Zick Y, Cesla R, Shaltiel S
Proc Natl Acad Sci U S A. 1980 Oct;77(10):5967-71. doi: 10.1073/pnas.77.10.5967.
N alpha-Toysl-L-lysine chloromethyl ketone (Tos-LysCH2Cl) was found to inhibit irreversibly the onset of the hormone-induced refractory state in intact thymocytes. When thymocytes (approximately 2 X 10(7) cells per ml) are treated with Tos-LysCH2Cl(10(-4) M, for 90 min at pH 7 and 37 degrees C) the cells retain their viability, including a full capacity to recognize and respond to hormonal stimuli, yet they selectively lose their ability to become desensitized to persistent triggering by a hormone, as reflected in the state of activation of intracellular cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). Whereas upon hormonal stimulation of untreated cells the immediate rise in the state of activation of this enzyme (up to an activity ratio of > 0.85) is followed by an exponential decline to basal values within approximately 60 min, in TosLysCH2Cl-treated cells the hormone-triggered elevation in the state of activation of the enzyme is maintained for > 60 min. Evidence is presented to suggest that in thymocytes TosLysCH2Cl inhibits the regulatory process that normally uncouples the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] system without interfering with previous or subsequent molecular events connected with the transfer of hormonal signals across the cell membrane. This technique allows, therefore, the preparation of viable thymocytes with a limited and distinct regulatory defect introduced by chemical (covalent) means. As such, it is most useful for studies aimed at the elucidation of the mechanism of cell desensitization and for further characterization and localization of key components responsible for cellular refractoriness.
发现Nα-对甲苯磺酰-L-赖氨酸氯甲基酮（Tos-LysCH2Cl）能不可逆地抑制完整胸腺细胞中激素诱导的不应期状态的起始。当胸腺细胞（每毫升约2×10⁷个细胞）用Tos-LysCH2Cl（10⁻⁴M，在pH 7和37℃下处理90分钟）处理时，细胞保持其活力，包括完全有能力识别和响应激素刺激，但它们选择性地丧失了对激素持续触发产生脱敏的能力，这反映在细胞内环磷酸腺苷依赖性蛋白激酶（ATP：蛋白质磷酸转移酶，EC 2.7.1.37）的激活状态上。未经处理的细胞在激素刺激后，该酶的激活状态立即升高（活性比>0.85），随后在约60分钟内呈指数下降至基础值，而在Tos-LysCH2Cl处理的细胞中，激素触发的酶激活状态升高持续>60分钟。有证据表明，在胸腺细胞中，Tos-LysCH2Cl抑制了正常情况下使腺苷酸环化酶[ATP焦磷酸裂解酶（环化），EC 4.6.1.1]系统解偶联的调节过程，而不干扰与激素信号跨细胞膜传递相关的先前或后续分子事件。因此，这种技术可以制备具有通过化学（共价）手段引入的有限且独特调节缺陷的活胸腺细胞。因此，它对于旨在阐明细胞脱敏机制以及进一步表征和定位负责细胞不应性的关键成分的研究非常有用。
