Detection of aldehyde dehydrogenase activity in human corneal extracts.
The major soluble protein in bovine corneal epithelial extracts is a 54 kD protein (BCP 54) which has recently been identified as the corneal aldehyde dehydrogenase. Although ALDH activity has been reported in human corneal extracts it was not yet clear whether this was identical with the 54 kD protein described in bovine corneas. To investigate this question, we studied human corneal extracts for the presence of ALDH using enzyme analysis, SDS-PAGE, native electrophoresis, isoelectric focusing and immunoblotting techniques. The corneal epithelium was the most active layer (8.46 +/- 1.9 IU/mg protein) followed by the stroma (2.83 +/- 0.56 IU/mg protein) and endothelium (0.06-3.6 IU/mg protein). When comparing substrate specificity between human and bovine corneal ALDH, using NADP as coenzyme, it was shown that the human enzyme preferred benzaldehyde whereas the bovine enzyme revealed the strongest enzymatic activity with hexanal. Human corneal ALDH was partly inhibited by disulfiram. Bovine and human cornea ALDH lost their enzymatic activity after heating at temperatures above 56 degrees C. Both human and bovine corneal extracts contained a prominent 54 kD protein which reacted with a rabbit anti BCP 54 antibody. Isoelectric focusing followed by enzyme staining in the gel revealed 5 human corneal isozyme species and 4 in bovine corneal extracts, migrating at a pH between 6.5 and 7.0. All isozymes could also be detected after immunoblotting with a rabbit anti BCP 54 antibody.
Gondhowiardjo
T D
TD
Department of Ophthalmology, University of Indonesia, Jakarta.
van Haeringen
N J
NJ
Hoekzema
R
R
Pels
L
L
Kijlstra
A
A
D000444:Aldehyde Dehydrogenase / Q000032:analysis*; D000818:Animals; D002417:Cattle; D003315:Cornea / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000201:enzymology; D004795:Enzyme Stability; D004848:Epithelium / Q000201:enzymology; D005136:Eye Proteins / Q000032:analysis; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing; D011817:Rabbits
D005136:Eye Proteins; C030863:corneal protein 54, bovine; D000444:Aldehyde Dehydrogenase