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M Klingenberg

Publications and source records attributed to M Klingenberg.

At least 19 recordsLinked to original sources
PubMed2001

Expression of the mitochondrial ADP/ATP carrier in Escherichia coli. Renaturation, reconstitution, and the effect of mutations on 10 positive residues.

Previously, the role of residues in the ADP/ATP carrier (AAC) from Saccharomyces cerevisiae has been studied by mutagenesis, but the dependence of mitochondrial biogenesis on functional AAC impedes segregation of the mutational effects on transport and biogenesis. Unlike other mitochondrial carriers, expression of the AAC from yeast or mammalians in Escherichia coli encountered difficulties because of disparate codon usage. Here we introduce the AAC from Neurospora crassa in E. coli, where it is accumulated in inclusion bodies and establish the reconstitution conditions. AAC expressed with heat shock vector gave higher activity than with pET-3a. Transport activity was absolutely dependent on cardiolipin. The 10 single mutations of intrahelical positive residues and of the matrix repeat (+X+) motif resulted in lower activity, except of R245A. R143A had decreased sensitivity toward carboxyatractylate. The ATP-linked exchange is generally more affected than ADP exchange. This reflects a charge network that propagates positive charge defects to ATP(4-) more strongly than to ADP(3-) transport. Comparison to the homologous mutants of yeast AAC2 permits attribution of the roles of these residues more to ADP/ATP transport or to AAC import into mitochondria.

Amino Acid Sequence↗
PubMed2000

Coenzyme Q is an obligatory cofactor for uncoupling protein function.

Uncoupling proteins (UCPs) are thought to be intricately controlled uncouplers that are responsible for the futile dissipation of mitochondrial chemiosmotic gradients, producing heat rather than ATP. They occur in many animal and plant cells and form a subfamily of the mitochondrial carrier family. Physiological uncoupling of oxidative phosphorylation must be strongly regulated to avoid deterioration of the energy supply and cell death, which is caused by toxic uncouplers. However, an H+ transporting uncoupling function is well established only for UCP1 from brown adipose tissue, and the regulation of UCP1 by fatty acids, nucleotides and pH remains controversial. The failure of UCP1 expressed in Escherichia coli inclusion bodies to carry out fatty-acid-dependent H+ transport activity inclusion bodies made us seek a native UCP cofactor. Here we report the identification of coenzyme Q (ubiquinone) as such a cofactor. On addition of CoQ10 to reconstituted UCP1 from inclusion bodies, fatty-acid-dependent H+ transport reached the same rate as with native UCP1. The H+ transport was highly sensitive to purine nucleotides, and activated only by oxidized but not reduced CoQ. H+ transport of native UCP1 correlated with the endogenous CoQ content.

Animals↗
PubMed2000

The bulk of UCP3 expressed in yeast cells is incompetent for a nucleotide regulated H+ transport.

The impact of uncoupling protein (UCP) 1, UCP3 and UCP3s expressed in yeast on oxidative phosphorylation, membrane potential and H+ transport is determined. Intracellular ATP synthesis is inhibited by UCP3, much more than by UCP1, while similar levels of UCP3 and UCP1 exist in the mitochondrial fractions. Measurements of membrane potential and H+ efflux in isolated mitochondria show that, different from UCP1, with UCP3 and UCP3s there is a priori a preponderant uncoupling not inhibited by GDP. The results are interpreted to show that UCP3 and UCP3s in yeast mitochondria are in a deranged state causing uncontrolled uncoupling, which does not represent their physiological function.

Animals↗
PubMed2000

Absence of cardiolipin in the crd1 null mutant results in decreased mitochondrial membrane potential and reduced mitochondrial function.

