title: Effect of pH and MgCl2 on the binding of purine nucleotides to the uncoupling protein in membrane particles from brown fat mitochondria.
abstract: Binding of purine nucleotides to the uncoupling protein (UCP) was investigated in membrane particles prepared from brown fat mitochondria of cold-acclimated rats. Mitochondrial membranes were separated from soluble protein with Lubrol WX and treated with 3 M urea at basic pH. The resulting membrane vesicles were permeable to GDP and contained up to 3 nmol UCP/mg protein with unchanged nucleotide binding, as compared to the mitochondria (GDP/UCP ratio = 1.0; pKd GDP = 6.0 at pH 7.0). UCP bound nucleotides to one type of specific binding sites, located exclusively on the cytosolic side of the mitochondrial membrane. The binding affinity of guanine nucleotides was 3-18-times higher than that of the corresponding adenine nucleotides, when measured in membrane particles from cold-acclimated rats, hamsters, and guinea pigs. The pH-dependent binding affinities of GDP and ADP attained a maximum at pH 5.0-6.0 (pKd GDP = 6.8, pKd ADP = 5.8) and were decreased by a factor of 10(2) at pH 4.0 and pH 8.0, respectively, whereas the binding affinity of ATP was maximal at pH 4.0 (pKd = 7.0) and was decreased by a factor of 10(3) at pH 7.5. Participation of the protein binding center in nucleotide interaction with UCP in the membrane was highly pH-dependent. Mg2+ modified the number of binding sites engaged at a given nucleotide concentration by complex binding of nucleotides; the Kd for Mg.GTP2- and Mg.GDP- was 20-50-times lower than that of the free nucleotides.
authors.affiliation: Institut für Biochemie I, Universität Heidelberg, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Rafael
authors.forename: I
authors.initials: I
authors.lastname: Pampel
authors.forename: X
authors.initials: X
authors.lastname: Wang
mesh_terms: D000064:Acclimatization / Q000502:physiology; D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D003080:Cold Temperature; D006153:Guanosine Diphosphate / Q000378:metabolism; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D007473:Ion Channels; D008024:Ligands; D015636:Magnesium Chloride / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D024101:Mitochondrial Proteins; D008956:Models, Chemical; D011685:Purine Nucleotides / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014475:Uncoupling Agents / Q000378:metabolism*; D000071256:Uncoupling Protein 1
chemical_list: D002352:Carrier Proteins; D007473:Ion Channels; D008024:Ligands; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011685:Purine Nucleotides; D014475:Uncoupling Agents; D000071256:Uncoupling Protein 1; D015636:Magnesium Chloride; D006153:Guanosine Diphosphate