The F-ATPases (also called the F1 Fo -ATPases or ATP synthases) are multi-subunit membrane-bound molecular machines that produce ATP in bacteria and in eukaryotic mitochondria and chloroplasts. The structures and enzymic mechanisms of their F1 -catalytic domains are highly conserved in all species investigated hitherto. However, there is evidence that the F-ATPases from the group of protozoa known as Euglenozoa have novel features. Therefore, we have isolated pure and active F1 -ATPase from the euglenozoan parasite, Trypanosoma brucei, and characterized it. All of the usual eukaryotic subunits (α, β, γ, δ, and ε) were present in the enzyme, and, in addition, two unique features were detected. First, each of the three α-subunits in the F1 -domain has been cleaved by proteolysis in vivo at two sites eight residues apart, producing two assembled fragments. Second, the T. brucei F1 -ATPase has an additional subunit, called p18, present in three copies per complex. Suppression of expression of p18 affected in vitro growth of both the insect and infectious mammalian forms of T. brucei. It also reduced the levels of monomeric and multimeric F-ATPase complexes and diminished the in vivo hydrolytic activity of the enzyme significantly. These observations imply that p18 plays a role in the assembly of the F1 domain. These unique features of the F1 -ATPase extend the list of special characteristics of the F-ATPase from T. brucei, and also, demonstrate that the architecture of the F1 -ATPase complex is not strictly conserved in eukaryotes.

• Klíčová slova
• Trypanosoma brucei, ATP synthase, F1-domain, p18-subunit, proteolysis of α-subunit, subunit composition,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• hydrolýza MeSH
• kinetika MeSH
• konformace proteinů MeSH
• konzervovaná sekvence MeSH
• membránový potenciál mitochondrií MeSH
• molekulární modely * MeSH
• multimerizace proteinu MeSH
• peptidové mapování MeSH
• podjednotky proteinů antagonisté a inhibitory   genetika   izolace a purifikace   metabolismus   MeSH
• proteolýza MeSH
• protonové ATPasy antagonisté a inhibitory   genetika   izolace a purifikace   metabolismus   MeSH
• protozoální proteiny antagonisté a inhibitory   genetika   izolace a purifikace   metabolismus   MeSH
• RNA interference MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• sekvenční seřazení MeSH
• stabilita enzymů MeSH
• Trypanosoma brucei brucei enzymologie   růst a vývoj   MeSH
• výpočetní biologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• podjednotky proteinů MeSH
• protonové ATPasy MeSH
• protozoální proteiny MeSH

A considerable amount of evidence supports the idea that lipid rafts are involved in many cellular processes, including protein sorting and trafficking. We show that, in this process, also a non-raft lipid, phosphatidylethanolamine (PE), has an indispensable function. The depletion of this phospholipid results in an accumulation of a typical raft-resident, the arginine transporter Can1p, in the membranes of Golgi, while the trafficking of another plasma membrane transporter, Pma1p, is interrupted at the level of the ER. Both these transporters associate with a Triton (TX-100) resistant membrane fraction before their intracellular transport is arrested in the respective organelles. The Can1p undelivered to the plasma membrane is fully active when reconstituted to a PE-containing vesicle system in vitro. We further demonstrate that, in addition to the TX-100 resistance at 4 degrees C, Can1p and Pma1pa exhibit different accessibility to nonyl glucoside (NG), which points to distinct intimate lipid surroundings of these two proteins. Also, at 20 degrees C, these two proteins are extracted by TX-100 differentially. The features above suggest that Pma1p and Can1p are associated with different compartments. This is independently supported by the observations made by confocal microscopy. In addition we show that PE is involved in the stability of Can1p-raft association.

• MeSH
• detergenty MeSH
• fosfatidylethanolaminy chemie   metabolismus   MeSH
• membránové mikrodomény chemie   metabolismus   MeSH
• membránové proteiny chemie   metabolismus   MeSH
• protonové ATPasy izolace a purifikace   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny izolace a purifikace   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sbalování proteinů MeSH
• transportní systémy pro bazické aminokyseliny izolace a purifikace   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CAN1 protein, S cerevisiae MeSH Prohlížeč
• detergenty MeSH
• fosfatidylethanolaminy MeSH
• membránové proteiny MeSH
• phosphatidylethanolamine MeSH Prohlížeč
• PMA1 protein, S cerevisiae MeSH Prohlížeč
• protonové ATPasy MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• transportní systémy pro bazické aminokyseliny MeSH

In an attempt to more closely define a protein basis of differences in ATPase and ATP synthase activities in a mutant of the methanoarchaeon Methanothermobacter thermautotrophicus resistant to the protonophoric uncoupler TCS (3,3',4',5-tetrachlorosalicylanilide), the composition of membrane associated proteins from the wild-type and mutant strains has been compared. The uncoupler-resistance in the mutant strain was not accompanied by changes in a protein size or changes in the level of subunits A, B and c (proteolipid) of the A1A0-type ATPase-synthase. On the other hand, we revealed a 670-kDa membrane-associated protein complex that is abundantly present only in the mutant strain; it is composed of at least 5 different subunits of 95, 52, 42, 29 and 22 kDa.

