PMID- 9204870
OWN - NLM
STAT- MEDLINE
DCOM- 19970731
LR  - 20161124
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 36
IP  - 27
DP  - 1997 Jul 8
TI  - Mutagenesis of the uncoupling protein of brown adipose tissue. Neutralization Of 
      E190 largely abolishes pH control of nucleotide binding.
PG  - 8253-60
AB  - For expression in Saccharomyces cerevisiae the cDNA of the uncoupling protein 
      (UCP) of brown adipose tissue from hamster has been isolated and used to 
      transform yeast cells. Optimized expression conditions yielded 2% of 
      mitochondrial protein as UCP. UCP was isolated, avoiding copurification of 
      ADP/ATP carrier and porin. Intrahelical E190, previously suggested to be the pH 
      sensor for nucleotide binding, was neutralized to glutamine by mutagenesis. In 
      binding titrations with [14C]guanosine 5'-triphosphate (GTP) and with fluorescent 
      dansyl-GTP, near equal binding capacity for GTP was measured in wild-type (wt) 
      and E190Q. The KD for GTP binding to UCP from yeast has the same strong pH 
      dependence as the original UCP from hamster. With both [14C]GTP and dansyl-GTP, 
      the KD in wt increased 16-19-fold from pH 6.0 to 7.5, while in E190Q this 
      increase was only 2.5-2.9-fold. As a result, at pH 7.5, both [14C]GTP and 
      dansyl-GTP bind 6-fold tighter to E190Q than to wt. The binding rate of GTP 
      decreased 10-fold from pH 6.0 to 7.5 in wt and only 4-fold in E190Q. Woodward 
      reagent K (WRK) known to interact specifically with E190 [Winkler, E., Wachter, 
      E., and Klingenberg, M. (1997) Biochemistry 36, 148-155] abolished [14C]GTP and 
      dansyl-GTP binding to wt UCP, whereas binding to E190Q was fully resistant to 
      WRK. H+ and Cl- transport activity in reconstituted vesicles were the same with 
      wt and E190Q. At pH 7.5, 5 microM GTP is unable to inhibit H+ and Cl- transport 
      in wt but inhibits in E190Q to maximum level. The different sensitivity toward 
      GTP versus GDP found in wt is absent in E190Q. Thus, the mutation E190Q results 
      in the predicted gain of function in binding and proves the role of the 
      intrahelical E190 as a pH sensor for nucleotide binding but excludes a role in 
      transport.
FAU - Echtay, K S
AU  - Echtay KS
AD  - Institute of Physical Biochemistry, University of Munich, Schillerstrasse 44, 
      80336 Munich, Germany.
FAU - Bienengraeber, M
AU  - Bienengraeber M
FAU - Klingenberg, M
AU  - Klingenberg M
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Carrier Proteins)
RN  - 0 (Dansyl Compounds)
RN  - 0 (Ion Channels)
RN  - 0 (Membrane Proteins)
RN  - 0 (Mitochondrial Proteins)
RN  - 0 (Nucleotides)
RN  - 0 (Uncoupling Protein 1)
RN  - 146-91-8 (Guanosine Diphosphate)
RN  - 86-01-1 (Guanosine Triphosphate)
RN  - X2RN3Q8DNE (Galactose)
SB  - IM
MH  - Adipose Tissue, Brown/*chemistry
MH  - Animals
MH  - Binding Sites
MH  - Carrier Proteins/*chemistry/*genetics/metabolism
MH  - Cricetinae
MH  - Dansyl Compounds/metabolism
MH  - *Exons
MH  - Galactose/pharmacology
MH  - Gene Transfer Techniques
MH  - Guanosine Diphosphate/metabolism
MH  - Guanosine Triphosphate/metabolism
MH  - Hydrogen-Ion Concentration
MH  - Immunoblotting
MH  - Ion Channels
MH  - Kinetics
MH  - Membrane Proteins/*chemistry/*genetics/metabolism
MH  - Mitochondria/chemistry
MH  - Mitochondrial Proteins
MH  - *Mutagenesis, Site-Directed
MH  - Nucleotides/*metabolism
MH  - Saccharomyces cerevisiae/ultrastructure
MH  - Structure-Activity Relationship
MH  - Uncoupling Protein 1
EDAT- 1997/07/08 00:00
MHDA- 1997/07/08 00:01
CRDT- 1997/07/08 00:00
PHST- 1997/07/08 00:00 [pubmed]
PHST- 1997/07/08 00:01 [medline]
PHST- 1997/07/08 00:00 [entrez]
AID - bi970513r [pii]
AID - 10.1021/bi970513r [doi]
PST - ppublish
SO  - Biochemistry. 1997 Jul 8;36(27):8253-60. doi: 10.1021/bi970513r.