Reconstitution of purified brown adipose tissue mitochondria uncoupling protein: demonstration of separate identity of nucleotide binding and proton translocation sites by chemical probes.
The reaction of two arginine-modifying reagents, phenylglyoxal and 2,3-butanedione, with a highly purified uncoupling protein of brown adipose tissue mitochondria decreased the GDP binding to the uncoupling protein. This inhibition was irreversible and dependent on time and concentration of the reagent. Complete inhibition of GDP binding by both reagents establishes that arginine is one of the critical amino acid residues involved in the binding of GDP. Reconstitution of the uncoupling protein (both unmodified and modified) into phospholipid vesicles by our procedure showed no effect of phenylglyoxal modification on the H+ conductance, thus demonstrating that the proton translocation site is structurally different and distinct from the GDP binding site.
Katiyar
S S
SS
Department of Medicine, University of Wisconsin, Madison 53706.
Shrago
E
E
D002001:Adipose Tissue, Brown / Q000502:physiology*; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D002352:Carrier Proteins; D002474:Cell-Free System; D006224:Cricetinae; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D007473:Ion Channels; D008081:Liposomes; D008297:Male; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D008928:Mitochondria / Q000502:physiology*; D024101:Mitochondrial Proteins; D011522:Protons; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D006150:Guanine Nucleotides; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011522:Protons; D000071256:Uncoupling Protein 1; D006153:Guanosine Diphosphate; D001120:Arginine