Factors affecting counteraction by methylamines of urea effects on aldose reductase

Abstract

The concentration of urea in renal medullary cells is high enough to affect enzymes seriously by reducing Vmax 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 Km and Vmax of aldose reductase (EC 1.1.1.21), 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, betaine, and trimethylamine N-oxide), substrates (DL-glyceraldehyde and D-xylose), and a mutation in recombinant aldose reductase protein (C298A). We find that Vmax of both wild-type and C298A mutant generally is reduced by urea and/or the methylamines. However, the effects on Km are much more complex, varying widely with the combination of conditions. At one extreme, we find a reduction of Km of wild-type enzyme by urea and/or methylamines that is partially additive, whereas at the other extreme we find that urea raises Km for D-xylose of the C298A mutant, betaine lowers the Km, 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 methylamine and substrate, and identity of even a single amino acid in the enzyme.

Figure 1

Effect of urea and TMAO on K_m of human aldose reductase for d-xylose (n = 3). ∗, Significantly different from control (P < 0.05). Additional significant differences with wild-type are TMAO versus urea and TMAO versus urea + TMAO. With C298A mutant, all other differences are also significant. Mean control values of K_m are wild-type, 6.9 mM, and C298A, 228 mM.

Figure 2

Effect of urea and betaine on K_m of human aldose reductase for d-xylose. ∗, Significantly different from control (P < 0.05). Additional significant differences with wild-type (n = 3) are urea versus betaine and urea versus urea + betaine. With C298A mutant (n = 5), all other differences are also significant. Mean control values of K_m are wild-type, 9.4 mM, and C298A, 229 mM.

Figure 3

Effect of urea and GPC on K_m of human aldose reductase for d-xylose (n = 3). ∗, Significantly different from control (P < 0.05). Additional significant differences with the C298A mutant are urea versus GPC and GPC versus urea + GPC. Mean control values of K_m are wild-type, 7.8 mM, and C298A, 191 mM.

Figure 4

Effect of urea and betaine on K_m of human aldose reductase for dl-glyceraldehyde (n = 3). ∗, Significantly different from control (P < 0.05). All other differences are also significant with wild type. With C298A, mutant urea versus betaine and urea versus urea + betaine are significantly different. Mean control values of K_m are wild type, 0.15 mM, and C298A, 1.12 mM.

Figure 5

Effect of urea and GPC on K_m of human aldose reductase for dl-glyceraldehyde (n = 3). ∗, Significantly different from control (P < 0.05). Additional significant differences with wild type are urea versus urea + GPC and GPC versus urea + GPC. Additional significant differences with C298A are urea versus GPC and urea versus urea + GPC. Mean control values of K_m are wild type, 0.17 mM, and C298A, 0.94 mM.