Determination of dimethylarginine dimethylaminohydrolase activity in the kidney.
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.
Tain
Y-L
YL
Department of Physiology and Functional Genomics, University of Florida, Gainesville, Florida 32610-0274, USA. tainyl@ufl.edu
Baylis
C
C
D000581:Amidohydrolases / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D002956:Citrulline / Q000032:analysis; D007668:Kidney / Q000737:chemistry* / Q000201:enzymology; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D014508:Urea / Q000737:chemistry
D002956:Citrulline; C018524:N,N-dimethylarginine; D014508:Urea; D001120:Arginine; D000581:Amidohydrolases; C059999:dimethylargininase