• MeSH
• antibiotická rezistence genetika   MeSH
• archeální proteiny analýza   izolace a purifikace   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• inhibitory enzymů farmakologie   MeSH
• membránové proteiny analýza   izolace a purifikace   MeSH
• Methanobacteriaceae účinky léků   enzymologie   genetika   MeSH
• molekulová hmotnost MeSH
• mutace * MeSH
• podjednotky proteinů analýza   izolace a purifikace   MeSH
• protonové ATPasy analýza   izolace a purifikace   MeSH
• salicylanilidy farmakologie   MeSH
• western blotting MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3,3',4',5-tetrachlorosalicylanilide MeSH Prohlížeč
• archeální proteiny MeSH
• inhibitory enzymů MeSH
• membránové proteiny MeSH
• podjednotky proteinů MeSH
• protonové ATPasy MeSH
• salicylanilidy MeSH

Plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae was isolated and purified in its two forms, the activated A-ATPase from glucose-metabolizing cells, and the basal-level B-ATPase from cells with endogenous metabolism only. Using two-dimensional gel electrophoretic analysis, we showed that both enzyme preparations are actually mixtures of the non-active, i.e. non-phosphorylated, and the active, i.e. phosphorylated, forms of the enzyme. Previous deliberations suggesting that the B-ATPase displays some activity which is lower than that of A-ATPase were apparently wrong. It seems that, molecularly speaking, the B-form is actually not active at all, and what activity we measure in our preparation is due to an admixture of the true active form (A-form). Fourier transform infrared spectroscopic study of the secondary structure and particularly thermal denaturation data suggest the possibility that the two enzyme forms interact to form complexes less stable than the single forms. On the whole then, there apparently is a different ratio of the active and inactive forms and/or complexes between the two forms present in all enzyme preparations.

• MeSH
• 2D gelová elektroforéza MeSH
• aktivace enzymů MeSH
• buněčná membrána enzymologie   MeSH
• fosforylace MeSH
• kvasinky enzymologie   MeSH
• protonové ATPasy chemie   izolace a purifikace   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protonové ATPasy MeSH

Classical isolation procedure for plasma membrane H(+)-ATPase of Saccharomyces cerevisiae based on fractional centrifugation yielded always a roughly two-fold greater amount of membranes when starting from glucitol-preincubated than from glucose-preincubated yeast. This difference persisted all the way to the purified plasma membranes and to the purified H(+)-ATPase. The ATP-hydrolyzing activity by plasma membranes was roughly twice greater in glucose-preincubated cells than in the D-glucitol-preincubated ones while the purified enzyme was 7 times more active after glucose than after glucitol. Effects of diethylstilbestrol, suloctidil, erythrosin B, vanadate and dicarbanonaboranuide were very similar on plasma membrane-localized and purified ATPases of both forms, suggesting that both preparations contain the two ATPase forms, the glucose-preincubated one being richer in the activated form while the glucitol-preincubated one contains less of it.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• buněčná membrána enzymologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• kultivační média MeSH
• protonové ATPasy antagonisté a inhibitory   izolace a purifikace   metabolismus   MeSH
• Saccharomyces cerevisiae enzymologie   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• inhibitory enzymů MeSH
• kultivační média MeSH
• protonové ATPasy MeSH

In order to characterize the biogenesis of unique thermogenic mitochondria of brown adipose tissue, differentiation of precursor cells isolated from mouse brown adipose tissue was studied in cell culture. Synthesis of mitochondrial uncoupling protein (UCP), F1-ATPase, and cytochrome oxidase was examined by L-[35S]methionine labeling and immunoblotting. For the first time, synthesis of physiological amounts of the UCP, a key and tissue-specific component of thermogenic mitochondria, was observed in cultures at about confluence (day 6), indicating that a complete differentiation of brown adipocytes was achieved in vitro. In postconfluent cells (day 8) the content of UCP decreased rapidly, in contrast to some other mitochondrial proteins (beta subunit of F1-ATPase, cytochrome oxidase). In these cells, it was possible, by using norepinephrine, to induce specifically the synthesis of the UCP but not of F1-ATPase or cytochrome oxidase. The maximal response was observed at 0.1 microM norepinephrine and the synthesis of UCP remained activated for at least 24 h. Detailed analysis revealed a major role of the beta-adrenergic receptors and elevated intracellular concentration of cAMP in stimulation of UCP synthesis. A quantitative recovery of the newly synthesized UCP in the mitochondrial fraction indicated completed biogenesis of functionally competent thermogenic mitochondria.

