Reconstitution of recombinant uncoupling proteins: UCP1, -2, and -3 have similar affinities for ATP and are unaffected by coenzyme Q10.
The successful development of recombinant expression and reconstitution protocols has enabled a detailed study of the transport properties and regulation of the uncoupling proteins (UCP). We optimized conditions of isolation and refolding of bacterially expressed uncoupling proteins and reexamined the transport properties and regulation of bacterially expressed UCP1, -2, and -3 reconstituted in liposomes. We show for the first time that ATP inhibits UCP1, -2, and -3 with similar affinities. The Ki values for ATP inhibition were 50 microm (UCP1), 70 microm (UCP2), and 120 microm (UCP3) at pH 7.2. These affinities for ATP are similar to those obtained with native UCP1 isolated from brown adipose tissue mitochondria (Ki = 65 microm at pH 7.2). The Vmax values for proton transport were also similar among the UCPs, ranging from 8 to 20 micromol.min(-1).mg(-1), depending on experimental conditions. We also examined the effect of coenzyme Q on fatty acid-catalyzed proton flux in liposomes containing recombinant UCP1, -2, and -3. We found that coenzyme Q had no effect on the fatty acid-dependent proton transport catalyzed by any of the UCPs nor did it affect nucleotide regulation of the UCPs. We conclude that coenzyme Q is not a cofactor of UCP-mediated proton transport.
Department of Biology, Portland State University, Portland, Oregon 97207, USA.
Martin
M
Jaburek
Keith D
KD
Garlid
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000273:Adipose Tissue / Q000378:metabolism; D002001:Adipose Tissue, Brown / Q000378:metabolism; D000818:Animals; D001672:Biocompatible Materials / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry*; D002384:Catalysis; D003067:Coenzymes; D018076:DNA, Complementary / Q000378:metabolism; D017886:Durapatite / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels; D007477:Ions; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins; D008752:Methylene Chloride / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism*; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2; D000071247:Uncoupling Protein 3
D001672:Biocompatible Materials; D002352:Carrier Proteins; D003067:Coenzymes; D018076:DNA, Complementary; D005227:Fatty Acids; D007473:Ion Channels; D007477:Ions; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D011522:Protons; D011994:Recombinant Proteins; C000604371:UCP1 protein, human; C000604389:UCP2 protein, human; C000604392:UCP3 protein, human; C000604373:Ucp1 protein, rat; C000604391:Ucp2 protein, rat; C000604394:Ucp3 protein, rat; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2; D000071247:Uncoupling Protein 3; D014451:Ubiquinone; D008752:Methylene Chloride; D000255:Adenosine Triphosphate; D017886:Durapatite; C024989:coenzyme Q10