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[58] Reconstitution of an H+ translocator, the “uncoupling protein” from brown adipose tissue mitochondria, in phospholipid vesicles

1986; Academic Press; Linguagem: Inglês

10.1016/0076-6879(86)27061-5

1557-7988

Martin Klingenberg, Edith Winkler,

Metabolomics and Mass Spectrometry Studies

This chapter describes the reconstitution methods of an H+ translocator, the uncoupling protein (UCP) from brown adipose tissue mitochondria in phospholipid vesicles, with special emphasis on its applications. The uncoupling protein (UCP) is isolated and purified from brown fat mitochondria where it can bind nucleotides. Only recently the purified protein has been reconstituted into phospholipid vesicles in such a manner that its catalytic activity as H+ conductor and the inhibition of this H+ conductance by GTP could be demonstrated. H+ conductivity is high and fully dependent on the membrane potential. For the UCP, it is important to determine the pH separately inside and outside, although with the inside pH, this is difficult because of the high buffer capacity of the phospholipids. The uncoupling protein is purified by using Triton X-100 as solubilizer. The methodology is suitable for most purposes and generates highly active proteoliposomes containing UCP at about 70% purity. It applies to mitochondria from brown fat (adipose tissue) of cold-adapted hamster, but can be extended to mitochondria from other brown fat sources.