Factors affecting counteraction by methylamines of urea effects on aldose reductase

摘要

The concentration of urea in renal medullary cells is high enough to affect enzymes seriously by reducing V_(max) or raising Km, yet the cells survive and function. The usual explanation is that the methylamines found in the renal medulla, namely glycerophosphocholine and betaine, have actions opposite to those of urea and thus counteract its effects. However, urea and methylamines have the similar (not counteracting) effects of reducing both the K_m and V_(max) of aldose reductase (EC 1.l.1.21), an enzyme whose function is important in renal medullas. Therefore, we examined factors that might determine whether counteraction occurs, namely different combinations of assay conditions (pH and salt concentration), methylamines (glycerophosphocholine, be- taine, 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 methylamines that is par- tially additive, whereas at the other extreme we find that urea 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 betaine to urea there is no net effect. We conclude that counteraction of urea effects on enzymes by methylamines can depend on ion concentration, PH, the specific m