Tremblay L, Gingras D, Boivin D, Béliveau R
Département de chimie-biochimie, Université du Québec à Montréal, Canada.
Biochim Biophys Acta. 1992 Jul 27;1108(2):183-9. doi: 10.1016/0005-2736(92)90024-g.
Tyrosine protein kinase (TPK) activity was detected in rat renal brush-border membranes (BBM) using poly(Glu80Na,Tyr20) as a substrate. Maximal TPK activity required prior detergent dispersion of the membranes with 0.05% Triton X-100 and the presence of vanadate, a potent inhibitor of phosphotyrosine protein phosphatases, in the phosphorylation medium. Optimal conditions for measurement of TPK activity were 10 mM of MgCl2 and MnCl2, at 30 degrees C and pH 7.0. TPK activity was inhibited by genistein, with a IC50 value of 15 microM, while no inhibition was observed in the presence of 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H7), an inhibitor of serine-threonine kinases. TPK activity was enriched 4-fold in the BBM fraction relative to cortex homogenate. It was co-enriched with BBM enzyme markers, but not with those of the basolateral membrane (BLM). The endogenous substrates of TPK in brush-border and basolateral membranes were determined by Western blot analysis using an antiphosphotyrosine monoclonal antibody (PY20). Various phosphotyrosine-containing proteins were found in the BBM (31, 34, 46, 50, 53, 72, 90, 118 and 170 kDa) and in the BLM (37, 48, 50, 53, 72, 90, 130 and 170 kDa). Addition of exogenous insulin receptor to BBM and BLM increased the phosphorylation of most of the substrates. Solubilization of the TPK activity from BBM with 0.5% CHAPS and subsequent gel filtration on Superdex 75 yielded two peaks of tyrosine protein kinase activity with apparent molecular masses of 49 and 66 kDa. These results provide evidence for a non-receptor TPK activity associated with the renal tubular luminal membrane.
以聚（Glu80Na，Tyr20）为底物，在大鼠肾刷状缘膜（BBM）中检测到酪氨酸蛋白激酶（TPK）活性。最大TPK活性需要先用0.05% Triton X - 100对膜进行去污剂分散处理，并在磷酸化介质中存在钒酸盐（一种有效的磷酸酪氨酸蛋白磷酸酶抑制剂）。测量TPK活性的最佳条件是10 mM的MgCl2和MnCl2，温度为30℃，pH值为7.0。染料木黄酮可抑制TPK活性，IC50值为15 μM，而在存在丝氨酸 - 苏氨酸激酶抑制剂1 -（5 - 异喹啉磺酰基）- 2 - 甲基 - 哌嗪二盐酸盐（H7）的情况下未观察到抑制作用。相对于皮质匀浆，BBM组分中的TPK活性富集了4倍。它与BBM酶标志物共同富集，但与基底外侧膜（BLM）的标志物不同。使用抗磷酸酪氨酸单克隆抗体（PY20）通过蛋白质免疫印迹分析确定刷状缘膜和基底外侧膜中TPK的内源性底物。在BBM（31、34、46、50、53、72、90、118和170 kDa）和BLM（37、48、50、53、72、90、130和170 kDa）中发现了各种含磷酸酪氨酸的蛋白质。向BBM和BLM中添加外源性胰岛素受体会增加大多数底物的磷酸化。用0.5% CHAPS从BBM中溶解TPK活性，随后在Superdex 75上进行凝胶过滤，得到两个酪氨酸蛋白激酶活性峰，表观分子量分别为49 kDa和66 kDa。这些结果为与肾小管腔膜相关的非受体TPK活性提供了证据。
