Previous studies in our laboratory reported L-malate as a free radical scavenger in aged rats. To investigate the antioxidant mechanism of L-malate in the mitochondria, we analyzed the change in gene expression of two malate-aspartate shuttle (MAS)-related carried proteins (AGC, aspartate/glutamate carrier and OMC, oxoglutarate/malate carrier) in the inner mitochondrial membrane, and three antioxidant enzymes (CAT, SOD, and GSH-Px) in the mitochondria. The changes in gene expression of these proteins and enzymes were examined by real-time RT-PCR in the heart and liver of aged rats treated with L-malate. L-malate was orally administered in rats continuously for 30 days using a feeding atraumatic needle. We found that the gene expression of OMC and GSH-Px mRNA in the liver increased by 39 % and 38 %, respectively, in the 0.630 g/kg L-malate treatment group than that in the control group. The expression levels of SOD mRNA in the liver increased by 39 %, 56 %, and 78 % in the 0.105, 0.210, and 0.630 g/kg L-malate treatment groups, respectively. No difference were observed in the expression levels of AGC, OMC, CAT, SOD, and GSH-Px mRNAs in the heart of rats between the L-malate treatment and control groups. These results predicted that L-malate may increase the antioxidant capacity of mitochondria by enhancing the expression of mRNAs involved in the MAS and the antioxidant enzymes.

• MeSH
• antioxidancia metabolismus   farmakologie   MeSH
• antiportéry účinky léků   metabolismus   MeSH
• enzymy genetika   metabolismus   MeSH
• glutathionperoxidasa metabolismus   MeSH
• jaterní mitochondrie účinky léků   enzymologie   MeSH
• játra účinky léků   enzymologie   MeSH
• katalasa metabolismus   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• maláty farmakologie   MeSH
• membránové transportní proteiny účinky léků   genetika   metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• potkani Sprague-Dawley MeSH
• regulace genové exprese enzymů MeSH
• stárnutí genetika   metabolismus   MeSH
• superoxiddismutasa metabolismus   MeSH
• transportní proteiny účinky léků   metabolismus   MeSH
• transportní systém pro kyselé aminokyseliny účinky léků   metabolismus   MeSH
• upregulace MeSH
• věkové faktory MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) from mature maize seeds (Zea mays L.) was purified to homogeneity and a final specific activity of 13.3 μmol min⁻¹ mg⁻¹. Purified PEPC was treated with phosphatase from bovine intestinal mucosa or protein kinase A to study its apparent phosphorylation level. Kinetic parameters of the enzyme reaction catalyzed by phosphorylated and dephosphorylated forms under different conditions were compared, as well as an effect of modulators. The enzyme dephosphorylation resulted in the change of hyperbolic kinetics to the sigmoidal one (with respect to PEP), following with the decrease of maximal reaction rate and the increase of sensitivity to L-malate inhibition. The hyperbolic kinetics of native PEPC present in dry maize seeds was not changed after the protein kinase A treatment, while it was converted to the sigmoidal one after dephosphorylation. Level of PEPC phosphorylation was not affected during seed imbibition.

• MeSH
• fosfoenolpyruvátkarboxylasa antagonisté a inhibitory   izolace a purifikace   metabolismus   MeSH
• fosforylace MeSH
• kinetika MeSH
• kukuřice setá enzymologie   MeSH
• maláty farmakologie   MeSH
• proteinkinasy závislé na cyklickém AMP farmakologie   MeSH
• semena rostlinná enzymologie   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Complex I, i.e. proton-pumping NADH:quinone oxidoreductase, is an essential component of the mitochondrial respiratory chain but produces superoxide as a side-reaction. However, conditions for maximum superoxide production or its attenuation are not well understood. Unlike for Complex III, it has not been clear whether a Complex I-derived superoxide generation at forward electron transport is sensitive to membrane potential or protonmotive force. In order to investigate this, we used Amplex Red for H(2)O(2) monitoring, assessing the total mitochondrial superoxide production in isolated rat liver mitochondria respiring at state 4 as well as at state 3, namely with exclusive Complex I substrates or with Complex I substrates plus succinate. We have shown for the first time, that uncoupling diminishes rotenone-induced H(2)O(2) production also in state 3, while similar attenuation was observed in state 4. Moreover, we have found that 5-(N-ethyl-N-isopropyl) amiloride is a real inhibitor of Complex I H(+) pumping (IC(50) of 27 microM) without affecting respiration. It also partially prevented suppression by FCCP of rotenone-induced H(2)O(2) production with Complex I substrates alone (glutamate and malate), but nearly completely with Complexes I and II substrates. Sole 5-(N-ethyl-N-isopropyl) amiloride alone suppressed 20% and 30% of total H(2)O(2) production, respectively, under these conditions. Our data suggest that Complex I mitochondrial superoxide production can be attenuated by uncoupling, which means by acceleration of Complex I H(+) pumping due to the respiratory control. However, when this acceleration is prevented by 5-(N-ethyl-N-isopropyl) amiloride inhibition, no attenuation of superoxide production takes place.

• MeSH
• amilorid analogy a deriváty   farmakologie   MeSH
• biologické modely MeSH
• buněčné dýchání účinky léků   MeSH
• financování organizované MeSH
• jaterní mitochondrie enzymologie   účinky léků   MeSH
• krysa rodu rattus MeSH
• kyselina glutamová farmakologie   MeSH
• kyselina jantarová farmakologie   MeSH
• maláty farmakologie   MeSH
• peroxid vodíku metabolismus   MeSH
• potkani Wistar MeSH
• protonové pumpy metabolismus   MeSH
• respirační komplex I metabolismus   MeSH
• rozpřahující látky farmakologie   MeSH
• superoxidy metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH