Tain Y-L, Baylis C
Department of Physiology and Functional Genomics, University of Florida, Gainesville, Florida 32610-0274, USA.
Kidney Int. 2007 Oct;72(7):886-9. doi: 10.1038/sj.ki.5002446. Epub 2007 Jul 25.
Dimethylarginine dimethylaminohydrolase (DDAH) metabolizes asymmetric dimethylarginine to generate L-citrulline and is present in large quantities in the kidney. We present a new study that optimizes the Prescott-Jones colorimetric assay to measure DDAH-dependent L-citrulline generation in kidney homogenates. We found that the removal of urea with urease is necessary since urea also produces a positive reaction. Deproteinization with sulfosalicylic acid was found to be optimal and that protease inhibitors were not necessary. All assays were conducted in phosphate buffer, since other common additives can create false positive and false negative reactions. Arginase or nitric oxide synthase isoenzymes were not found to influence L-citrulline production. Our optimized L-citrulline production assay to measure DDAH activity correlated closely with the direct measure of the rate of asymmetric dimethylarginine consumption. Using this assay, we found that both superoxide and nitric oxide inhibit renal cortical DDAH activity in vitro.
二甲基精氨酸二甲胺水解酶（DDAH）将不对称二甲基精氨酸代谢生成L-瓜氨酸，且在肾脏中大量存在。我们开展了一项新研究，对普雷斯科特-琼斯比色法进行优化，以测定肾匀浆中依赖DDAH的L-瓜氨酸生成量。我们发现用脲酶去除尿素是必要的，因为尿素也会产生阳性反应。结果表明用磺基水杨酸进行脱蛋白效果最佳，且无需使用蛋白酶抑制剂。所有测定均在磷酸盐缓冲液中进行，因为其他常见添加剂会产生假阳性和假阴性反应。未发现精氨酸酶或一氧化氮合酶同工酶会影响L-瓜氨酸的生成。我们优化的用于测定DDAH活性的L-瓜氨酸生成测定法与不对称二甲基精氨酸消耗速率的直接测量结果密切相关。使用该测定法，我们发现超氧化物和一氧化氮在体外均会抑制肾皮质DDAH活性。
