Factors affecting counteraction by methylamines of urea effects on aldose reductase

The concentration of urea in renal medullary cells is high enough to affect enzymes seriously by reducing V-max or raising K-m, yet the cells survive and function, The usual explanation is that the methylamines found in the r enal medulla, namely glycerophosphocholine and betaine, have actions opposi te to those of urea and thus counteract its effects. However, urea and meth ylamines have the similar (not counteracting) effects of reducing both the K-m and V-max of aldose reductase (EC 1.1.1.21), an enzyme whose function i s important in renal medullas. Therefore, we examined factors that might de termine whether counteraction occurs, namely different combinations of assa y conditions (pH and salt concentration), methylamines (glycerophosphocholi ne, betaine, and trimethylamine N-oxide), substrates (DL-glyceraldehyde and D-xylose), and a mutation in recombinant aldose reductase protein (C298A), We find that V-max of both wild-type and C298A mutant generally is reduced by urea and/or the methylamines. However, the effects on K-m are much more complex, varying widely with the combination of conditions. At one extreme , we find a reduction of K-m of wild-type enzyme by urea and/or methylamine s that is partially additive, whereas at the other extreme we find that ure a raises K-m for D-xylose of the C298A mutant, betaine lowers the K-m, and the two counteract in a classical fashion so that at a 2:1 molar ratio of b etaine to urea there is no net effect. We conclude that counteraction of ur ea effects on enzymes by methylamines can depend on ion concentration, pH, the specific methylamine and substrate, and identity of even a single amino acid in the enzyme.