• MeSH
• 2D gelová elektroforéza MeSH
• buněčná diferenciace MeSH
• hnědá tuková tkáň cytologie   metabolismus   MeSH
• iontové kanály MeSH
• kinetika MeSH
• kultivované buňky MeSH
• membránové proteiny biosyntéza   izolace a purifikace   MeSH
• methionin metabolismus   MeSH
• mitochondriální proteiny MeSH
• mitochondrie metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• protonové ATPasy biosyntéza   izolace a purifikace   MeSH
• radioizotopy síry MeSH
• respirační komplex IV biosyntéza   izolace a purifikace   MeSH
• transportní proteiny * MeSH
• uncoupling protein 1 MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• iontové kanály MeSH
• membránové proteiny MeSH
• methionin MeSH
• mitochondriální proteiny MeSH
• protonové ATPasy MeSH
• radioizotopy síry MeSH
• respirační komplex IV MeSH
• transportní proteiny * MeSH
• uncoupling protein 1 MeSH

Embryonic development of mouse and rat brown adipose tissue was characterized by electron microscopy and by quantifying the mitochondrial oxidative, phosphorylating and thermogenic capacities immunochemically, using antibodies against cytochrome oxidase, F1-ATPase and uncoupling protein, respectively. Mitochondria and cytochrome oxidase were detected from the 15-16th day of pregnancy and their amounts continuously increased toward birth. F1-ATPase was also found on the 15th day but it reached a maximum level already on the 19th day when the uncoupling protein appeared and rapidly increased during further maturation of brown adipose tissue. It thus appears that mitochondria in early prenatal brown adipose tissue lack completely uncoupling protein and are nonthermogenic. They transform into typical thermogenic mitochondria abruptly only 2 days before birth.

• MeSH
• buněčná diferenciace MeSH
• embryo savčí MeSH
• embryonální a fetální vývoj * MeSH
• hnědá tuková tkáň enzymologie   fyziologie   ultrastruktura   MeSH
• inbrední kmeny potkanů MeSH
• iontové kanály MeSH
• krysa rodu Rattus MeSH
• membránové proteiny izolace a purifikace   fyziologie   MeSH
• mitochondriální proteiny MeSH
• mitochondrie enzymologie   fyziologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• protonové ATPasy izolace a purifikace   MeSH
• respirační komplex IV izolace a purifikace   MeSH
• termoregulace * MeSH
• transportní proteiny * MeSH
• uncoupling protein 1 MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• iontové kanály MeSH
• membránové proteiny MeSH
• mitochondriální proteiny MeSH
• protonové ATPasy MeSH
• respirační komplex IV MeSH
• transportní proteiny * MeSH
• uncoupling protein 1 MeSH

Using isolated polypeptides of the F0 sector of bovine heart mitochondrial H+-ATPase, antisera were developed detecting specifically two components of F0. These two components were identified as F0I and oligomycin-sensitivity-conferring protein (OSCP) respectively. Both F0I and OSCP were digested by mild trypsin treatment of submitochondrial particles depleted of the catalytic part of H+-ATPase (USMP). Proteolysis was largely prevented by binding of F1 to F0. Proteolysis of F0I resulted in the formation of three immunoreactive, membrane-bound fragments of apparently 26 kDa, 25.5 kDa and 18 kDa, respectively, indicating that F0I contains trypsin-accessible Arg or Lys residues located close to the end and the middle part of the protein, respectively, which are in intimate contact with F1. Digestion of USMP with trypsin resulted in depression of passive H+ conduction through F0 which could be ascribed to proteolysis of F0I.

• MeSH
• antigeny analýza   MeSH
• antisérum MeSH
• biologický transport účinky léků   MeSH
• buněčná membrána enzymologie   metabolismus   MeSH
• králíci MeSH
• molekulová hmotnost MeSH
• protonové ATPasy imunologie   izolace a purifikace   metabolismus   MeSH
• skot MeSH
• srdeční mitochondrie enzymologie   MeSH
• submitochondriální částice enzymologie   imunologie   MeSH
• trypsin farmakologie   MeSH
• vazebná místa účinky léků   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny MeSH
• antisérum MeSH
• protonové ATPasy MeSH
• trypsin MeSH