Cardiolipin (CL) is a unique phospholipid which is present throughout the eukaryotic kingdom and is localized in mitochondrial membranes. Saccharomyces cerevisiae cells containing a disruption of CRD1, the structural gene encoding CL synthase, have no CL in mitochondrial membranes. To elucidate the physiological role of CL, we compared mitochondrial functions in the crd1Delta mutant and isogenic wild type. The crd1Delta mutant loses viability at elevated temperature, and prolonged culture at 37 degrees C leads to loss of the mitochondrial genome. Mutant membranes have increased phosphatidylglycerol (PG) when grown in a nonfermentable carbon source but have almost no detectable PG in medium containing glucose. In glucose-grown cells, maximum respiratory rate, ATPase and cytochrome oxidase activities, and protein import are deficient in the mutant. The ADP/ATP carrier is defective even during growth in a nonfermentable carbon source. The mitochondrial membrane potential is decreased in mutant cells. The decrease is more pronounced in glucose-grown cells, which lack PG, but is also apparent in membranes containing PG (i.e. in nonfermentable carbon sources). We propose that CL is required for maintaining the mitochondrial membrane potential and that reduced membrane potential in the absence of CL leads to defects in protein import and other mitochondrial functions.

Cardiolipins↗
PubMed2000

Site-directed mutagenesis identifies residues in uncoupling protein (UCP1) involved in three different functions.

Using site-specific mutagenesis, we have constructed several mutants of uncoupling protein (UCP1) from brown adipose tissue to investigate the function of acidic side chains at positions 27, 167, 209, and 210 in H(+) and Cl(-) transport as well as in nucleotide binding. The H(+) transport activity was measured with mitochondria and with reconstituted vesicles. These mutant UCPs (D27N, D27E, E167Q, D209N, D210N, and D209N + D210N) are expressed at near wt levels in yeast. Their H(+) transport activity in mitochondria correlates well with the reconstituted protein except for D27N (intrahelical), which shows strong inhibition of H(+) transport in the reconstituted system and only 50% decrease of uncoupled respiration in mitochondria. In the double adjacent acidic residues (between helix 4 and helix 5), mutation of D210 and of D209 decreases H(+) transport 80% and only 20%, respectively. These mutants retain full Cl(-) transport activity. The results indicate that D210 participates in H(+) uptake at the cytosolic side and D27 in H(+) translocation through the membrane. Differently, E167Q has lost Cl(-) transport activity but retains the ability to transport H(+). The separate inactivation of H(+) and Cl(-) transport argues against the fatty acid anion transport mechanism of H(+) transport by UCP. The mutation of the double adjacent acidic residues (D209, D210) decreases pH dependency for only nucleoside triphosphate (NTP) but not diphosphate (NDP) binding. The results identify D209 and D210 in accordance with the previous model as those residues which control the location of H214 in the binding pocket, and thus contribute to the pH control of NTP but not of NDP binding.

Adipose Tissue, Brown↗
PubMed1999

Regulation of UCP3 by nucleotides is different from regulation of UCP1.

UCP3 is an isoform of UCP1, expressed primarily in skeletal muscle. Functional properties of UCP3 are still largely unknown. Here, we report about the expression of UCP3 and of UCP1 in inclusion bodies of Escherichia coli. On solubilization and reconstitution into proteoliposomes, both UCP3 and UCP1 transport Cl- at rates equal to the reconstituted native UCP1. Cl- transport is inhibited by low concentrations of ATP, ADP, GTP and GDP. However, no H+ transport activity is found possibly due to the lack of a cofactor presents in UCP from mitochondria. The specificity of inhibition by nucleoside tri- and diphosphate is different between UCP1 and UCP3. UCP1 is more sensitive to tri- than diphosphate whereas in UCP3, the gradient is reverse. These results show a new paradigm for the regulation of thermogenesis at various tissues by the ATP/ADP ratio. In brown adipose tissue, the thermogenesis is correlated with a low ATP/ADP whereas in skeletal muscle, non-shivering thermogenesis is active at a high ATP/ADP ratio, i.e. in the resting state.

Adipose Tissue↗
PubMed1999

Binding of nucleotides by the mitochondrial ADP/ATP carrier as studied by 1H nuclear magnetic resonance spectroscopy.

