸蛋白激酶活性的三个峰（TK-I、TK-II和TK-III）。 TK-I 和 TK-II 对 EGF 和胰岛素均不敏感，已通过 Sephacryl S200 凝胶过滤进一步纯化并进行表征。 TK-I 的表观分子量为 65000，迄今为止更喜欢使用 Mn2+ 而非 Mg2+ 作为激活剂，除 ATP 外还可以使用 GTP 作为磷酸盐供体，并被聚赖氨酸刺激 2-3 倍。 TK-II 的分子量约为 50000，对 Mg2+ 和 Mn2+ 具有同等的激活作用，不使用 GTP，对聚赖氨酸不敏感。 TK-I 和 TK-II 可以磷酸化合成肽 Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly（及其用 Orn 代替 Arg 的衍生物）、血管紧张素 II 和聚（Glu，Tyr）4:1，与 TK-I 和 TK-II 表现出不同的 km 值（分别为 100 和 10 μM）。当 TK-I 与 [γ-32P]ATP 和 MnCl2 一起孵育时，观察到分子量为 55 和 60 kDa 的碱稳定放射性标记带的双峰。在相同条件下，TK-II 产生 51 kDa 的单个碱稳定放射性标记带，这可能代表 TK-II 本身的自磷酸化产物。
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 apparentmrof 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, whosemrapproximates 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 differentkm, values with TK-I and TK-II, (100 and 10 μM, respectively). When TK-I was incubated with [γ-32P]ATP and MnCl2a 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.