The reconstituted isolated uncoupling protein is a membrane potential driven H+ translocator.

Abstract

The isolated uncoupling protein (UCP) from brown fat adipose tissue mitochondria has been reconstituted into artificial phospholipid vesicles. Because of the high lability of H+ transport, several new steps have been introduced in the reconstitution; the detergent octyl‐POE, the addition of phospholipids to mitochondria prior to solubilization and purification, the vesicle formation by rapid removal of detergent with polystyrene beads and of external salts by a mixed ion exchange. In the K+‐loaded proteoliposomes, H+ influx can be induced by a diffusion potential on addition of valinomycin. H+ influx is inhibited to more than 90% by GTP addition, in the assay for UCP activity. By reversing delta psi with external K+, H+ efflux is measured, however, at a four times lower rate. In vesicles loaded with internal GTP, H+ influx is fully inhibited but can be activated by Dowex‐OH treatment to an even higher rate than that found in the GTP‐free vesicles. Binding studies with GTP show that most of the active UCP are oriented with the binding site outside as in mitochondria, and that in GTP‐loaded vesicles GTP is also bound at the outside. The rate of H+ transport is linearly dependent on the membrane potential. Despite the ordered orientation, there is no ‘valve’ mechanism, since there is H+ efflux with a reversed potential. pH dependency is only small between pH 6.5 and 7.5, indicating that the H+‐translocating site differs from the highly pH‐dependent nucleotide‐binding site. The turnover number of reconstituted UCP is commensurate with mitochondrial function and indicates a carrier instead of a channel‐type H+ transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of uncoupling protein in brown‐fat mitochondria by purine nucleotides

“…While under our conditions of measurement (KCI medium), transport of both H ' and C1-was analyzed simultaneously, each of the transport activities in mitochondria was measured separately and under different conditions in a previous study [38]. As for the liposome system, there was an apparent loss of CI--translocating activity, in contrast to highly retained H + translocation [18]. It might be speculated, therefore, that the sensitivity of H ' transport is modulated by the simultaneous C1-transport via UP, due to the mechanism suggested above.…”

The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP

“…Before the publication of these data, a 32-kDa protein abundant in the membranes of BAT mitochondria was described : this protein was absent from liver mitochondria, induced during exposure of rats to the cold for several days, and down-regulated after readaptation to room temperature [36]. Following these studies, the 32-kDa protein was purified from hamster and rat [37,38], sequenced [39], cloned as cDNAs [40][41][42][43], and its protontranslocating activity reconstituted in liposomes [44][45][46][47]. It was therefore accepted that this protein was responsible for the regulated loose coupling of BAT mitochondria, and it was generally referred to as the uncoupling protein, UCP (UCP1).…”

Carnitine cycle in brown adipose tissue mitochondria as a tool for studying the regulatory role of fatty acids in the uncoupling protein function

“…Apart from other types of mitochondria they possess the uncoupling protein (UP), a unique proton translocator [2,3], converting A/~H + into heat. H + fluxes through the UP are inhibited by purine nucleotides [4][5][6][7][8] and activated by fatty acids [3,[8][9][10].…”

“…H + fluxes through the UP are inhibited by purine nucleotides [4][5][6][7][8] and activated by fatty acids [3,[8][9][10]. Both regulatory mechanisms were also demonstrated in a reconstituted system [3], but the activation by fatty acids was disputed in another laboratory [2]. The direct proof of the existence of the putative FA-binding site on UP is still lacking.…”