title: A facile electrophoretic technique to monitor phosphoenolpyruvate-dependent kinases.
abstract: Phosphoenolpyruvate (PEP)-dependent kinases are central to numerous metabolic processes and mediate the production of adenosine triphosphate (ATP) by substrate-level phosphorylation (SLP). While pyruvate kinase (PK, EC: 2.7.1.40), the final enzyme of the glycolytic pathway is critical in the anaerobic synthesis of ATP from ADP, pyruvate phosphate dikinase (PPDK, EC: 2.7.9.1), and phosphoenolpyruvate synthase (PEPS, EC: 2.7.9.2) help generate ATP from AMP coupled to PEP as a substrate. Here we demonstrate an inexpensive and effective electrophoretic technology to determine the activities of these enzymes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). The generation of pyruvate is linked to exogenous lactate dehydrogenase (LDH), and the oxidation of reduced nicotinamide adenine dinucleotide (NADH) coupled to 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium chloride (INT) results in a formazan precipitate which is easily quantifiable. The selectivity of the enzymes is ensured by including either AMP or ADP and pyrophosphate (PP(i) ) or inorganic phosphate (P(i) ). Activity bands were readily obtained after incubation in the respective reaction mixtures for 20-30 min. Cell-free extract concentrations as low as 20 μg protein equivalent yielded activity bands and substrate levels were manipulated to optimize sensitivity of this analytical technique. High-pressure liquid chromatography (HPLC), two-dimensional (2-D) SDS-PAGE (where SDS is sodium dodecyl sulfate), and immunoblot studies of the excised activity band help further characterize these PEP-dependent kinases. Furthermore, these enzymes were readily identified on the same gel by incubating it sequentially in the respective reaction mixtures. This technique provides a facile method to elucidate these kinases in biological systems.
authors.affiliation: Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada.
authors.forename: Christopher
authors.initials: C
authors.lastname: Auger
authors.forename: Varun
authors.initials: V
authors.lastname: Appanna
authors.forename: Zachary
authors.initials: Z
authors.lastname: Castonguay
authors.forename: Sungwon
authors.initials: S
authors.lastname: Han
authors.forename: Vasu D
authors.initials: VD
authors.lastname: Appanna
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D015151:Immunoblotting; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D017866:Phosphotransferases (Paired Acceptors) / Q000737:chemistry / Q000378:metabolism*; D011551:Pseudomonas fluorescens / Q000201:enzymology; D011770:Pyruvate Kinase / Q000737:chemistry / Q000378:metabolism; D011772:Pyruvate, Orthophosphate Dikinase / Q000737:chemistry / Q000378:metabolism*; D019289:Pyruvic Acid / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D011770:Pyruvate Kinase; D017866:Phosphotransferases (Paired Acceptors); D011772:Pyruvate, Orthophosphate Dikinase; C022509:pyruvate, water dikinase