title: A blue native polyacrylamide gel electrophoretic technology to probe the functional proteomics mediating nitrogen homeostasis in Pseudomonas fluorescens.
abstract: As glutamate and ammonia play a pivotal role in nitrogen homeostasis, their production is mediated by various enzymes that are widespread in living organisms. Here, we report on an effective electrophoretic method to monitor these enzymes. The in gel activity visualization is based on the interaction of the products, glutamate and ammonia, with glutamate dehydrogenase (GDH, EC: 1.4.1.2) in the presence of either phenazine methosulfate (PMS) or 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium (INT). The intensity of the activity bands was dependent on the amount of proteins loaded, the incubation time and the concentration of the respective substrates. The following enzymes were readily identified: glutaminase (EC: 3.5.1.2), alanine transaminase (EC: 2.6.1.2), aspartate transaminase (EC: 2.6.1.1), glycine transaminase (EC: 2.6.1.4), ornithine oxoacid aminotransferase (EC: 2.6.1.13), and carbamoyl phosphate synthase I (EC: 6.3.4.16). The specificity of the activity band was confirmed by high pressure liquid chromatography (HPLC) following incubation of the excised band with the corresponding substrates. These bands are amenable to further molecular characterization by a variety of analytical methods. This electrophoretic technology provides a powerful tool to screen these enzymes that contribute to nitrogen homeostasis in Pseudomonas fluorescens and possibly in other microbial systems.
authors.affiliation: Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada.
authors.forename: Sungwon
authors.initials: S
authors.lastname: Han
authors.forename: Christopher
authors.initials: C
authors.lastname: Auger
authors.forename: Varun P
authors.initials: VP
authors.lastname: Appanna
authors.forename: Joseph
authors.initials: J
authors.lastname: Lemire
authors.forename: Zachary
authors.initials: Z
authors.lastname: Castonguay
authors.forename: Elchin
authors.initials: E
authors.lastname: Akbarov
authors.forename: Vasu D
authors.initials: VD
authors.lastname: Appanna
mesh_terms: D015086:2,6-Dichloroindophenol / Q000737:chemistry; D000410:Alanine Transaminase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000641:Ammonia / Q000737:chemistry; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D057075:Enzyme Assays; D005969:Glutamate Dehydrogenase / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D005972:Glutaminase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D051308:Glycine Transaminase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006706:Homeostasis; D008773:Methylphenazonium Methosulfate / Q000737:chemistry; D009584:Nitrogen / Q000378:metabolism*; D009953:Ornithine-Oxo-Acid Transaminase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000378:metabolism*; D013778:Tetrazolium Salts / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D013778:Tetrazolium Salts; C013574:iodonitrotetrazolium; D008773:Methylphenazonium Methosulfate; D018698:Glutamic Acid; D000641:Ammonia; D015086:2,6-Dichloroindophenol; D005969:Glutamate Dehydrogenase; D001219:Aspartate Aminotransferases; D009953:Ornithine-Oxo-Acid Transaminase; D000410:Alanine Transaminase; D051308:Glycine Transaminase; D005972:Glutaminase; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D009584:Nitrogen