Reconstitution of purified brown adipose tissue mitochondria uncoupling protein: demonstration of separate identity of nucleotide binding and proton translocation sites by chemical probes
Katiyar, S.S.; Shrago, E.
Proceedings of the National Academy of Sciences of the United States of America 86(8): 2559-2562
ISSN/ISBN: 0027-8424 PMID: 2539594 DOI: 10.2307/34088
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.