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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.

Aldehyde dehydrogenase has recently been shown to be one of the major soluble proteins in the mammalian cornea. The enzyme has a subunit molecular weight of 54 kd and gel filtration experiments indicate that a dimer molecule is the enzymatically active species. The purpose of the studies described here was to investigate whether oligomeric forms of this enzyme could also be detected using the much faster SDS-PAGE mini-gel electrophoresis technique combined with immunoblotting and "in gel" enzyme detection. Low temperature treatment of samples prior to electrophoresis revealed that both human and bovine corneal ALDH are mainly present as a 54 kd and as a dimer molecule with an apparent molecular weight of 88 kd. Bovine corneal ALDH also contained larger oligomers with a molecular weight of 110, 154 and 210 kd respectively. The classical 3 minutes boiling procedure prior to SDS-PAGE dissociated the oligomers into the 54 kd subunit. Zymography experiments showed that enzyme activity was only present in the 88 kd form of corneal ALDH. Pretreatment of corneal ALDH at various temperatures showed that the temperature induced shift of the 88 kd species to the 54 kd subunit paralleled the decrease in enzymatic activity. The fact that reduction of samples with DTT did not dissociate the 88 kd form suggests that disulfide bridge formation is not involved in the oligomerisation of corneal ALDH.

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