Nucleotide binding to the cytosolic binding site of the mitochondrial ADP/ATP carrier (AAC) was studied by 1H-nuclear magnetic resonance spectroscopy. Binding (as opposed to translocation) could be identified as a result of the rapid ligand on/off kinetics, using the cytosolic side specific inhibitor carboxyatractyloside (CAT) for the distinction from nonspecific interactions. The off rate constant of the nonhydrolyzable ATP analogue AMP-PCP was more than 3 orders of magnitude larger than the transport rate. The nucleotides adopt an anti conformation in the carrier binding site as shown by measurements of the transferred nuclear overhauser effect (TRNOE). A thermal transition around 14 degreesC that had been previously detected in transport studies [Klingenberg, M., Grebe, K., and Appel, M. (1982) Eur. J. Biochem. 126, 263-269] was reflected by the inhibitor sensitive line broadening, indicating that this transition also affects nucleotide binding. Nucleotide monophosphates were employed to study the relation between nucleotide structure and affinity, using selective excitation, sample spinning with digital suppression of spinning sidebands, and line shape simulation. The binding of purines depends on the distribution of the electrical potential and on the position of ring substituents, while pyrimidines are barely recognized at all by the AAC. It is also shown that the photocleavable "caged" derivatives are more tightly bound than the original nucleotides. A two step model of carrier catalysis will be discussed on the basis of these results.

Adenosine Diphosphate↗
PubMed1999

Kinetics of electrogenic transport by the ADP/ATP carrier.

The electrogenic transport of ATP and ADP by the mitochondrial ADP/ATP carrier (AAC) was investigated by recording transient currents with two different techniques for performing concentration jump experiments: 1) the fast fluid injection method: AAC-containing proteoliposomes were adsorbed to a solid supported membrane (SSM), and the carrier was activated via ATP or ADP concentration jumps. 2) BLM (black lipid membrane) technique: proteoliposomes were adsorbed to a planar lipid bilayer, while the carrier was activated via the photolysis of caged ATP or caged ADP with a UV laser pulse. Two transport modes of the AAC were investigated, ATP(ex)-0(in) and ADP(ex)-0(in). Liposomes not loaded with nucleotides allowed half-cycles of the ADP/ATP exchange to be studied. Under these conditions the AAC transports ADP and ATP electrogenically. Mg(2+) inhibits the nucleotide transport, and the specific inhibitors carboxyatractylate (CAT) and bongkrekate (BKA) prevent the binding of the substrate. The evaluation of the transient currents yielded rate constants of 160 s(-1) for ATP and >/=400 s(-1) for ADP translocation. The function of the carrier is approximately symmetrical, i.e., the kinetic properties are similar in the inside-out and right-side-out orientations. The assumption from previous investigations, that the deprotonated nucleotides are exclusively transported by the AAC, is supported by further experimental evidence. In addition, caged ATP and caged ADP bind to the carrier with similar affinities as the free nucleotides. An inhibitory effect of anions (200-300 mM) was observed, which can be explained as a competitive effect at the binding site. The results are summarized in a transport model.

Adenosine Diphosphate↗
PubMed1999

Uncoupling protein--a useful energy dissipator.

The structure/function relationship in the uncoupling proteins (UCP) is reviewed, stressing UCP from brown adipose tissue (UCP1) since, so far, nearly no biochemistry is known for the UCP variants UCP2, UCP3, and UCP4. The transport for H+ and Cl- and its dependence on fatty acids in reconstituted vesicles is described. The inhibition and binding of nucleotides to UCP1, in particular, the pH dependence and two-stage binding are analyzed. A model for the role of fatty acid in H+ transport is shown. The role of specific residues in UCP1 is analyzed by directed mutagenesis in a yeast expression system. The different regulation by the cellular energy potential of UCP1 versus UCP3 is discussed.

Adipose Tissue, Brown↗
PubMed1999

Structure-function relationship in UCP1.

The function of uncoupling protein (UCP1) as a H+ transporter regulated by nucleotide binding is elucidated. H+ transport requires fatty acids (FA) with relatively wide structural tolerance. The nucleotide binding site is specific for purine nucleotides and tolerates a number of derivatives. The strong pH dependency facilitates regulation of nucleotide binding and thus H+ translocation. The structure-function relationship of UCP1 has been analysed by various probes and by mutagenesis. According to our model, FA are a cofactor in H+ transport, providing H+ shuttling carboxyl groups in the translocation channel. By mutagenesis, additional H+ translocating groups at both sides of the translocation channel were found. Two pH sensors, controlling nucleotide binding, were identified in accordance with earlier postulates deduced from the pH dependence of nucleoside diphosphate (NDP) and nucleoside triphosphate (NTP). A common pH sensor E190 and a specific pH sensor H214 for triphosphates only, control access to the phosphate binding moiety. The three mitochondrial carrier family characteristic intrahelical arginines are essential for nucleotide binding. Mutagenesis of other charged residues reveals their role in structure stabilisation and/or has more generalised effects due to charge relay networks in UCP1.

