Determination of enzyme activity by high-performance liquid chromatography

Welling, Gjalt W.; Scheffer, Albert Jan; Welling‐Wester, Sytske

doi:10.1016/0378-4347(94)00154-5

Cited by 13 publications

(3 citation statements)

References 108 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Identification of the metabolized products of the angiotensins was performed (a) by coelution with marker peptides in reversed-phase HPLC [13], (b) by reaction with specific antisera in immunoassays (DIA and/or ELISA) and (c) by Edman degradation on a pulse-liquid Results obtained with head membrane preparations are identical to those obtained with membrane preparations of CNS. However, in order to have sufficient material for these studies, head parts including the whole of the CNS were used.…”

Section: Analysis Of Peptide Productsmentioning

confidence: 99%

Metabolism of angiotensins by head membranes of the leech Theromyzon tessulatum

Laurent

1996

FEBS Letters

View full text Add to dashboard Cite

Angiotensins (angiotensin I, angiotensin II, angiotensin II-amide) have been isolated in leeches and such peptides are involved in diuresis in these animals. To explore possible inactivation mechanisms of these peptides, angiotensins were incubated with head membranes of the leech T. tessulatum. Membranes derived from head parts of this leech are very rich in peptidases. They contain endopeptidase-24.11-1ike enzyme (NEP-like) associated with a battery of exopeptidase. The way that angiotensins are degraded by the combined attack of these membrane peptidases has been investigated. The contribution of individual peptidases was assessed by adding inhibitors (phosphoramidon, captopril and amastatin) to the membrane fractions, when they were incubated with the peptides. In the case of angiotensin I, the primary attack was performed by a combined action of the NEP-like and the ACE-like enzymes, followed by aminopeptidase attacks. Angiotensin II and III were hydrolyzed by NEP-like enzyme at the same Tyr-Ile bond, whereas the Nterminal arginine residue of angiotensin IH was removed by an arginyl aminopeptidase. These results show that angiotensins are efficiently degraded by membranes and that NEP-like enzyme plays a key role in this process.

show abstract

Section: Analysis Of Peptide Productsmentioning

confidence: 99%

Metabolism of angiotensins by head membranes of the leech Theromyzon tessulatum

Laurent

1996

FEBS Letters

View full text Add to dashboard Cite

show abstract

“…As a second alternative, if no convenient spectrophotometric assay is available, metabolites can also be measured with (high performance) liquid chromatography [11], either on its own, e.g., using detection by UV-light absorbance, or in combination with mass spectrometry. In contrast to spectrophotometric measurements, this is a discontinuous assay, requiring that the reaction be quenched at different time points before the substrates and products are analysed in order to obtain a time-course.…”

Section: Introductionmentioning

confidence: 99%

Workflow for Data Analysis in Experimental and Computational Systems Biology: Using Python as ‘Glue’

et al. 2019

View full text Add to dashboard Cite

Bottom-up systems biology entails the construction of kinetic models of cellular pathways by collecting kinetic information on the pathway components (e.g., enzymes) and collating this into a kinetic model, based for example on ordinary differential equations. This requires integration and data transfer between a variety of tools, ranging from data acquisition in kinetics experiments, to fitting and parameter estimation, to model construction, evaluation and validation. Here, we present a workflow that uses the Python programming language, specifically the modules from the SciPy stack, to facilitate this task. Starting from raw kinetics data, acquired either from spectrophotometric assays with microtitre plates or from Nuclear Magnetic Resonance (NMR) spectroscopy time-courses, we demonstrate the fitting and construction of a kinetic model using scientific Python tools. The analysis takes place in a Jupyter notebook, which keeps all information related to a particular experiment together in one place and thus serves as an e-labbook, enhancing reproducibility and traceability. The Python programming language serves as an ideal foundation for this framework because it is powerful yet relatively easy to learn for the non-programmer, has a large library of scientific routines and active user community, is open-source and extensible, and many computational systems biology software tools are written in Python or have a Python Application Programming Interface (API). Our workflow thus enables investigators to focus on the scientific problem at hand rather than worrying about data integration between disparate platforms.

show abstract

“…Yet, the method does not simplify the generation of kinetic data based on initial rate measurements, because only a single activity data point per plate can be obtained at a time. Employing HPLC as analytic instrument has been published and reviewed earlier for various sample matrixes and the attractiveness of such systems is based on the automated injection capability, modularity and analytical versatility.…”

Section: Introductionmentioning

confidence: 99%

Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP‐dependent enzymes

Börner

Grey

Adlercreutz

2016

Biotechnology Journal

View full text Add to dashboard Cite

Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes.

show abstract

Determination of enzyme activity by high-performance liquid chromatography

Cited by 13 publications

References 108 publications

Metabolism of angiotensins by head membranes of the leech Theromyzon tessulatum

Metabolism of angiotensins by head membranes of the leech Theromyzon tessulatum

Workflow for Data Analysis in Experimental and Computational Systems Biology: Using Python as ‘Glue’

Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP‐dependent enzymes

Contact Info

Product

Resources

About