Whether active UCP1 can reduce ROS production in brown-fat mitochondria is presently not settled. The issue is of principal significance, as it can be seen as a proof- or disproof-of-principle concerning the ability of any protein to diminish ROS production through membrane depolarization. We therefore undertook a comprehensive investigation of the significance of UCP1 for ROS production, by comparing the ROS production in brown-fat mitochondria isolated from wildtype mice (that display membrane depolarization) or from UCP1(-/-) mice (with a high membrane potential). We tested the significance of UCP1 for glycerol-3-phosphate-supported ROS production by three methods (fluorescent dihydroethidium and the ESR probe PHH for superoxide, and fluorescent Amplex Red for hydrogen peroxide), and followed ROS production also with succinate, acyl-CoA or pyruvate as substrate. We studied the effects of the reverse electron flow inhibitor rotenone, the UCP1 activity inhibitor GDP, and the uncoupler FCCP. We also examined the effect of a physiologically induced increase in UCP1 amount. We noted GDP effects that were not UCP1-related. We conclude that only ROS production supported by exogenously added succinate was affected by the presence of active UCP1; ROS production supported by any other tested substrate (including endogenously generated succinate) was unaffected. This conclusion indicates that UCP1 is not involved in control of ROS production in brown-fat mitochondria. Extrapolation of these data to other tissues would imply that membrane depolarization may not necessarily decrease physiologically relevant ROS production. This article is a part of a Special Issue entitled: 18th European Bioenergetics Conference (Biochim. Biophys. Acta, Volume 1837, Issue 7, July 2014).

• Klíčová slova
• Brown adipose tissue mitochondria, Cold acclimation, Glycerol-3-phosphate dehydrogenase, Reactive oxygen species, Succinate, Uncoupling protein 1,
• MeSH
• elektronová paramagnetická rezonance MeSH
• glycerolfosfáty farmakologie   MeSH
• guanosindifosfát farmakologie   MeSH
• hnědá tuková tkáň metabolismus   MeSH
• imunoblotting MeSH
• iontové kanály genetika   metabolismus   MeSH
• karbonylkyanid-p-trifluormethoxyfenylhydrazon farmakologie   MeSH
• kyselina jantarová farmakologie   MeSH
• kyselina pyrohroznová farmakologie   MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie účinky léků   metabolismus   fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• nízká teplota MeSH
• peroxid vodíku metabolismus   MeSH
• protonové ionofory farmakologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• spotřeba kyslíku účinky léků   MeSH
• superoxidy metabolismus   MeSH
• uncoupling protein 1 MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alpha-glycerophosphoric acid MeSH Prohlížeč
• glycerolfosfáty MeSH
• guanosindifosfát MeSH
• iontové kanály MeSH
• karbonylkyanid-p-trifluormethoxyfenylhydrazon MeSH
• kyselina jantarová MeSH
• kyselina pyrohroznová MeSH
• mitochondriální proteiny MeSH
• peroxid vodíku MeSH
• protonové ionofory MeSH
• reaktivní formy kyslíku MeSH
• superoxidy MeSH
• Ucp1 protein, mouse MeSH Prohlížeč
• uncoupling protein 1 MeSH

The physiological role of monocarboxylate transport in brown adipose tissue mitochondria has been reevaluated. We studied pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and phenylpyruvate uniport via the uncoupling protein (UCP1) as a GDP-sensitive swelling in K+ salts induced by valinomycin or by monensin and carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone in Na+ salts. We have demonstrated that this uniport is inhibited by fatty acids. GDP inhibition in K+ salts was not abolished by an uncoupler, indicating a negligible monocarboxylic acid penetration via the lipid bilayer. In contrast, the electroneutral pyruvate uptake (swelling in ammonium pyruvate or potassium pyruvate induced by change in pH) mediated by the pyruvate carrier was inhibited by its specific inhibitor alpha-cyano-4-hydroxycinnamate but not by fatty acids. Moreover, alpha-cyano-4-hydroxycinnamate enhanced the energization of brown adipose tissue mitochondria, which was monitored fluorometrically by 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide and safranin O. Consequently, we suggest that UCP1 might participate in futile cycling of unipolar ketocarboxylates under certain physiological conditions while expelling these anions from the matrix. The cycle is completed on their return via the pyruvate carrier in an H+ symport mode.

