Synthesis of [(32)P]phosphoramidate for use as a low molecular weight phosphodonor reagent.
Phosphoramidate serves as a useful phosphodonor reagent in protein and peptide phosphorylation, notably in studying two-component signal transduction systems in which low molecular weight phosphodonors can substitute for the phosphodonor function of histidine protein kinases in in vitro phosphorylation studies of response regulator proteins. A convenient method for the synthesis of radiolabeled phosphoramidate has not been developed, and this has limited its broader use. Here we report the synthesis of radiolabeled ammonium hydrogen phosphoramidate [(NH(4))H(32)PO(3)NH(2)] which is achieved by activation of [(32)P]orthophosphate with ethyl isocyanate followed by aminolysis with ammonium hydroxide to form the desired phosphoramidate. The procedure is conveniently carried out in a microfuge tube and requires only two successive precipitation steps to obtain pure ammonium hydrogen phosphoramidate. Molar yields of 15-30% and specific activities of 10-20 Ci/mol are readily achieved. Phosphorylation of microgram quantities of response regulator proteins CheY, CheB, and DrrA is shown. Low level, but detectable, nonspecific phosphorylation was observed for reactions near ambient temperatures when substrate response regulators lacking the active site aspartate but containing histidine residues are used. More significant levels of nonspecific phosphorylation were observed for reactions at elevated temperatures when using a nonresponse regulator control protein (RNase A).
Buckler
D R
DR
Department of Biochemistry, Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854, USA.
Stock
A M
AM
D000577:Amides / Q000138:chemical synthesis* / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D002855:Chromatography, Thin Layer / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry; D000072236:Methyl-Accepting Chemotaxis Proteins; D008970:Molecular Weight; D010756:Phosphoric Acids / Q000138:chemical synthesis* / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D012486:Salmonella typhimurium / Q000737:chemistry
D000577:Amides; D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010756:Phosphoric Acids; D010761:Phosphorus Radioisotopes; C069656:CheB protein, Bacteria; C011067:phosphoramidic acid