Brunati A M, Marchiori F, Pinna L A
FEBS Lett. 1985 Sep 2;188(2):321-5. doi: 10.1016/0014-5793(85)80395-1.
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 mR 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 mR 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 km values with TK-I and TK-II, (100 and 10 microM, respectively). When TK-I was incubated with [gamma-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.
当大鼠脾脏颗粒部分的提取物进行DEAE - 纤维素梯度色谱分析时，可以分辨出酪氨酸蛋白激酶活性的三个峰（TK - I、TK - II和TK - III）。对表皮生长因子（EGF）和胰岛素均不敏感的TK - I和TK - II，已通过Sephacryl S200凝胶过滤进一步纯化并进行了特性鉴定。TK - I的表观相对分子质量为65000，作为激活剂，它远比Mg2 +更喜欢Mn2 +，除了ATP外还能使用GTP作为磷酸供体，并受到多聚赖氨酸2 - 3倍的刺激。TK - II的相对分子质量约为50000，被Mg2 +和Mn2 +同等激活，不使用GTP，对多聚赖氨酸不敏感。TK - I和TK - II可以磷酸化合成肽Asp - Ala - Glu - Tyr - Ala - Ala - Arg - Arg - Arg - Gly（以及用鸟氨酸取代精氨酸的衍生物）、血管紧张素II和聚（Glu，Tyr）4:1，它们与TK - I和TK - II表现出不同的米氏常数（分别为100和10微摩尔）。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的一对碱稳定的放射性标记条带。在相同条件下，TK - II产生一条51 kDa的单一碱稳定放射性标记条带，这可能代表TK - II自身的自磷酸化产物。
