Activity Assays for Rhomboid Proteases

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F17%3A00483419" target="_blank" >RIV/61388963:_____/17:00483419 - isvavai.cz</a>

Výsledek na webu

<a href="http://dx.doi.org/10.1016/bs.mie.2016.11.002" target="_blank" >http://dx.doi.org/10.1016/bs.mie.2016.11.002</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/bs.mie.2016.11.002" target="_blank" >10.1016/bs.mie.2016.11.002</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Activity Assays for Rhomboid Proteases

Popis výsledku v původním jazyce

Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis, however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of KM and V-max to measure catalytic parameters, using detergent solubilized rhomboids with TatA, the only known substrate for bacterial rhomboids, and the model substrate fluorescently labeled casein.

Název v anglickém jazyce

Activity Assays for Rhomboid Proteases

Popis výsledku anglicky

Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis, however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of KM and V-max to measure catalytic parameters, using detergent solubilized rhomboids with TatA, the only known substrate for bacterial rhomboids, and the model substrate fluorescently labeled casein.

Druh

C - Kapitola v odborné knize

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2017

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název knihy nebo sborníku

Enzymology at the Membrane Interface: Intramembrane Proteases

ISBN

978-0-12-812213-6

Počet stran výsledku

43

Strana od-do

395-437

Počet stran knihy

474

Název nakladatele

Academic Press

Místo vydání

Cambridge

Kód UT WoS kapitoly

000403271000016