• MeSH
• biologický transport MeSH
• guanosindifosfát farmakologie   MeSH
• hnědá tuková tkáň metabolismus   MeSH
• iontové kanály MeSH
• karbonylkyanid-p-trifluormethoxyfenylhydrazon farmakologie   MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• křečci praví MeSH
• křeček rodu Mesocricetus MeSH
• kyseliny fenylpyrohroznové farmakologie   MeSH
• kyseliny karboxylové metabolismus   MeSH
• kyseliny kumarové farmakologie   MeSH
• lipidové dvojvrstvy MeSH
• membránové proteiny metabolismus   MeSH
• mitochondriální proteiny MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• monensin farmakologie   MeSH
• pyruváty metabolismus   MeSH
• rotenon farmakologie   MeSH
• transportní proteiny metabolismus   MeSH
• uncoupling protein 1 MeSH
• valinomycin farmakologie   MeSH
• zduření mitochondrií účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alpha-cyano-4-hydroxycinnamate MeSH Prohlížeč
• guanosindifosfát MeSH
• iontové kanály MeSH
• karbonylkyanid-p-trifluormethoxyfenylhydrazon MeSH
• kyseliny fenylpyrohroznové MeSH
• kyseliny karboxylové MeSH
• kyseliny kumarové MeSH
• lipidové dvojvrstvy MeSH
• membránové proteiny MeSH
• mitochondriální proteiny MeSH
• monensin MeSH
• phenylpyruvic acid MeSH Prohlížeč
• pyruváty MeSH
• rotenon MeSH
• transportní proteiny MeSH
• uncoupling protein 1 MeSH
• valinomycin MeSH

Fatty acid (FA) uniport via mitochondrial uncoupling protein (UcP) was detected fluorometrically with PBFI, potassium-binding benzofuran phthalate and SPQ, 6-methoxy-N-(3-sulfopropyl)-quinolinium, indicating K+ and H+, respectively. The FA structural patterns required for FA flip-flop, UcP-mediated FA uniport, activation of UcP-mediated H+ transport in proteoliposomes, and inhibition of UcP-mediated Cl- uniport by FA, were identical. Positive responses were found exclusively with FA which were able to flip-flop in a protonated form across the membrane and no responses were found with 'inactive' FA lacking the flip-flop ability. The findings support the existence of FA cycling mechanism.

• MeSH
• benzoáty metabolismus   MeSH
• benzofurany metabolismus   MeSH
• biologický transport MeSH
• chinolinové sloučeniny metabolismus   MeSH
• chloridy metabolismus   MeSH
• draslík metabolismus   MeSH
• ethery cyklické metabolismus   MeSH
• fluorescenční barviva metabolismus   MeSH
• fluorometrie MeSH
• guanosindifosfát farmakologie   MeSH
• ionofory farmakologie   MeSH
• iontové kanály MeSH
• koncentrace vodíkových iontů MeSH
• kyseliny laurové metabolismus   MeSH
• liposomy metabolismus   MeSH
• mastné kyseliny chemie   metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• mitochondriální proteiny MeSH
• protony MeSH
• transportní proteiny metabolismus   MeSH
• uncoupling protein 1 MeSH
• valinomycin farmakologie   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• 12-hydroxydodecanoic acid MeSH Prohlížeč
• 6-methoxy-N-(3-sulfopropyl)quinolinium MeSH Prohlížeč
• benzoáty MeSH
• benzofurany MeSH
• chinolinové sloučeniny MeSH
• chloridy MeSH
• draslík MeSH
• ethery cyklické MeSH
• fluorescenční barviva MeSH
• guanosindifosfát MeSH
• ionofory MeSH
• iontové kanály MeSH
• kyseliny laurové MeSH
• liposomy MeSH
• mastné kyseliny MeSH
• membránové proteiny MeSH
• mitochondriální proteiny MeSH
• potassium-binding benzofuran isophthalate MeSH Prohlížeč
• protony MeSH
• transportní proteiny MeSH
• uncoupling protein 1 MeSH
• valinomycin MeSH

