Related Experiment Video

Updated: Feb 6, 2026

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium
09:45

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium

Published on: May 18, 2021

2.6K

Continuous assays for meprin alpha and beta using prolyl tripeptidyl aminopeptidase (PtP) from Porphyromonas

Anja Schulze1, Michael Wermann1, Hans-Ulrich Demuth1

  • 1Fraunhofer Institute for Cell Therapy and Immunology, Department of Drug Design and Target Validation (IZI-MWT), Halle (Saale), Germany.

Analytical Biochemistry
|August 13, 2018
PubMed

Insights

Novel continuous assays for meprin alpha and beta endoprotease activity were developed using an auxiliary enzyme and chromogenic or fluorogenic substrates. These methods overcome limitations of existing assays and enable potent inhibitor analysis.

Area of Science:

  • Biochemistry
  • Enzymology

Background:

  • Current assays for meprin α and β endoprotease activity rely on internally quenched substrates, which have limitations.
  • These limitations include significant substrate inhibition and potential fluorescence quenching by inhibitor compounds.

Purpose of the Study:

  • To develop novel, continuous assays for meprin α and β endoprotease activity.
  • To overcome the disadvantages associated with existing assay methods.

Main Methods:

  • A novel continuous assay was established using an auxiliary enzyme, prolyl tripeptidyl aminopeptidase (PtP), and the chromogenic substrate KKGYVADAP-p-nitroanilide.
  • The auxiliary enzyme PtP was expressed and purified in a single step.
  • Conditions for using the fluorogenic substrate KKGYVADAP-AMC to assess meprin β activity were also established.

Main Results:

  • Enzyme kinetic data for meprin α and β exhibited hyperbolic v/S-characteristics.
  • Kinetic parameters for meprin β were determined: Km = 184 ± 32 μM and kcat = 20 ± 4 s-1.
  • The novel assays were successfully applied to determine inhibitory parameters for actinonin and two hydroxamates.

Conclusions:

  • Two novel continuous assay methods for meprin α and β endoprotease activity have been developed.
  • These assays facilitate the assessment of inhibitory mechanisms and potency against these drug targets.
  • The assay methodology may also be applicable to other endoproteases and their inhibitors.
Keywords:
AstacinsAuxiliary enzymeContinuous assayMeprin α/βProlyl tripeptidyl aminopeptidase (PtP)

More Related Videos

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells12:16

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Published on: December 17, 2015

27.1K
Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo09:44

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Published on: June 2, 2019

22.6K

Related Concept Videos

Continuing Care01:25

Continuing Care

Continuing care describes the variety of health, personal, and social services provided over a prolonged period. The need for continuing care is increasing because people are living longer. Many people do not have families or others to care for them. Continuing care is mainly for patients who are disabled, functionally dependent, or suffering from a terminal disease. It is available within institutional settings or in homes. Examples include nursing centers or facilities, assisted living,...
2.0K
Continuity of a Function01:23

Continuity of a Function

A function is continuous at a point a if three conditions are met: the function is defined at a, the limit of the function as x approaches a exists, and this limit equals the function’s value. Mathematically, this is written asThis definition ensures the graph of the function does not exhibit any breaks, holes, or jumps at that point. Discontinuities occur when any of these conditions fail. A removable discontinuity exists when the two-sided limit exists but the function is either...
249
Continuity Equation01:28

Continuity Equation

The continuity equation asserts that the mass flow rate must remain constant for a steady flow of an incompressible fluid within a confined system. This principle applies to systems where fluid passes through varying cross-sectional areas, such as nozzles, syringes, and pipes.
The mass flow rate is expressed as:
3.3K
Continuity Equation01:20

Continuity Equation

The total amount of current flowing per unit cross-sectional area is called the current density. Hence, the current passing through a cross-sectional area can be written as the surface integral of the current density.
1.5K
Equation of Continuity01:12

Equation of Continuity

Fluid motion is represented by either velocity vectors or streamlines. The volume of a fluid flowing past a given location through an area during a period of time is called the flow rate Q, or more precisely, the volume flow rate. Flow rate and velocity are related—for instance, a river has a greater flow rate if the velocity of the water in it is greater. However, the flow rate also depends on the size and shape of the river. The relationship between flow rate (Q) and average speed (v)...
11.1K
Continuous Charge Distributions01:17

Continuous Charge Distributions

Imagine a bucket of water. It contains many molecules, of the order of 1026 molecules. Thus, although it contains discrete elements (molecules) at the microscopic level, macroscopically, it can be considered continuous. Small volume elements of water, infinitesimal compared to the bulk of the bucket's volume, still contain many molecules. Under this framework, quantized matter is approximated as continuous for practical purposes.
The electric charge can also be subjected to an analogical...
8.4K