Lytle C
Division of Biomedical Sciences, University of California, Riverside 92521, USA.
Am J Physiol. 1998 Apr;274(4):C1002-10. doi: 10.1152/ajpcell.1998.274.4.C1002.
When Na-K-2Cl cotransport is activated in duck red blood cells by either osmotic cell shrinkage, norepinephrine, fluoride, or calyculin A, phosphorylation of the transporter occurs at a common set of serine/threonine sites. To examine the kinetics and regulation of the activating kinase, phosphatase activity was inhibited abruptly with calyculin A and the subsequent changes in transporter phosphorylation and activity were determined. Increases in fractional incorporation of 32P into the transporter and uptake of 86Rb by the cells were closely correlated, suggesting that the phosphorylation event is rate determining in the activation process. Observed in this manner, the activating kinase was 1) stimulated by cell shrinkage, 2) inhibited by cell swelling, staurosporine, or N-ethylmaleimide, and 3) unaffected by norepinephrine or fluoride. The inhibitory effect of swelling on kinase activity was progressively relieved by calyculin A, suggesting that the kinase itself is switched on by phosphorylation. The kinetics of activation by calyculin A conformed to an autocatalytic model in which the volume-sensitive kinase is stimulated by a product of its own reaction (e.g., via autophosphorylation).
当通过渗透性细胞皱缩、去甲肾上腺素、氟化物或花萼海绵诱癌素A激活鸭红细胞中的钠 - 钾 - 2氯协同转运蛋白时，转运蛋白在一组共同的丝氨酸/苏氨酸位点发生磷酸化。为了研究激活激酶的动力学和调节机制，用花萼海绵诱癌素A突然抑制磷酸酶活性，并测定随后转运蛋白磷酸化和活性的变化。32P掺入转运蛋白的分数增加与细胞对86Rb的摄取密切相关，这表明磷酸化事件是激活过程中的速率决定因素。以这种方式观察到，激活激酶：1）受细胞皱缩刺激；2）受细胞肿胀、星形孢菌素或N - 乙基马来酰亚胺抑制；3）不受去甲肾上腺素或氟化物影响。花萼海绵诱癌素A可逐渐缓解肿胀对激酶活性的抑制作用，这表明激酶本身通过磷酸化而被激活。花萼海绵诱癌素A的激活动力学符合自催化模型，其中体积敏感激酶受其自身反应产物（例如，通过自磷酸化）刺激。
