Enzymatic determination of subpicomole quantities of phospho-L-arginine in nerve cytoplasm.
An ultramicro phospho-L-arginine (Arg-P) assay for nanoliter-volume biological samples is described. The assay and method is demonstrated to be reliable and reproducible for the detection of subpicomole quantities of both Arg-P and ATP in serial samples of cytoplasm from a single, isolated giant nerve fiber from the crayfish Procambarus clarkii. The assay for Arg-P is based on the ATP:L-arginine Nw-phosphotransferase (EC 2.7.3.3.)-catalyzed phosphorylation of ADP to ATP. The generated ATP is measured with a standard luciferin-luciferase bioluminescence method. Arg-P and ATP concentrations of pure axoplasmic samples from resting nerve were found to be approximately 13 and 2 mM, respectively. There was no evidence that substances present in the biological sample interfered with the assay. Recovery of added Arg-P and ATP were approximately 100%. Arg-P and ATP concentrations of isolated biological samples were found to be stable for 1 h at room temperature. The techniques described here are useful for the study of energy metabolism in single, isolated giant cells, and could be easily modified for the study of a variety of metabolic intermediates in situ in functioning tissues and organs.
D000244:Adenosine Diphosphate / Q000302:isolation & purification; D000255:Adenosine Triphosphate / Q000302:isolation & purification; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000302:isolation & purification; D001122:Arginine Kinase; D003400:Astacoidea; D001369:Axons / Q000032:analysis; D003593:Cytoplasm / Q000032:analysis; D004734:Energy Metabolism; D008832:Microchemistry; D009417:Nerve Tissue / Q000032:analysis*; D009943:Organophosphorus Compounds / Q000302:isolation & purification; D010770:Phosphotransferases
D009943:Organophosphorus Compounds; C015441:phospho-L-arginine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D010770:Phosphotransferases; D001122:Arginine Kinase