The monitoring of nucleotide diphosphate kinase activity by blue native polyacrylamide gel electrophoresis

Abstract

Nucleoside diphosphate kinase (NDPK) has been shown to play a pivotal role in modulating a plethora of cellular processes. In this study, we report on a blue native (BN) PAGE technique which allows the facile assessment of NDPK activity and expression. The in-gel detection of NDPK relies on the precipitation of formazan at the site of immobilized enzyme activity. This is achieved by coupling the formation of ATP, as a consequence of gamma-phosphate transfer from NTP to ADP, to hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), oxidized nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate (PMS), and iodonitrotetrazolium chloride (INT). 2-D denaturing gel analysis confirmed that the activity bands corresponded to NDPK as indicated by subunit composition. Furthermore, the sensitivity and specificity of this readily accessible procedure was assessed by monitoring the in-gel activity of NDPK using different concentrations of GTP and CTP as well as deoxynucleoside triphosphates. This electrophoretic technique allows the quick and easy detection of NDPK, a housekeeping enzyme crucial to cell survival.

A Novel Strategy Involved Anti-Oxidative Defense: The Conversion of NADH into NADPH by a Metabolic Network

“…Blue Native polyacrylamide gel electrophoresis (BN PAGE) and in-gel activity stains were performed as described previously [11], [13]. Membrane proteins were prepared in a Native buffer (50 mM BisTris, 500 mM ε-aminocaproic acid (pH 7.0)) containing 1% maltoside at a final concentration of 4 mg/mL.…”

An ATP and Oxalate Generating Variant Tricarboxylic Acid Cycle Counters Aluminum Toxicity in Pseudomonas fluorescens

“…Blue Native polyacrylamide gel electrophoresis (BN PAGE) and in-gel activity stains were performed as described previously [16]. Membrane proteins were prepared in blue native (BN) buffer (50 mM Bis-Tris, 500 mM 6-aminohexanoic acid, pH 7.0, 4°C) and 1% β-dodecyl-D-maltoside.…”

“…SCS was detected by adding 1 mM succinyl-CoA, and 0.1 mM ADP. The formation of ATP was coupled to 5 mM glucose, 5 units of hexokinase, 5 units of glucose-6-phosphate, 5 units of glucose-6-phosphate dehydrogenase, 0.5 mM NADP, INT, PMS, and reaction buffer [16]. OCT activity was ascertained by adding 1 mM succinyl-CoA, 1 mM oxalate, 15 µg of protein equivalent from P.fluorescens membrane fraction (which contains SDH), INT, PMS, and reaction buffer.…”

Research of beer quality with the addition of elder sap

“…Пиво з використанням спецій. В Японії пиво представлено такими видами: Wasabi dry зі спеціями васабі, коріандр, гвоздика, імбир, гірчиця тощо [6].…”