Amino Acid Sequence↗
PubMed1998

In the uncoupling protein (UCP-1) His-214 is involved in the regulation of purine nucleoside triphosphate but not diphosphate binding.

The nucleotide binding to uncoupling protein (UCP-1) of brown adipose tissue is regulated by pH. The binding pocket of the nucleotide phosphate moiety has been proposed to be controlled by the protonization of a carboxyl group (pK approximately 4.5) for both nucleoside diphosphates (NDP) and nucleoside triphosphates (NTP) (identified as Glu-190) and of a histidine (pK approximately 7. 2) for NTP only. Here we identify His-214 as a pH sensor specific for NTP binding only. In reconstituted UCP-1 from hamster, DEPC diminishes binding of NTP but not of NDP. It also prevents inhibition of H+ transport by NTP but not by NDP. Hamster UCP-1 expressed in Saccharomyces cerevisiae was mutated to H214N resulting in only moderate change of the binding affinity for NTP (GTP) but a 10-fold affinity decrease with the bulkier substituent in H214W, whereas the affinity for NDP (ADP) was largely unchanged. The steep decrease with pH of the binding affinity for NTP in wild type (from pH 6.0 to 7.5) was much flatter in the mutants. Also, the pH dependence of binding and dissociation rates was diminished in these mutants. The transport of H+ and Cl- was not affected. Thus, His-214 is only involved in nucleotide binding, whereas, as previously shown, His-145 and His-147 are involved only in H+ transport. The results validate the earlier proposal of a histidine regulating the NTP binding in addition to a carboxyl group controlling both NTP and NDP binding. It is proposed that His-214 protrudes into the binding pocket for the gamma-phosphate thus inhibiting NTP binding and that His214H+ is retracted by a background -CO2- group to give way for the gamma-phosphate.

Adipose Tissue, Brown↗
PubMed1998

Slow-phase kinetics of nucleotide binding to the uncoupling protein from brown adipose tissue mitochondria.

The kinetics of nucleotide binding to the uncoupling protein (UCP) from brown adipose tissue mitochondria were studied with a filter binding method. Fast and slow phases of binding were observed, corresponding to the two-stage binding model based on equilibrium binding studies (Huang, S. G., and Klingenberg, M. (1996) Biochemistry 35, 7846-7854) (Reaction 1). [reaction: see text] Although this method determines total binding, only the slow phase can be resolved. The fast unresolved phase represents the formation of the initial loose UCP-nucleotide complex (UN; Kd approximately 2 microM), whereas the slow phase reflects the tight binding (U*N) associated with a conformational change induced by the bound nucleotide. Best fits of the binding data yielded, for the slow phase, k+1 values of 3.0 x 10(-3) s-1 for GTP, 4.8 x 10(-3) s-1 for ATP, 0.13 s-1 for GDP, and >0.7 s-1 for ADP and dissociation rate constants (k-1) of 0.10 x 10(-3) s-1 for GTP, 0.58 x 10(-3) s-1 for ATP, 8.8 x 10(-3) s-1 for GDP, and >0.3 s-1 for ADP at pH 6.7 and 4 degrees C. The rates were fairly pH- and temperature-dependent. The distribution constant Kc' (=k+1/k-1) between the tight and loose complexes ranged between 2 and 30, suggesting formation of 71-97% of the tight complex at equilibrium. The Kc' decreases with increasing pH, indicating a progressively less tight complex population. Anions (SO42-) form a loose complex with UCP, thus affecting the initial association step, but not the subsequent transition step. While the kinetic constants were verified by dilution and chase experiments as well as in mass action plots, they were further corroborated with data obtained by fluorescence competition measurements. Taken together, our results show that nucleotide binding to UCP occurs via a two-stage mechanism in which the initial loose complex rearranges slowly into a tight complex.

Adenine Nucleotides↗
PubMed1998

H+ transport by uncoupling protein (UCP-1) is dependent on a histidine pair, absent in UCP-2 and UCP-3.

