title: Analysis of Ethylene Receptors: Assay for Histidine Kinase Activity.
abstract: The ethylene receptors of plants exist in two subfamilies. Members of subfamily-1 have functional histidine kinase domains, whereas members of subfamily-2 have diverged histidine-kinase-like domains that in some cases have been shown to exhibit Ser/Thr kinase activity. Here, we describe a method to biochemically characterize the enzymatic activity of these kinase domains in vitro. For this purpose, the histidine kinase domain of the receptors is transgenically expressed in yeast as a fusion to glutathione-S-transferase (GST) for subsequent affinity purification. Autophosphorylation activity is assessed by the use of an in vitro kinase assay with the purified protein. Acid/base stability of the incorporated phosphate can then be used as a diagnostic for whether His, Asp, or Ser/Thr/Tyr is phosphorylated.
authors.affiliation: Department of Biological Sciences, Life Sciences Center, Dartmouth College, 78 College Street, Hanover, NH, 03755, USA. George.e.schaller@dartmouth.edu.
authors.forename: G Eric
authors.initials: GE
authors.lastname: Schaller
authors.affiliation: Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, M407 Walters Life Sciences, Knoxville, TN, 37996, USA.
authors.forename: Brad M
authors.initials: BM
authors.lastname: Binder
mesh_terms: D004789:Enzyme Activation; D000071677:Histidine Kinase / Q000235:genetics / Q000378:metabolism*; D007553:Isotope Labeling; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C083750:ethylene receptors, plant; D000071677:Histidine Kinase
keywords: Arabidopsis; Fusion protein; Glutathione-S-transferase; Histidine kinase; Kinase assay; Yeast