In Vitro Kinetic Characterization of Transporter-Mediated Permeability

2013 ◽  
pp. 23-35
Author(s):  
Bente Steffansen ◽  
Anne Sophie Grandvuinet
Keyword(s):  
Kinetic Characterization

scholarly journals Kinetic characterization of inhibitors of histone deacetylases (HDACs) and sirtuins

2019 ◽  
Author(s):  
Carlos Moreno-Yruela ◽  
Andreas Stahl Madsen ◽  
Christian A. Olsen
Keyword(s):  
Histone Deacetylases ◽  
Hdac Inhibitors ◽  
Kinetic Properties ◽  
Kinetic Characterization ◽  
Class Iii ◽  
Vitro Assay ◽  
Substrate Conversion ◽  
Fluorogenic Peptide Substrate

Abstract Histone deacetylase (HDAC) inhibitors are employed for the treatment of lymphoma and are under development against multiple other types of cancer and neurodegenerative diseases. Here, we describe a robust and uncomplicated in vitro assay for HDAC inhibitor kinetic profiling. Enzyme and fluorogenic peptide substrate are incubated together with a small amount of protease “assay developer”, which enables continuous recording of substrate conversion under steady-state conditions. Assay progression curves upon addition of an inhibitors at varying concentrations permit determination of kinetic constants and overall inhibitor potency. This assay helped provide new insight into the kinetic properties of known HDAC inhibitors as well as the kinetic characterization of both inhibitors and substrates of sirtuin enzymes, which are class III HDACs involved in metabolic control and oncogene regulation.

Kinetic characterization of CYP2E1 inhibition in vivo and in vitro by the chloroethylenes

1998 ◽  
Vol 72 (10) ◽  
pp. 609-621 ◽  
Author(s):  
Patrick D. Lilly ◽  
Janice R. Thornton-Manning ◽  
Michael L. Gargas ◽  
Harvey J. Clewell ◽  
Melvin E. Andersen ◽  
...  
Keyword(s):  
Kinetic Characterization

Activation of JNK3α1 Requires both MKK4 and MKK7:  Kinetic Characterization of in Vitro Phosphorylated JNK3α1

Biochemistry ◽  
2000 ◽  
Vol 39 (11) ◽  
pp. 3141-3148 ◽  
Author(s):  
JeanMarie Lisnock ◽  
Patrick Griffin ◽  
Jimmy Calaycay ◽  
Betsy Frantz ◽  
Janey Parsons ◽  
...  
Keyword(s):  
Kinetic Characterization

scholarly journals A Basic Post-SET Extension of NSDs Is Essential for Nucleosome Binding In Vitro

2014 ◽  
Vol 19 (6) ◽  
pp. 928-935 ◽  
Author(s):  
Abdellah Allali-Hassani ◽  
Ekaterina Kuznetsova ◽  
Taraneh Hajian ◽  
Hong Wu ◽  
Ludmila Dombrovski ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Substrate Specificity ◽  
Nuclear Receptor ◽  
Histone H3 ◽  
In Vitro Assays ◽  
Kinetic Characterization ◽  
Set Domain ◽  
Nucleosome Binding

The nuclear receptor SET domain-containing family of proteins (NSD1, NSD2, and NSD3) is known to mono- and dimethylate lysine 36 of histone H3 (H3K36). Overexpression and translocation of NSDs have been widely implicated in a variety of diseases including cancers. Although the substrate specificity of NSDs has been a subject of many valuable studies, the activity of these proteins has never been fully characterized in vitro. In this study, we present full kinetic characterization of NSD1, NSD2, and NSD3 and provide robust in vitro assays suitable for screening these proteins in a 384-well format using nucleosome as a substrate. Through monitoring the changes in substrate specificity of a series of NSD constructs and using molecular modeling, we show that a basic post-SET extension common to all three NSDs (corresponding to residues 1209 to 1226 of NSD2) is essential for proper positioning on nucleosome substrates.

In Vitro Reconstitution and Kinetic Characterization of the Essential Condensation Enzymes Involved in Prodiginine and Tambjamine Biosynthesis

2019 ◽  
Author(s):  
Katherine J. Picott ◽  
Julie A. Deichert ◽  
Ella M. deKemp ◽  
Avena Ross
Keyword(s):  
Substrate Inhibition ◽  
Kinetic Characterization ◽  
Reaction Sequence ◽  
Ping Pong ◽  
In Vitro Reconstitution

We have reconstituted<i> </i>the <i>in vitro</i> activity of the purified condensation enzymes PigC and TamQ, from prodiginine and tambjamine pathways respectively. Upon analysis and comparison of their kinetic profiles, a ping-pong reaction sequence with substrate inhibition was identified for each condensation enzyme.

Kinetic characterization of apoptotic Ras signaling through Nore1-MST1 complex formation

2017 ◽  
Vol 398 (5-6) ◽  
pp. 701-707 ◽  
Author(s):  
Agne Koturenkiene ◽  
Cihan Makbul ◽  
Christian Herrmann ◽  
Diana Constantinescu-Aruxandei
Keyword(s):  
Complex Formation ◽  
Ternary Complex ◽  
External Factors ◽  
Kinetic Constants ◽  
Ras Signaling ◽  
Kinetic Characterization ◽  
Apoptotic Signaling ◽  
First Time

Abstract Ras-mediated apoptotic signaling is expected to be mediated via Rassf-MST complexes, but the system has been poorly characterized in vitro until now. Here we demonstrate that active H-Ras, Nore1A and MST1 form a stable ternary complex in vitro without other external factors, Nore1A interacting simultaneously with H-Ras and MST1 via its RBD and SARAH domain, respectively. Moreover, our data show for the first time that the SARAH domain of Nore1A plays a role in the Nore1A binding to H-Ras. Finally, we analyze the relation between the electrostatic and hydrophobic forces and kinetic constants of the Nore1A – H-Ras complex.

scholarly journals Kinetic Characterization of the Glycosyltransferase Module of Staphylococcus aureus PBP2

2005 ◽  
Vol 187 (6) ◽  
pp. 2215-2217 ◽  
Author(s):  
Dianah Barrett ◽  
Catherine Leimkuhler ◽  
Lan Chen ◽  
Deborah Walker ◽  
Daniel Kahne ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Important Class ◽  
Kinetic Characterization ◽  
Penicillin Binding Protein ◽  
Gram Positive ◽  
Class A ◽  
Lipid Ii ◽  
Positive Class

ABSTRACT We report the heterologous overexpression and purification of Staphylococcus aureus PBP2 and demonstrate efficient glycan polymerization from lipid II in vitro. S. aureus PBP2 is the first purified gram-positive class A penicillin-binding protein to show good transglycosylase activity. This enables further studies on this important class of enzymes.

scholarly journals Kinetic characterization of selective peroxisome-proliferator-activated receptor gamma modulators in vitro

Critical Care ◽  
2012 ◽  
Vol 16 (S3) ◽  
Author(s):  
T Knape ◽  
LK Eifler ◽  
A Heeg ◽  
L Kuchler ◽  
B Brüne ◽  
...  
Keyword(s):  
Peroxisome Proliferator ◽  
Kinetic Characterization ◽  
Peroxisome Proliferator Activated Receptor
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