UCP from brown adipose tissue of hamster (now UCP-1) expressed in Saccharomyces cerevisiae was used to examine the role of a conspicuous histidine pair H145 and H147 which is conserved among UCP-1 from various animals. Single and double mutants were generated by converting H145 and H147 into neutral residues (H145Q and H147N). As measured by fluorescence of dansyl-GTP binding, the level of expression of the mutant UCP was the same as wild-type (wt) in the isolated mitochondria. With the isolated and reconstituted UCP, transport of H+ and Cl- were measured. The fatty acid dependent H+ transport was reduced to about 10% in the single mutant H145Q and H147N and almost abolished in the double mutant, whereas Cl- transport into these vesicles was not affected as compared to wt. The possible involvement of the His pair in nucleotide binding and its pH dependence were examined by determining the KD and the kinetics for [14C]GTP and [14C]ADP binding. There were no marked changes in the affinity as well as in the binding and dissociation rates toward both these nucleotides in the mutant versus wt. Thus, the involvement in nucleotide binding can be excluded. The His pair is localized on the matrix side, probably at the entrance of the H+ translocation channel in UCP-1. It is absent in the recently discovered UCP-2, and therefore, UCP-2 might be predicted not to be a H+ transporter or to use a different mechanism. UCP-3 is deficient only in the equivalent H145 and thus can be predicted to still sustain a reduced H+ transport. The data support our contention that H+-dissociation side chains of UCP-1 are involved in H+ transport in cooperation with fatty acid carboxyl groups.

Amino Acid Sequence↗
PubMed1997

Mutagenesis of some positive and negative residues occurring in repeat triad residues in the ADP/ATP carrier from yeast.

In AAC2 from Saccharomyces cerevisiae, nine additional charged residues (six positive, three negative) were neutralized by mutagenesis following the previous mutation of six arginines. Oxidative phosphorylation (OxPhos) in cells and mitochondria, the expression level of AAC protein, and the various transport modes of AAC in the reconstituted system were measured. Mutations are: within the first helix at K38A which is exclusive for AAC; K48A, and R152A, part of a positive triad occurring in the matrix portion of each repeat; two matrix lysines, K179M and K182I, and the negative triad helix-terminating residues, E45G, D149S, D249S. Cellular ATP synthesis (OxPhos) is nearly completely inhibited in K48A, R152A, D149S, and D249S, but still amounts to 10% in K38A and between 30% and 90% in the gly+ mutants K179M, K179I + K182I, and E45G. Comparison of the AAC content measured by ELISA and the binding of [3H]CAT and [3H]BKA reveals discrepancies in K48A, D149S, and D249S mitochondria, which provide evidence that these mutations largely abolish inhibitor binding. Also these mitochondria have undetectable OxPhos. Differently in K38A, CAT and BKA binding are retained at high AAC levels but OxPhos is very low. This reveals a special functional role of K38, different from the more structural role of R152, K48, D149, and D249. Transport activity was measured with reconstituted AAC. The electroneutral ADP/ADP exchange of gly- mutants is largely or fully suppressed in K48A, D149S, and D249S. K38A and R152A are still active at 18% and 30% of wt. The other three exchange modes, ATP/ADP, ADP/ATP, and ATP/ATP, are nearly suppressed in all gly- mutants but remain high in gly+ mutants. ATP-linked modes are higher than the ADP/ADP mode in gly+ but lower in gly- mutants, resulting in an exchange mode inversion (EMI). In the competition for AAC2 transport capacity, the weak ATP exporting modes are suppressed by the much stronger unproductive ADP/ADP mode causing inhibition of OxPhos. Together with previous results all members of three charge triads are now mutagenized, revealing drastic functional rotatory asymmetries within the three repeat domains. In the intrahelical arginine triad the third (R294A), in the positive matrix triad the second (R152A), and in the helix-terminating negative triad the first (E45G) still show high activity.

Adenosine Triphosphate↗
PubMed1997

Biochemical and physical parameters of the electrical currents measured with the ADP/ATP carrier by photolysis of caged ADP and ATP.

