The reconstituted isolated uncoupling protein is a membrane potential driven H+ translocator.
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)
Klingenberg
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Winkler
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D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D003080:Cold Temperature; D006224:Cricetinae; D004553:Electric Conductivity; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014634:Valinomycin / Q000494:pharmacology
D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; D014634:Valinomycin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D006160:Guanosine Triphosphate; D006180:Proton-Translocating ATPases