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

B. G. Rosser, S. P. Powers, Gregory James Gores

Research output: Contribution to journalArticle

103 Citations (Scopus)

Abstract

Our aim was to measure calpain protease activity during increases in cytosolic free calcium (Ca 2+(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 Ca 2+(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 Ca 2+(i) for 2-4 min. Thereafter, Ca 2+(i) decreased to values of 1.5-fold above basal and protease activity returned to normal. Incubation of cells in Ca 2+-free buffer abolished the rise in Ca 2+(i) and calpain protease activity. Calpain protease activity increases concomitantly with increases of Ca 2+(i) supporting the hypothesis that calpain proteases participate in Ca 2+-mediated signal transduction.

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

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leucylmethionine
Calpain
Hepatocytes
Peptide Hydrolases
Demonstrations
Adenosine Triphosphate
Fluorescence
Glutathione
Video Microscopy
Signal transduction
Substrates
Rats
Hydrolysis
Signal Transduction
Microscopic examination
Buffers
Cells
Calcium
Membranes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Calpain activity increases in hepatocytes following addition of ATP. Demonstration by a novel fluorescent approach. / Rosser, B. G.; Powers, S. P.; Gores, Gregory James.

In: Journal of Biological Chemistry, Vol. 268, No. 31, 1993, p. 23593-23600.

Research output: Contribution to journalArticle

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