Effect of pH and MgCl2 on the binding of purine nucleotides to the uncoupling protein in membrane particles from brown fat mitochondria.
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.
Rafael
J
J
Institut für Biochemie I, Universität Heidelberg, Germany.
Pampel
I
I
Wang
X
X
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
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