Control of uncoupling protein in brown-fat mitochondria by purine nucleotides. Chemical modification by diazobenzenesulfonate.
The uncoupling protein (UP) of isolated brown adipose tissue mitochondria was studied with respect to the mechanism of control of UP function by purine nucleotides. Passive transport of H+ and Cl- was followed simultaneously in a KCl medium. With both GDP and ATP a higher sensitivity of Cl- transport (apparent Ki = 2.2 microM and 4.7 microM respectively) than of H+ transport (apparent Ki = 7.7 microM and 34 microM respectively) was observed. Chemical modification of isolated mitochondria by diazobenzenesulfonate (DABS) up to 75 mumol/mg protein did not affect the transport, its ionic selectivity and regulation by endogenous free fatty acids. In contrast, the sensitivity to purine nucleotides of both H+ and Cl- translocation was decreased (apparent Ki increased 71 and 47 times respectively). DABS decreased the affinity of [3H]GDP for the specific nucleotide-binding site on mitochondria (Kd increased from 2.7 microM to 13 microM) and depressed, to a smaller extent, the GDP-binding capacity. Correlation between occupancy of the specific nucleotide-binding site by GDP and inhibition of transport yielded a linear relationship for Cl- transport in control mitochondria. For H+ transport in the control, and for both H+ and Cl- transports in DABS-treated mitochondria, a biphasic correlation was obtained. The results show that different structural parts of UP are involved in transport and its control by the regulatory ligands and that, in addition to binding of purine nucleotides to UP, the inhibition of ion transport by purine nucleotides depends on an intrinsic factor modulating the inhibitory effect.
D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins; D002712:Chlorides / Q000378:metabolism; D006224:Cricetinae; D003979:Diazonium Compounds / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism; D007473:Ion Channels; D008565:Membrane Proteins / Q000378:metabolism*; D008647:Mesocricetus; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D011522:Protons; D011685:Purine Nucleotides / Q000494:pharmacology*; D013425:Sulfanilic Acids / Q000494:pharmacology; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D002712:Chlorides; D003979:Diazonium Compounds; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011522:Protons; D011685:Purine Nucleotides; D013425:Sulfanilic Acids; D000071256:Uncoupling Protein 1; D006153:Guanosine Diphosphate; C000469:diazobenzenesulfonic acid