Capillary electrophoresis-based nanoscale assays for monitoring ecto-5′-nucleotidase activity and inhibition in preparations of recombinant enzyme and melanoma cell membranes

Iqbal, Jamshed; Jirovský, David; Lee, Sang-Yong; Zimmermann, Herbert; Müller, Christian

doi:10.1016/j.ab.2007.09.028

Cited by 41 publications

(41 citation statements)

References 36 publications

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“…Our group has long-standing experience in the quantification of nucleotides by CE, and especially in enzyme assays involving the formation or transformation of nucleotides [15][16][17][18][19][20][21][22]. Thus we decided to quantify the nucleotide PAP which is formed from the co-substrate PAPS during the CST reaction.…”

Section: Development and Optimization Of Ce Methods For Monitoring Cstmentioning

confidence: 99%

A capillary electrophoresis method with dynamic pH junction stacking for the monitoring of cerebroside sulfotransferase

Zech

Gieselmann

et al. 2015

Journal of Chromatography A

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Section: Development and Optimization Of Ce Methods For Monitoring Cstmentioning

confidence: 99%

A capillary electrophoresis method with dynamic pH junction stacking for the monitoring of cerebroside sulfotransferase

Zech

Gieselmann

et al. 2015

Journal of Chromatography A

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“…CE-based on-column enzymatic reaction methods [34] have been developed previously for the quantitative determination of nucleotides using polyacrylamide-coated fused-silica capillaries [35]. Thus, it is very easy to study the effect of the on-column DAAO catalytic reaction with D,L-Trp because the method of immobilizing enzyme on the HDBcoated capillary [29] is very simple and the immobilized enzyme is stable.…”

Section: On-column Aao Catalytic Reactionmentioning

confidence: 99%

Chiral ligand‐exchange CE assays for separation of amino acid enantiomers and determination of enzyme kinetic constant

Qiao

Yang

et al. 2009

Electrophoresis

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This paper deals with studies on the use of Zn(II)-L-ornithine complex as a chiral selecting system for the enantioseparation and UV detection of amino acids (AAs) by using the principle of ligand-exchange CE. Successful enantioseparation of three pairs of label-free aromatic AAs and four pairs of labeled AA enantiomers have been achieved with a buffer of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4 and 6.0 mM L-Orn at pH 8.2. This new method was shown to be applicable to the quantitative analysis of D- and L-aromatic AAs, with a linear range between 12.5 and 800.0 microg/mL, and a correlation coefficient above 0.99. Thus this assay, which is facile and relatively rapid, allows us to measure the enzyme catalytic activity in the incubation of D,L-AAs with D-AA oxidase. Using this new method, we can determine the enzyme kinetic constant, lending insight into potential enzyme mechanism.

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“…Recently, we have developed CE-based enzyme assays for the determination of Michaelis-Menten constants (K m values), maximal velocities (V max ) for substrates, and inhibition constants (IC 50 or K i values) for enzyme inhibitors of various nucleotide and nucleoside-metabolizing enzymes, namely ecto-5 0 -nucleotidase [21], nucleoside triphosphate diphosphohydrolase (ecto-NTPDase) [22], adenosine kinase [23], and herpes simplex type 1 (HSV-1) thymidine kinase [24]. However, the previously established methods were not sensitive enough to quantify the reaction products of NPP reactions, because their detection limits for nucleotides such as AMP, the product of NPP-catalyzed hydrolysis of ATP, are only in the range of 1.6-2.3 mM.…”

Section: H]-or [mentioning

confidence: 99%

A highly sensitive CE‐UV method with dynamic coating of silica‐fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions

et al. 2008

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A new highly sensitive capillary electrophoresis (CE) method applying dynamic coating and on-line stacking for the monitoring of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and the screening of inhibitors was developed. NPP1 and NPP3 are membrane glycoproteins that catalyze the hydrolysis nucleotides, e.g. convert adenosine 5'-triphosphate to adenosine 5'-monophosphate (AMP) and pyrophosphate. Enzymatic reactions were performed and directly subjected to CE analysis. Since the enzymatic activity was low, standard methods were insufficient. The detection of nanomolar AMP and other nucleotides could be achieved by field-enhanced sample injection and the addition of polybrene to the running buffer. The polycationic polymer caused a dynamic coating of the silica-fused capillary, resulting in a reversed electroosmotic flow. The nucleotides migrated in the direction of the electroosmotic flow, whereas the positively charged polybrene molecules moved in the opposite direction, resulting in a narrow sample zone over a long injection time. Using this on-line sensitivity enhancement technique, a more than 70-fold enrichment was achieved for AMP (limit of detection, 46 nM) along with a short migration time (5 min) without compromising separation efficiency and peak shape. The optimized CE conditions were as follows: fused-silica capillary (30 cm effective lengthx75 mum), electrokinetic injection for 60 s, 50 mM phosphate buffer pH 6.5, 0.002% polybrene, constant current of -60 microA, UV detection at 210 nm, uridine 5'-monophosphate as the internal standard. The new method was used to study enzyme kinetics and inhibitors. It opens an easy way to determine the activities of slowly metabolizing enzymes such as NPPs, which are of considerable interest as novel drug targets.

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Capillary electrophoresis-based nanoscale assays for monitoring ecto-5′-nucleotidase activity and inhibition in preparations of recombinant enzyme and melanoma cell membranes

Cited by 41 publications

References 36 publications

A capillary electrophoresis method with dynamic pH junction stacking for the monitoring of cerebroside sulfotransferase

A capillary electrophoresis method with dynamic pH junction stacking for the monitoring of cerebroside sulfotransferase

Chiral ligand‐exchange CE assays for separation of amino acid enantiomers and determination of enzyme kinetic constant

A highly sensitive CE‐UV method with dynamic coating of silica‐fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions

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