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Calcium‐Activated Neutral Protease Activity Is Decreased in PC12 Cells After Ethanol Exposure
Author(s) -
DePetrillo Paolo B.
Publication year - 1997
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1997.68051863.x
Subject(s) - calpain , ionomycin , chemistry , calcium , protease , ethanol , neutral protease , biochemistry , microbiology and biotechnology , enzyme , biology , organic chemistry
Calcium‐activated neutral protease activity was determined in PC12 cells exposed to ethanol for 96 h using a fluorescence‐based assay with N ‐succinyl‐Leu‐Tyr 7‐amido‐4‐methylcoumarin as the substrate. Stimulated activity was measured at high (1,400 µ M ) or low (140 µ M ) Ca 2+ concentrations in the presence of 20 µ M ionomycin. Kinetic parameters were derived by fitting a model relating fluorescence intensity to time: F t = F final *(1 − e − k obs t ). Cell extracts were subjected to nondenaturing gel electrophoresis and casein zymography with quantification of the activity of the two calpain isoforms. Exposure to ethanol significantly decreased whole cell calpain activity measured by k obs beginning at 20 m M , to 27.8% of control at 1,400 µ M Ca 2+ and 29.2% of control at 140 µ M Ca 2+ in the presence of 20 µ M ionomycin. No changes in μ‐calpain or m‐calpain activities were found in cell extracts from cells exposed to 20 m M ethanol, whereas at 40 and 80 m M ethanol, significant decreases in both μ‐calpain and m‐calpain activities were discovered.