The major form of protein tyrosine kinase in the dog prostate is expressed by a 50 kDa polypeptide
The major form of protein tyrosine kinase in the dog prostate is expressed by a 50 kDa polypeptide
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狗前列腺中蛋白酪氨酸激酶的主要形式由 50 kDa 多肽表达
DOI：
10.1016/0014-5793(92)80382-q
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发表时间：
1992
期刊：
影响因子：
3.5
通讯作者：
S. Chevalier
中科院分区：
文献类型：
--
作者：
L. Nguyễn;Y. Durocher;A. Chapdelaine;S. Chevalier
我们已经报道，狗前列腺中的蛋白酪氨酸激酶(PTK)活性分布在胞质(75%)和颗粒(Triton X-100增溶)组分中，并且在凝胶过滤后，这两种PTK都作为MR44000[(1991)生物化学]的实体迁移。牢房。比奥尔。69、146-153]。在这里，我们用抗磷酸酪氨酸抗体的免疫沉淀法证明，可溶性的PTK具有自我磷酸化的能力。此外，负责该酶活性的多肽已通过两种方法鉴定：(1)双向电泳，第一维在非变性条件下进行，允许定位天然酶，而第二维(SDSPAGE)允许分析与其活性相对应的耐碱性磷酸蛋白；(2)蛋白质经十二烷基硫酸钠-PAGE复性后，与酶制剂一起进行多聚GT的原位磷酸化；(2)蛋白质复性后，与酶制剂一起进行的多聚GT的原位磷酸化；放射性标记的磷酸酪氨酸在复性蛋白中的唯一存在，证实了它的酶性质。利用这些方法，狗前列腺中PTK的主要形式被证明是由一个50 kDa的多肽表达的，该多肽具有自磷酸化位点，并以活性单体的形式存在于细胞质中。
We have already reported that the protein tyrosine kinase (PTK) activity in the dog prostate is distributed in cytosolic (75%) and particulate (Triton X-100-solubilized) fractions and that upon gel filtration, both PTKs migrate as entities ofMr44 000 [(1991) Biochem. Cell. Biol. 69, 146–153]. Herein we demonstrate by immunoprecipitation with anti-phosphotyrosine antibodies that the soluble PTK has the ability to undergo self-phosphorylation. In addition, the polypeptide responsible for that enzymatic activity has been identified by 2 approaches; (1) a two-dimensional electrophoresis, in which the first dimension performed in non-denaturing conditions allowed the localization of the native enzyme, while the second dimension (SDS-PAGE) permitted the analysis of alkali-resistant phosphoproteins corresponding to the activity; (2) protein renaturation after SDS-PAGE followed by in situ phosphorylation (with [γ-32P]ATP) of polyGT electrophoresed together with the enzyme preparation; the exclusive presence of the radiolabeled phosphotyrosine in the renatured protein confirmed its enzymatic nature. Using these methods, the major form of PTK in the dog prostate was shown to be expressed by a 50 kDa polypeptide which possesses autophosphorylation sites and which is present in the cytosol as an active monomer.