Isolated uncoupling protein (UCP) can be cross-linked, by various disulfide-forming reagents, to dimers. The best cross-linking is achieved with Cu2 +-phenanthroline oxidation. Because cross-linking is independent of UCP concentration and prevented by SDS addition, a disulfide bridge must be formed between the two subunits of the native dimer. Cross-linking is prevented by SH reagent and reversed by SH-reducing reagents. In mitochondria, cross-linking of UCP with disulfide-forming agents is even more efficient than in isolated state. It proves that UCP is a dimer in mitochondria, before isolation. Disulfide-bridge formation does not inhibit GTP-binding to UCP. Cross-linked UCP re-incorporated in proteoliposomes either before or after cross-linking fully retains the H +-transport function.Rapid cross-linking by membrane impermeant reagents indicates a surface localization of the C-terminus in soluble UCP and projection to the outer surface in mitochondria. Intermolecular disulfide-bridge formation in a dimer requires juxtaposition of identical cysteines at the twofold symmetry axis. A rigid juxtaposition of cysteines is unlikely, unless intended for a native disulfide bridge. The absence of such a bridge in UCP suggests that juxtaposition of cysteines is generated by high mobility. In order to localize the cysteine involved, cross-linked UCP was cleaved by BrCN. The CB-7 C-terminal peptide, which contains cysteines at positions 287 and 304, disappears. Limited trypsinolytic cleavage, previously shown to occur at Lys-292, removed cross-linking in UCP both in the solubilized and mitochondrially bound state. The cleaved C-terminal peptide of 11 residues contains only cystein-304 which, thus, should be the only one (out of 7 cysteines in UCP) involved in the S -S bridge formation. Obviously, the C-terminal location of the cysteine, because of its high mobility, permits juxtapositioning for cross-linking. This agrees with predictions from hydrophobicity analysis that the last 14 residues in UCP protrude from the membrane.The uncoupling protein (UCP) from brown adipose tissue is considered to be thc key element in the thermogenesis of this highly specialized brown adipose cell. The main function of UCP is to transfer protons through the membrane in a voltage-dependent transport in order to short-circuit the electrogenic protons produced by the respiratory chain [I]. UCP is a relatively small protein of about 33 kDa which is assumed to be a deeply embedded intrinsic membrane protein.In the isolated functionally intact form, UCP forms a dimer within the detergent micelle, as evaluated from hydrodynamic studies [2]. A dimer also has to be postulated on the basis of the 'half-site reactivity' of nucleotide binding to UCP, i. e. the binding capacity for only one nucleotide molecule/two UCP peptide molecules [3].The primary sequence of UCP from hamster was first determined by amino acid analysis [4] and then, for UCP from rat, by cDNA analysis [5, 61. Secondary structure predictions have been made on the basis of hydrophobi...
The binding of bongkrekate to mitochondrial membrane was investigated using [3H]bongkrekate. These measurements were designed to examine the previously derived reorienting site mechanism which implies that bongkrekate binds to the single carrier site only from the inner face of the mitochondrial membrane. The binding studies confirm pH‐dependent accumulation of [3H]bongkrekate inside the mitochondria which superimposes on to binding of carrier sites. By breaking the membrane with Lubrol or sonication, binding to the carrier sites can be titrated and Kd∼ 5 × 10−8 M is determined. ADP or ATP increases the amount of binding but does not change the Kd. Reciprocally bongkrekate increases ADP binding in those sections of a tritation curve where bonkrekate binding is increased by ADP. [35S]Atractylate is displaced by [3H]bongkrekate at a 1:1 molar ratio. This displacement is dependent on ADP concentration with the Km= 0.5 × 10−6 M.
The earlier described isomer, isobongkrekate, also binds specifically to the carrier sites. From competition with bogkrekate a ratio KisoBKAd/KBKAd= 0.10 is determined. [35S]Carboxyatractylate displaces most of [3H]isobongkrekate but only little of [3H]bongkrekate. The rate of displacement is more than 10‐times faster for isobongkrekate than for bongkrekate. The displacement is dependent on ADP with a Km= 5 × 10−6 M.
All these resuits are fully consistent with the single site reorienting mechanism. In no instant do bongkrekate and atractylate as well as ADP or ATP bind simultaneously to the carrier.
Pentacarbonyl(tetrafluoroborato)rhenium setzt sich mit Trimethyl(1‐propinyl)stannan zu (OC)5Re–CC–CH3 (1), mit Ethinyltrimethylsilan zum σ,π‐Ethinid‐verbrückten Komplex [(OC)5Re(μ‐η1,η2‐CCH)Re(CO)5]+ BF–4 (2) um. Aus 2 und Natriumethanolat entsteht unter HBF4‐Abspaltung der σ,σ‐Ethindiid‐verbrückte Komplex (OC)5Re–CC–Re(CO)5 (3). 3 läßt sich mit HBF4·Et2O wieder zu 2 protonieren.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.