Application of commercial enzymes to measure the activity of the arginine pathway-urea cycle in intact cells.
The activity of the complete arginine pathway-urea cycle was assessed in intact plant cells by employing the commercial enzymes arginase (EC 3.5.3.1) and urease (EC 3.5.1.5) to determine the amount of NaH14CO3 incorporated into [guanido-14C]arginine and/or into [14C]urea during a 3-h labeling period. Recovery of [guanido-14C]arginine was linear from 5 to 1000 nmol/g tissue and averaged 80 +/- 5% (mean +/- SE, N = 3). The procedure is reliable, inexpensive, well suited to the simultaneous analysis of numerous samples, and significantly more sensitive than existing methods. The method is ideally suited for assessing the activity of the complete arginine biosynthetic pathway in intact cells. In addition, the method has the distinct advantage of providing simultaneous measurement of the amount of NaH14CO3 accumulating in arginine relative to the amount accumulating as urea. Evidence is presented demonstrating that both the activity of the arginine pathway and the relative amounts of [guanido-14C]arginine and [14C]urea synthesized from NaH14CO3 were influenced by changes in the level of ornithine, NH+4, or phosphorus available to plant tissues.
D001119:Arginase / Q000378:metabolism*; D001120:Arginine / Q000096:biosynthesis*; D001639:Bicarbonates / Q000378:metabolism; D010758:Phosphorus / Q000502:physiology; D010944:Plants / Q000378:metabolism; D012639:Seeds / Q000378:metabolism; D017693:Sodium Bicarbonate; D014508:Urea / Q000096:biosynthesis*; D014510:Urease / Q000378:metabolism*
D001639:Bicarbonates; D010758:Phosphorus; D017693:Sodium Bicarbonate; D014508:Urea; D001120:Arginine; D014510:Urease; D001119:Arginase