ATPase
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Acta physiologica Scandinavica ; Vol. 154 Supplement 624
146 s. : tab., grafy ; 24 cm
- MeSH
- kardiomyocyty enzymologie fyziologie MeSH
- protein - isoformy klasifikace MeSH
- sodíko-draslíková ATPasa fyziologie MeSH
- Publikační typ
- přehledy MeSH
F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme's protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F1-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F1-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F1-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F1-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F1-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F1-ATPase isolation from other organisms.
- MeSH
- protonové ATPasy chemie MeSH
- Trypanosoma brucei brucei metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- audiovizuální média MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
Acta pharmacologica et toxicologica, ISSN 0065-1508 vol. 52, suppl. I, 1983
19 s. : grafy ; 24 cm
Dysfunctions of the F(1)F(o)-ATPase complex cause severe mitochondrial diseases affecting primarily the paediatric population. While in the maternally inherited ATPase defects due to mtDNA mutations in the ATP6 gene the enzyme is structurally and functionally modified, in ATPase defects of nuclear origin mitochondria contain a decreased amount of otherwise normal enzyme. In this case biosynthesis of ATPase is down-regulated due to a block at the early stage of enzyme assembly-formation of the F(1) catalytic part. The pathogenetic mechanism implicates dysfunction of Atp12 or other F(1)-specific assembly factors. For cellular energetics, however, the negative consequences may be quite similar irrespective of whether the ATPase dysfunction is of mitochondrial or nuclear origin.
- MeSH
- adenosintrifosfatasy genetika MeSH
- buněčné jádro enzymologie metabolismus MeSH
- fibroblasty metabolismus MeSH
- lidé MeSH
- mitochondriální DNA genetika metabolismus MeSH
- mitochondriální nemoci enzymologie genetika MeSH
- mitochondriální protonové ATPasy biosyntéza genetika nedostatek MeSH
- mitochondrie enzymologie MeSH
- mutace MeSH
- reaktivní formy kyslíku analýza metabolismus MeSH
- vakuolární protonové ATPasy * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
- srovnávací studie MeSH
ATP synthases are present in every life form being the key enzymes of cellular bioenergetics. The enzyme from the Archaea forms a new class of ATPases, A1A0 ATP synthase. This enzyme has unusual structural and functional features, which separate it from F1F0 and V1V0 ATPases as a distinct enzyme class ? A1A0 ATPase/ synthase. It contains the transmembrane A0 domain and the cytoplasmatic A1 domain, including a specific site for ATP synthesis. The A1 domain is linked to the A0 part by D-subunit, a structural and functional analog of the ?- subunit of F1F0 ATPase. The genomic approach to the study of this enzyme combined with methods of molecular biology, biochemistry and structural biology, will extend the study of A1A0 ATPase/synthase and ATP synthesis to the molecular level.
The yeast Saccharomyces cerevisiae accumulates the high levels of inorganic polyphosphates (polyPs) performing in the cells numerous functions, including phosphate and energy storage. The effects of vacuolar membrane ATPase (V-ATPase) dysfunction were studied on polyP accumulation under short-term cultivation in the Pi-excess media after Pi starvation. The addition of bafilomycin A1, a specific inhibitor of V-ATPase, to the medium with glucose resulted in strong inhibition of the synthesis of long-chain polyP and in substantial suppression of short-chain polyP. The addition of bafilomycin to the medium with ethanol resulted in decreased accumulation of high-molecular polyP, while the accumulation of low-molecular polyP was not affected. The levels of polyP synthesis in the mutant strain with a deletion in the vma2 gene encoding a V-ATPase subunit were significantly lower than in the parent strain in the media with glucose and with ethanol. The synthesis of the longest chain polyP was not observed in the mutant cells. The synthesis of only the low-polymer acid-soluble polyP fraction occurred in the cells of the mutant strain. However, the level of polyP1 was nearly tenfold lower than compared to the cells of the parent strain. Both bafilomycin A1 and the mutation in vacuolar ATPase subunit vma2 lead to a considerable decrease of cellular polyP accumulation. Thus, the defects in ΔμH(+) formation on the vacuolar membrane resulted in the decrease of polyP biosynthesis in S. cerevisiae.
- MeSH
- delece genu MeSH
- ethanol metabolismus MeSH
- inhibitory enzymů metabolismus MeSH
- kultivační média chemie MeSH
- makrolidy metabolismus MeSH
- podjednotky proteinů genetika metabolismus MeSH
- polyfosfáty metabolismus MeSH
- Saccharomyces cerevisiae enzymologie metabolismus MeSH
- vakuolární protonové ATPasy genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Na(+)/K(+)-ATPase (NKA) is an essential cation pump protein responsible for the maintenance of the sodium and potassium gradients across the plasma membrane. Recently published high-resolution structures revealed amino acids forming the cation binding sites (CBS) in the transmembrane domain and variable position of the domains in the cytoplasmic headpiece. Here we report molecular dynamic simulations of the human NKA α1β1 isoform embedded into DOPC bilayer. We have analyzed the NKA conformational changes in the presence of Na(+)- or K(+)-cations in the CBS, for various combinations of the cytoplasmic ligands, and the two major enzyme conformations in the 100 ns runs (more than 2.5 μs of simulations in total). We identified two novel cytoplasmic pathways along the pairs of transmembrane helices TM3/TM7 or TM6/TM9 that allow hydration of the CBS or transport of cations from/to the bulk. These findings can provide a structural explanation for previous mutagenesis studies, where mutation of residues that are distal from the CBS resulted in the alteration of the enzyme affinity to the transported cations or change in the enzyme activity.
- MeSH
- draslík metabolismus MeSH
- fosfatidylcholiny metabolismus MeSH
- kationty metabolismus MeSH
- konformace proteinů MeSH
- lidé MeSH
- lipidové dvojvrstvy metabolismus MeSH
- simulace molekulární dynamiky MeSH
- sodík metabolismus MeSH
- sodíko-draslíková ATPasa chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
