Calpain activity increases in hepatocytes following addition of ATP. Demonstration by a novel fluorescent approach

B. G. Rosser, S. P. Powers, G. J. Gores

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Abstract

Our aim was to measure calpain protease activity during increases in cytosolic free calcium (Ca2+(i)) after addition of extracellular ATP. The calpain protease substrate t-butoxycarbonyl-Leu-Met-7-amino-4- chloromethylcoumarin was synthesized. Nonfluorescent t-butoxycarbonyl-Leu- Met-7-amino-4-chloromethylcoumarin diffuses into the cell where it is conjugated to glutathione forming t-butoxycarbonyl-Leu-Met-7-amino-4- methylcoumarin glutathione conjugate (Boc-Leu-Met-MAC-SG). The nonfluorescent, membrane impermeant Boc-Leu-Met-MAC-SG accumulates in the cell. Intracellular proteolytic hydrolysis of Boc-Leu-Met-MAC-SG releases and unquenches the fluorescence of MAC-SG. Intracellular fluorescence of MAC-SG was quantitated in single, cultured rat hepatocytes using digitized video fluorescent microscopy. Enhancement of intracellular fluorescence generation by increases in Ca2+(i) and inhibition by a calpain inhibitor indicated the probe was a calpain substrate. After addition of ATP, calpain protease activity increased to 156 ± 13% of basal concurrent with a 3-fold rise of Ca2+(i) for 2-4 min. Thereafter, Ca2+(i) decreased to values of 1.5-fold above basal and protease activity returned to normal. Incubation of cells in Ca2+-free buffer abolished the rise in Ca2+(i) and calpain protease activity. Calpain protease activity increases concomitantly with increases of Ca2+(i) supporting the hypothesis that calpain proteases participate in Ca2+-mediated signal transduction.

Original languageEnglish (US)
Pages (from-to)23593-23600
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number31
StatePublished - Jan 1 1993

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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