Calpain and calpastatin in rabbit corneal epithelium.
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 demonstrated immunoreactive peaks for calpains I and II. Calpain II from the soluble fraction of corneal epithelium eluted at a similar NaCl concentration (260 mM) 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.
Shearer
T R
TR
Department of Clinical Science and Laboratory Medicine, Faculty of Medicine, Kyoto University, Japan.
Azuma
M
M
David
L L
LL
Yamagata
Y
Y
Murachi
T
T
D000818:Animals; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D002154:Calpain / Q000032:analysis / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003315:Cornea / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004848:Epithelium / Q000201:enzymology; D015151:Immunoblotting; D011817:Rabbits
D002135:Calcium-Binding Proteins; C033668:calpastatin; D002154:Calpain