DETECTION OF C-ABL TYROSINE KINASE-ACTIVITY INVITRO PERMITS DIRECT COMPARISON OF NORMAL AND ALTERED ABL GENE-PRODUCTS
DETECTION OF C-ABL TYROSINE KINASE-ACTIVITY INVITRO PERMITS DIRECT COMPARISON OF NORMAL AND ALTERED ABL GENE-PRODUCTS
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DOI：
10.1128/mcb.5.11.3116
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发表时间：
1985-01-01
影响因子：
5.3
通讯作者：
WITTE, ON
中科院分区：
文献类型：
--
作者：
KONOPKA, JB;WITTE, ON
费城染色体阳性慢性粒细胞白血病细胞中c-abl基因易位的v-abl转化蛋白P160 v-abl和P210 c-abl基因产物具有酪氨酸特异性蛋白激酶活性。在类似的测定条件下，正常的c-abl基因产物，鼠P150 c-abl和人P145 c-abl，缺乏可检测的激酶活性。改变反应条件以鉴定允许检测c-abl酪氨酸激酶活性的条件。发现以前用于免疫沉淀的福尔马林固定的金黄色葡萄球菌抑制体外abl激酶活性。此外，以前在细胞裂解缓冲液中使用的十二烷基硫酸钠和脱氧胆酸盐去污剂用于降低回收的abl激酶活性。c-abl激酶活性测定条件的发现，使得将P150 c-abl和P145 c-abl激酶活性与改变的abl蛋白P160 v-abl和P210 c-abl进行比较成为可能。比较abl蛋白在体外和体内的酪氨酸磷酸化位点，表明它们在体内的功能不同。c-abl激酶测定条件的发展应有助于阐明c-abl功能。
The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were used to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.