TY - JOUR
T1 - Calpain and calpastatin in rabbit corneal epithelium
AU - Shearer, Thomas R.
AU - Azuma, Mitsuyoshi
AU - David, Larry L.
AU - Yamagata, Yoko
AU - Murachi, Takashi
N1 - Funding Information:
ACKNOWLEDGEMENTS Supported by grants EY-03600 and EY-05786 from the National Eye Institute, National Institutes of Health (USA); and a fellowship from the Japan Society for Promotion of Science to T.R.S.
PY - 1990
Y1 - 1990
N2 - The purpose of this study was to provide a direct assay for calpain and its endogenous inhibitor calpastatin in normal rabbit epithelium. Corneal epithelial extracts were fractionated by DEAE (1) chromatography on HPLC. Fractions were analyzed for calpain by ELISA, immunoblotting, and caseinolytic enzyme activity with FITC-labeled casein. Results demonstated Immunoreactive peaks for calpains I and II. Calpain II from the soluble fraction of corneal epithelium eluted at a similar NaCl concentration (260 raM) as calpain II from other tissues, was inhibited by both E64 and the removal of Ca, contained an 80 kDa subunit in immunoblots, and was present at specific activity of 220 units/g protein (in a crude homogenate). Calpain antigen was also present in the EDTA/EGTA washed insoluble fraction of corneal epithelium. Calpastatin in corneal epithelium eluted at 130 -160 mM NaCl on DEAE, coeluted with calpain I, and was present at 330 units/g protein (crude homogenate). The results demonstrated a calpain/calpastatin system in corneal epithelium, where it is speculated to play a role in epithelial cell turnover and wound healing.
AB - The purpose of this study was to provide a direct assay for calpain and its endogenous inhibitor calpastatin in normal rabbit epithelium. Corneal epithelial extracts were fractionated by DEAE (1) chromatography on HPLC. Fractions were analyzed for calpain by ELISA, immunoblotting, and caseinolytic enzyme activity with FITC-labeled casein. Results demonstated Immunoreactive peaks for calpains I and II. Calpain II from the soluble fraction of corneal epithelium eluted at a similar NaCl concentration (260 raM) as calpain II from other tissues, was inhibited by both E64 and the removal of Ca, contained an 80 kDa subunit in immunoblots, and was present at specific activity of 220 units/g protein (in a crude homogenate). Calpain antigen was also present in the EDTA/EGTA washed insoluble fraction of corneal epithelium. Calpastatin in corneal epithelium eluted at 130 -160 mM NaCl on DEAE, coeluted with calpain I, and was present at 330 units/g protein (crude homogenate). The results demonstrated a calpain/calpastatin system in corneal epithelium, where it is speculated to play a role in epithelial cell turnover and wound healing.
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U2 - 10.3109/02713689009000053
DO - 10.3109/02713689009000053
M3 - Article
C2 - 2311414
AN - SCOPUS:0025318116
SN - 0271-3683
VL - 9
SP - 39
EP - 44
JO - Current Eye Research
JF - Current Eye Research
IS - 1
ER -