Molecular weight forms of corneal aldehyde dehydrogenase.
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.
Gondhowiardjo
T D
TD
Department of Ophthalmology, University of Indonesia, Jakarta.
van Haeringen
N J
NJ
Kijlstra
A
A
D000444:Aldehyde Dehydrogenase / Q000032:analysis* / Q000737:chemistry; D000818:Animals; D002417:Cattle; D003315:Cornea / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005136:Eye Proteins / Q000032:analysis; D006358:Hot Temperature; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000032:analysis; D008970:Molecular Weight; D013696:Temperature
D005136:Eye Proteins; D007527:Isoenzymes; C030863:corneal protein 54, bovine; D000444:Aldehyde Dehydrogenase