Na /K -ATPase interaction with methylglyoxal as reactive metabolic side product

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61989592%3A15110%2F17%3A73582007" target="_blank" >RIV/61989592:15110/17:73582007 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00209805:_____/17:00077887

Výsledek na webu

<a href="https://ac.els-cdn.com/S0891584917301739/1-s2.0-S0891584917301739-main.pdf?_tid=37409eb4-c2e3-11e7-a507-00000aacb362&acdnat=1509966860_7109c31ebd4d3a12da2c5260fc2e404b" target="_blank" >https://ac.els-cdn.com/S0891584917301739/1-s2.0-S0891584917301739-main.pdf?_tid=37409eb4-c2e3-11e7-a507-00000aacb362&acdnat=1509966860_7109c31ebd4d3a12da2c5260fc2e404b</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.freeradbiomed.2017.03.024" target="_blank" >10.1016/j.freeradbiomed.2017.03.024</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Na /K -ATPase interaction with methylglyoxal as reactive metabolic side product

Popis výsledku v původním jazyce

Proteins are subject to oxidative modification and the formation of adducts with a broad spectrum of reactive species via enzymatic and non-enzymatic mechanisms. Here we report that in vitro non-enzymatic methylglyoxal (MGO) binding causes the inhibition and formation of MGO advanced glycation end-products (MAGEs) in Na+/K+-ATPase (NKA). Concretely, MGO adducts with NKA amino acid residues (mainly Arg) and Nε-(carboxymethyl)lysine (CML) formation were found. MGO is not only an inhibitor for solubilized NKA (IC50=91±16μM), but also for reconstituted NKA in the lipid bilayer environment, which was clearly demonstrated using a DPPC/DPPE liposome model in the presence or absence of the NKA-selective inhibitor ouabain. High-resolution mass spectrometric analysis of a tryptic digest of NKA isolated from pig (Sus scrofa) kidney indicates that the intracellular α-subunit is naturally (post-translationally) modified by MGO in vivo. In contrast to this, the β-subunit could only be modified by MGO artificially, and the transmembrane part of the protein did not undergo MGO binding under the experimental setup used. As with bovine serum albumin, serving as the water-soluble model, we also demonstrated a high binding capacity of MGO to water-poorly soluble NKA using a multi-spectral methodology based on electroanalytical, immunochemical and fluorimetric tools. In addition, a partial suppression of the MGO-mediated inhibitory effect could be observed in the presence of aminoguanidine (pimagedine), a glycation suppressor and MGO-scavenger. All the results here were obtained with the X-ray structure of NKA in the E1 conformation (3WGV) and could be used in the further interpretation of the functionality of this key enzyme in the presence of highly-reactive metabolic side-products, glycation agents and generally under oxidative stress conditions.

Název v anglickém jazyce

Na /K -ATPase interaction with methylglyoxal as reactive metabolic side product

Popis výsledku anglicky

Proteins are subject to oxidative modification and the formation of adducts with a broad spectrum of reactive species via enzymatic and non-enzymatic mechanisms. Here we report that in vitro non-enzymatic methylglyoxal (MGO) binding causes the inhibition and formation of MGO advanced glycation end-products (MAGEs) in Na+/K+-ATPase (NKA). Concretely, MGO adducts with NKA amino acid residues (mainly Arg) and Nε-(carboxymethyl)lysine (CML) formation were found. MGO is not only an inhibitor for solubilized NKA (IC50=91±16μM), but also for reconstituted NKA in the lipid bilayer environment, which was clearly demonstrated using a DPPC/DPPE liposome model in the presence or absence of the NKA-selective inhibitor ouabain. High-resolution mass spectrometric analysis of a tryptic digest of NKA isolated from pig (Sus scrofa) kidney indicates that the intracellular α-subunit is naturally (post-translationally) modified by MGO in vivo. In contrast to this, the β-subunit could only be modified by MGO artificially, and the transmembrane part of the protein did not undergo MGO binding under the experimental setup used. As with bovine serum albumin, serving as the water-soluble model, we also demonstrated a high binding capacity of MGO to water-poorly soluble NKA using a multi-spectral methodology based on electroanalytical, immunochemical and fluorimetric tools. In addition, a partial suppression of the MGO-mediated inhibitory effect could be observed in the presence of aminoguanidine (pimagedine), a glycation suppressor and MGO-scavenger. All the results here were obtained with the X-ray structure of NKA in the E1 conformation (3WGV) and could be used in the further interpretation of the functionality of this key enzyme in the presence of highly-reactive metabolic side-products, glycation agents and generally under oxidative stress conditions.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

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

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2017

Kód důvěrnosti údajů

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

Název periodika

Free Radical Biology &amp; Medicine

ISSN

0891-5849

e-ISSN

—

Svazek periodika

108

Číslo periodika v rámci svazku

July

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

9

Strana od-do

146-154

Kód UT WoS článku

000403463500014

EID výsledku v databázi Scopus

—