Hormone-binding assay using living bacteria expressing eukaryotic receptors.
Studies on hormone-receptor interaction include, as a rule, isolation and extensive purification of the receptor protein or a particular receptor-containing fraction. To bypass these time- and resource-consuming procedures, we proposed a live cell-based assay using transgenic bacteria expressing single eukaryotic receptors. We describe here 3H-cytokinin binding to corresponding plant receptors as an example. The method includes procedures of bacteria growing, incubation with labeled hormone, separation of bound from unbound ligand, determination of radioactivity in bacterial precipitates, and mathematical analysis of primary data. The established simple protocol for specific labeling hormone-binding sites in intact bacteria allows determination of the main parameters of the ligand-receptor interaction.
Romanov
Georgy A
GA
Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia.
Lomin
Sergey N
SN
D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D000071677:Histidine Kinase; D030781:Organisms, Genetically Modified; D010937:Plant Growth Regulators / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D029681:Arabidopsis Proteins; D010937:Plant Growth Regulators; D011956:Receptors, Cell Surface; D011494:Protein Kinases; D000071677:Histidine Kinase; C421783:WOL protein, Arabidopsis