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

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