A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes.
Phosphoglycosyltransferases (PGTs) are families of integral membrane proteins with intriguingly diverse architectures. These enzymes function to initiate many important biosynthetic pathways including those leading to peptidoglycan, N-linked glycoproteins and lipopolysaccharide O-antigen. In spite of tremendous efforts, characterization of these enzymes remains a challenge not only due to the inherent difficulties associated with the purification of integral membrane proteins but also due to the limited availability of convenient assays. Current PGT assays include radioactivity-based methods, which rely on liquid-liquid or solid-liquid extractions, multienzyme systems linked to lactate dehydrogenase and NAD(+) generation, and HPLC-based approaches, all of which may suffer from low sensitivity and low throughput. Herein, we present the validation of a new luminescence-based assay (UMP-Glo) for measuring activities of PGT enzymes. This assay measures UMP, the by-product of PGT reactions, in a sensitive and quantitative manner by measuring the luminescence output in a discontinuous coupled assay system. The assay is rapid and robust in nature, and also compatible with microtiter plate formats. Activity and kinetic parameters of PglC, a PGT from Campylobacter jejuni, were quickly established using this assay. The efficacy of the assay was further corroborated using two different PGTs; PglC from Helicobacter pullorum and WecA from Thermatoga maritima.
Department of Biology and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Debasis
D
Das
Department of Biology and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Marthe T C
MT
Walvoort
Department of Biology and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Vinita
V
Lukose
Department of Biology and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Barbara
B
Imperiali
D001426:Bacterial Proteins / Q000378:metabolism; D016123:Campylobacter jejuni / Q000201:enzymology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis / Q000494:pharmacology; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016998:Helicobacter / Q000201:enzymology; D007700:Kinetics; D049449:Luminescence; D017830:Octoxynol / Q000494:pharmacology; D010766:Phosphorylation; D020124:Thermotoga maritima / Q000201:enzymology; D013997:Time Factors; D014529:Uridine / Q000494:pharmacology; D014542:Uridine Monophosphate / Q000378:metabolism
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D017830:Octoxynol; D014542:Uridine Monophosphate; D005964:Glucosyltransferases; D014529:Uridine; D004121:Dimethyl Sulfoxide