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. 1980 Oct;77(10):5967–5971. doi: 10.1073/pnas.77.10.5967

Inhibiting the onset of hormone-induced desentiziation of viable thymocytes by N alpha-tosyl-L-lysine chloromethyl ketone.

Y Zick, R Cesla, S Shaltiel
PMCID: PMC350193  PMID: 6255472

Abstract

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.

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Selected References

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