The uncoupling protein generates heat by catalyzing electrophoretic proton transport across the inner membrane of brown adipose tissue mitochondria. It also transports Cl- and other monovalent anions, and both proton and anion transport are inhibited by purine nucleotides. Several long-standing hypotheses bear on specific aspects of Cl- transport, H+ transport, and nucleotide gating mechanisms in uncoupling protein. We reevaluated these hypotheses in mitochondria and liposomes reconstituted with purified uncoupling protein; GDP inhibition is strictly noncompetitive with Cl- and unaffected by either transmembrane electrical potential or fatty acids. The Km and Vmax values for Cl- are independent of pH, arguing against a common binding site for Cl- and OH- ions. Cl- transport was inhibited by fatty acids and stimulated by fatty acid removal, refuting the consensus hypothesis that there is no interaction between fatty acids and anion transport through uncoupling protein. These results support a mechanism in which the transport pathway for anions is identical with the fatty acid binding site and distinct from the nucleotide binding site.

• MeSH
• biologický transport MeSH
• chloridy metabolismus   MeSH
• guanosindifosfát farmakologie   MeSH
• hnědá tuková tkáň metabolismus   MeSH
• iontové kanály MeSH
• koncentrace vodíkových iontů MeSH
• křečci praví MeSH
• křeček rodu Mesocricetus MeSH
• mastné kyseliny farmakologie   MeSH
• membránové proteiny farmakologie   MeSH
• mitochondriální proteiny MeSH
• mitochondrie metabolismus   MeSH
• rozpřahující látky farmakologie   MeSH
• sérový albumin hovězí farmakologie   MeSH
• transportní proteiny farmakologie   MeSH
• uncoupling protein 1 MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• chloridy MeSH
• guanosindifosfát MeSH
• iontové kanály MeSH
• mastné kyseliny MeSH
• membránové proteiny MeSH
• mitochondriální proteiny MeSH
• rozpřahující látky MeSH
• sérový albumin hovězí MeSH
• transportní proteiny MeSH
• uncoupling protein 1 MeSH

As it has been found, the incubation of [gamma-32P]ATP with elongation factor--1 alpha purified from rabbit reticulocytes resulted in the phosphorylation of several substrate proteins /Tuhácková, Z. (1992) In: Rec. Adv. Cell. Mol. Biol. 4, 79-86, Peeters Press, Leuven/ (1). In the present paper chromatofocusing of the purified eEF-1 alpha demonstrates that the ATP-dependent protein kinase activity is associated with a single protein catalyzing the GTP-dependent binding of aminoacyl-tRNA to ribosomes. Both of these activities are inhibited by staurosporine and gossypol. The inhibition by GDP but not by GTP indicates a possible involvement of conformation changes also in the modulation of the protein kinase activity displayed by eEF-1 alpha.

• MeSH
• alkaloidy farmakologie   MeSH
• chromatografie afinitní MeSH
• chromatografie iontoměničová MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• elongační faktor 1 MeSH
• elongační faktory antagonisté a inhibitory   krev   izolace a purifikace   MeSH
• fosforylace MeSH
• gossypol farmakologie   MeSH
• guanosindifosfát farmakologie   MeSH
• histony metabolismus   MeSH
• kinetika MeSH
• králíci MeSH
• molekulová hmotnost MeSH
• proteinkinasa C antagonisté a inhibitory   MeSH
• retikulocyty metabolismus   MeSH
• ribozomy metabolismus   MeSH
• staurosporin MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alkaloidy MeSH
• elongační faktor 1 MeSH
• elongační faktory MeSH
• gossypol MeSH
• guanosindifosfát MeSH
• histony MeSH
• proteinkinasa C MeSH
• staurosporin MeSH

