Factors affecting counteraction by methylamines of urea effects on aldose reductase.
Factors affecting counteraction by methylamines of urea effects on aldose reductase.
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DOI：
10.1073/pnas.96.11.6517
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发表时间：
1999-05
影响因子：
11.1
通讯作者：
M. Burg;E. Peters;K. Bohren;K. Gabbay
中科院分区：
文献类型：
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作者：
M. Burg;E. Peters;K. Bohren;K. Gabbay
肾髓质细胞中的尿素浓度高到足以通过降低Vmax或升高Km来严重影响酶，但细胞存活并发挥功能。通常的解释是，在肾髓质中发现的甲胺，即甘油磷酸胆碱和甜菜碱，具有与尿素相反的作用，因此抵消了尿素的作用。然而，尿素和甲胺具有降低醛糖还原酶（EC 1.1.1.21）的Km和Vmax的类似（非抵消）作用，醛糖还原酶是一种在肾髓质中具有重要功能的酶。因此，我们研究了可能决定是否发生抵消作用的因素，即测定条件（pH值和盐浓度）、甲胺（甘油磷酸胆碱、甜菜碱和三甲胺N-氧化物）、底物（DL-甘油醛和D-木糖）和重组醛糖还原酶蛋白（C298 A）突变的不同组合。我们发现，无论是野生型和C298 A突变体的Vmax通常是由尿素和/或甲胺降低。然而，对Km的影响要复杂得多，随着条件的组合而变化很大。在一个极端中，我们发现尿素和/或甲胺降低了野生型酶的Km，这是部分加和的，而在另一个极端中，我们发现尿素提高了C298 A突变体的D-木糖的Km，甜菜碱降低了Km，并且两者以经典方式抵消，使得在甜菜碱与尿素的摩尔比为2：1时，没有净效应。我们的结论是，尿素对酶的影响，甲胺的抵消作用可以取决于离子浓度，pH值，特定的甲胺和底物，甚至是一个单一的氨基酸在酶的身份。
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.