Rouimi P, Loomes K, Jörnvall H
Department of Chemistry I, Karolinska Institutet, Stockholm, Sweden.
Eur J Biochem. 1993 Apr 1;213(1):487-92. doi: 10.1111/j.1432-1033.1993.tb17785.x.
Mammalian alcohol and sorbitol dehydrogenases, with distantly related subunits but different substrates, quaternary structures and zinc contents, were evaluated by comparison of their sensitivities to proteases. Sorbitol dehydrogenase is more sensitive to proteolysis than alcohol dehydrogenase, but both enzymes show limited cleavage with Lys-specific and Glu-specific proteases. With the former, the major cleavage in both proteins involves Lys-Lys-Pro segments, at positions 247-248 in alcohol dehydrogenase, surface-positioned after the most distal beta-strand in the coenzyme-binding domain, and at 61-62 in sorbitol dehydrogenase, at another surface in the catalytic domain. Further cleavages affect these two and a third surface. A non-surface cleavage was obtained with the Glu-specific protease and sorbitol dehydrogenase, after insufficient protease inhibition before analysis by SDS/PAGE. It probably reflects non-native conditions, and the fact that this protease is active in strong SDS, necessitating pre-analytical use of a specific inhibitor. Differences in cleavage patterns between the two proteins do not involve areas corresponding to the dimer interactions in alcohol dehydrogenase. Hence, these areas are likely to be the same in sorbitol dehydrogenase. However, major differences involve regions that flank the surface which has a 20-residue loop segment in alcohol dehydrogenase that is missing in sorbitol dehydrogenase. This segment, previously highlighted from comparisons, is therefore also emphasized by experimental results with proteolysis. The surface exposed by the missing segment is likely to correlate with the separate quaternary structures and to indicate possible sites for the additional subunit interactions in the sorbitol dehydrogenase tetramer versus the alcohol dehydrogenase dimer.