Mersalyl, 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) and fluorescent Thiolyte DB react with SH groups in the H+ channel (SHc) of the uncoupling protein of brown adipose tissue mitochondria, as inferred from their inhibition of H+ transport. Cl- transport by the uncoupling protein was unaffected. Using these modifiers and N-ethylmaleimide (MalNEt), distinct SH groups (SHB) in the purine nucleotide binding site were identified. Nbs2 reacts more readily with the SHB than with the SHc groups, but mersalyl and Thiolyte DB are more reactive with the SHc groups. MalNEt reacts exclusively with the SHB. GDP inhibition is fully prevented after sufficient modification of the SHB. Pretreatment with p-diazobenzenesulfonate (N2PhSO2) suppresses only 20-25% of fluorescence of Thiolyte-DB-labeled uncoupling protein on SDS/PAGE gels, while MalNEt suppresses 66% and Nbs2 80-90%. Since N2PhSO2 also affects the GDP binding site, these results demonstrate that the N2PhSO2-reactive residue is not identical with the SHB.

• MeSH
• biologický transport účinky léků   MeSH
• chloridy analýza   MeSH
• fluorescenční barviva MeSH
• fluorescenční spektrometrie MeSH
• guanosindifosfát farmakologie   MeSH
• hnědá tuková tkáň analýza   enzymologie   MeSH
• iontové kanály účinky léků   MeSH
• mersalyl MeSH
• mitochondrie analýza   enzymologie   MeSH
• nitrobenzoany MeSH
• protonové ATPasy analýza   MeSH
• purinové nukleotidy analýza   MeSH
• rozpřahující látky analýza   fyziologie   MeSH
• sulfhydrylové sloučeniny analýza   farmakologie   MeSH
• vazebná místa účinky léků   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chloridy MeSH
• fluorescenční barviva MeSH
• guanosindifosfát MeSH
• iontové kanály MeSH
• mersalyl MeSH
• nitrobenzoany MeSH
• protonové ATPasy MeSH
• purinové nukleotidy MeSH
• rozpřahující látky MeSH
• sulfhydrylové sloučeniny MeSH

A concerted function of purine nucleotide (PN) binding and fatty acid (FA) release from the uncoupling protein (UP) resulting in the maximum coupling (potential) of brown adipose tissue (BAT) mitochondria was demonstrated. The uncoupling effect of FA was studied (at 4 mM MgCl2): 17 nmol oleate per mg protein caused a slight uncoupling with 8.9 mM ATP but with ATP below 3.6 mM almost total uncoupling was achieved. This shows that the PN-controlled gate can be stabilized in the closed conformation (with 8.9 mM ATP), also when FA is bound to UP. The sensitivity of the FA effect to ATP proves that oleate directly interacts with UP. The closed conformation of the H+ channel of UP is then abolished by oleate when a lower free ATP concentration is maintained outside.

• MeSH
• adenosindifosfát farmakologie   MeSH
• adenosintrifosfát farmakologie   MeSH
• guanosindifosfát farmakologie   MeSH
• hnědá tuková tkáň metabolismus   MeSH
• homeostáza MeSH
• iontové kanály MeSH
• karnitin metabolismus   MeSH
• kinetika MeSH
• křečci praví MeSH
• kyselina olejová MeSH
• kyseliny mastné neesterifikované fyziologie   MeSH
• kyseliny olejové farmakologie   MeSH
• membránové proteiny metabolismus   MeSH
• mitochondriální proteiny MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• rozpřahující látky farmakologie   MeSH
• spotřeba kyslíku účinky léků   MeSH
• transportní proteiny * MeSH
• uncoupling protein 1 MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosindifosfát MeSH
• adenosintrifosfát MeSH
• guanosindifosfát MeSH
• iontové kanály MeSH
• karnitin MeSH
• kyselina olejová MeSH
• kyseliny mastné neesterifikované MeSH
• kyseliny olejové MeSH
• membránové proteiny MeSH
• mitochondriální proteiny MeSH
• rozpřahující látky MeSH
• transportní proteiny * MeSH
• uncoupling protein 1 MeSH