The transport by the mitochondrial ADP/ATP carrier (AAC) has been shown in a preliminary communication to produce electrical capacitive currents on photolysis of caged ATP or ADP with reconstituted AAC liposomes attached to black lipid membranes [Brustovetsky, N., Becker, A., Klingenberg, M., and Bamberg, E. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 664-668]. Here we study the relation of the currents to ADP/ATP fluxes, the interaction of caged ADP and ATP with AAC and other basic facets of this method. Caged ADP and ATP are not transported by the AAC, as shown in mitochondria. Flux measurements with reconstituted AAC show that caged nucleotides are competitive inhibitors (Ki = 5 microM for caged ADP and 1 microM for caged ATP). Caged ATP competes with photolyzed ATP as shown by the dependence of the currents on the caged ATP concentration and on the light intensity. A competition of added ADP with caged ATP on the currents yields Ki = 50 microM for ADP. We conclude that caged ADP and ATP bind tighter to AAC than ADP or ATP, allowing immediate initiation of translocation by in situ photolysis. The caged compounds bind preferentially at the cytosolic side of AAC. With a regenerative hexokinase + glucose system, the currents are stabilized in repetitive flashes and can be used for applying inhibitors etc. during a flash series. The currents are completely inhibited by the combined addition of the AAC inhibitors bongkrekate (BKA) and carboxyatractylate (CAT). The partial inhibition by CAT or BKA is dependent on the number of flash cycles increasing from 60% to 90%, and by replacing chloride with gluconate from only 30% to 90%. The current are increased by a K+ diffusion potential (valinomycin + KCl) and decreased by the permeant anion TPB-. The pH dependence of the currents and of the parallel flux measurements indicates that only the fully charged ATP4- and ADP3- are transported. A strong temperature dependence of the currents with a break at 15 degrees C (EA = 95 and 28 kJ) agrees with former measurements of flux rates in mitochondria. In conclusion, the capacitive currents faithfully reflect AAC transport function and are a powerful tool for investigating the charge transfer in transport.

Adenosine Diphosphate↗
PubMed1997

Mutagenesis of the uncoupling protein of brown adipose tissue. Neutralization Of E190 largely abolishes pH control of nucleotide binding.

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.

Adipose Tissue, Brown↗
PubMed1997

Identification of the pH sensor for nucleotide binding in the uncoupling protein from brown adipose tissue.

The transport inhibiting nucleotide binding to the uncoupling protein (UCP) has a unique pH dependence and has been postulated to be controlled by the dissociation state of a carboxyl group in UCP with pK 4.5 and, in addition only for the nucleoside triphosphate, by a group with pK 7.2. To prove this assumption and to identify the carboxyl group, Woodward reagent K (WRK) was applied to UCP. In mitochondria, WRK was found to inhibit binding of GTP in a noncompetitive manner using WRK in the millimolar range. In isolated UCP, GTP binding is inhibited by WRK at a 1 to 2 ratio to UCP, suggesting that WRK primarily reacts with only one carboxyl group. Prebound GTP protects against WRK reaction as monitored by GTP binding. The protection decreases from pH 5 to 7 due to better reactivity of WRK and less tight GTP binding. WRK does not inhibit H+ transport by UCP but prevents GTP inhibition of H+ transport. For elucidating the WRK target residue, the WRK derivatized group was labeled with [3H] by reduction with [3H]NaBH4. Both GTP and GDP largely protected against WRK-dependent [3H] labeling. CNBr fragmentation identified the region T121-M197 as the [3H] incorporation site. Combined CNBr and tryptophane cleavage by the reagent 3-bromo-3-methyl-2-((2-nitrophenyl) thio)-3H-indole (BNPS) allowed to further delimit the 2.8 kDa peptide W173-M197 as the [3H] label carrier which contains two acid residues E190 and D195. To further identify the residue, limited tryptic digestion in sarcosyl-treated UCP was performed, and a tryptic fragment enclosing E190 and D195 was isolated which carried most of the [3H] label. Edman degradation showed the major [3H] label at the eighth position corresponding to E190 and no peak at D195. Thus, the original postulate of the pH-sensing carboxyl group regulating both the nucleoside di- and triphosphate binding has been verified. It is identified as E190 situated in the fourth transmembrane helix. In total, now four residues close to the nucleotide binding sites in UCP have been determined.

Adipose Tissue, Brown↗