Nguyen L T, Durocher Y, Chapdelaine A, Chevalier S
Department of Biochemistry, University of Montreal, Canada.
FEBS Lett. 1992 Jan 20;296(2):215-8. doi: 10.1016/0014-5793(92)80382-q.
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 of Mr 44,000 [(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 [gamma-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.
我们已经报道过，犬前列腺中的蛋白酪氨酸激酶（PTK）活性分布于胞质（75%）和颗粒（经Triton X-100溶解）组分中，并且在凝胶过滤时，两种PTK均以44,000的Mr迁移[(1991) Biochem. Cell. Biol. 69, 146 - 153]。在此，我们通过用抗磷酸酪氨酸抗体进行免疫沉淀证明，可溶性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）二维电泳，其中在非变性条件下进行的第一维电泳可定位天然酶，而第二维（SDS-PAGE）允许分析与该活性相对应的耐碱磷蛋白；（2）SDS-PAGE后蛋白质复性，然后与酶制剂一起对电泳的聚GT进行原位磷酸化（用[γ-32P]ATP）；复性蛋白中放射性标记的磷酸酪氨酸的唯一存在证实了其酶的性质。使用这些方法，犬前列腺中PTK的主要形式被证明由一种50 kDa的多肽表达，该多肽具有自磷酸化位点，并且作为活性单体存在于胞质溶胶中。
