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M Klingenberg

Publications and source records attributed to M Klingenberg.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1996

Chloride channel properties of the uncoupling protein from brown adipose tissue mitochondria: a patch-clamp study.

The uncoupling protein (UCP) from brown adipose tissue mitochondria possesses H+ and Cl- transport activities [reviewed in Klingenberg, M. (1990) Trends Biochem. Sci. 15, 108-112]. Being a member of a mitochondrial carrier family, the transport of H+ and Cl- is carrier-like, i.e., much slower as compared to channels. Here we report that UCP reconstituted into giant liposomes displays stable chloride channel properties under patch-clamp conditions. The transport inhibitors (GTP, GDP, ATP, and ADP) also inhibit this channel in a reversible way, showing that the channel activity is associated with UCP. The slightly inward-rectifying chloride channel has a unit conductance of approximately 75 pS in symmetrical 100 mM KCl and closes at high positive potentials on the matrix side of UCP. Channel gatings switch from slow open-closure transitions to fast flickerings as the holding potential increases over +60 mV. Substitution experiments reveal a strong discrimination against cations [P(Cl-)/P(K+) approximately 17] and a permeability ratio order of Cl- > Br- > F- > SCN- > I- > NO3- > SO4(2-) > HPO4(2-) > gluconate. Nucleotide inhibition studies indicate that 70% UCP molecules had its matrix side oriented outside in the giant liposomes. Fatty acids, pH, divalent cations (Ca2+ and Mg2+), and mersalyl do not influence these Cl- currents. The Cl- channel can be blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) from the matrix side of UCP. The data are consistent with a dimer consisting of two monomeric 75-pS Cl- channels or with a monomeric 150-pS channel having a 50% subconductance state. The channel current increases with Cl- concentration showing a typical saturation curve with Km approximately 63 mM and gmax approximately 120 pS (100 mM KCl in the pipet). The Cl- conductance measured under these conditions is 6 orders of magnitude higher than the Cl- transport activity reported earlier, suggesting that the UCP has the potential of behaving as an anion channel.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

Probing the role of positive residues in the ADP/ATP carrier from yeast. The effect of six arginine mutations of oxidative phosphorylation and AAC expression.

ADP/ATP transport is the terminal step of oxidative phosphorylation in mitochondria. In this paper seven mutants of AAC2 from Saccharomyces cerevisiae are studied on the cellular and mitochondrial level. Six conspicuously located arginines were mutated into mostly neutral residues [Nelson, D. R., Lawson, J. E., Klingenberg, M., & Douglas, M. G. (1993) J. Mol. Biol. 230, 1159-1170]. R96A, R96H, R204L, and R294A are located in the second transmembrane helix of each repeat while R252I, R253I, and R254I are in the arginine triplet of the last domain. All six arginine residues are conserved in all known ADP/ATP carrier sequences. At the cellular level, oxidative phosphorylation in R96H and R294A retains 8% of the wild-type rate, but it is virtually zero in the other mutants. However, cytochrome c, a parameter of oxidative capacity, remains at 4-42% of wt. The weak coordination of respiratory chain and AAC expression indicates that respiration is needed also for other purposes. In mitochondria the AAC-linked ATP synthesis is measured and segregated by using the AAC inhibitor bongkrekate (BKA). Only the R96H and R294A mutants express a significant rate of AAC-dependent ATP synthesis amounting to 2-18% of the plasmid-borne wild-type AAC2 mitochondria. In all other mutants it is virtually zero. However, respiratory capacity and cytochrome c content are reduced only by 20-70%. Whereas in immunoblots the presence of AAC is detected in all mutant mitochondria, by quantitative ELISA no AAC can be measured down to 0.05 mumol of AAC dimer/g of protein in R96A and R204L, whereas in R96H, R2521, R2531, and R254I the content is around 0.2 and in R294A the content is 0.46 as compared to 0.6 in the plasmid wild type. Also the [3H]CAT and [3H]BKA binding is virtually zero in some mutants and closely parallels the ELISA-determined AAC content, indicating that the mutations did not affect the inhibitor binding site. The turnover of AAC [V(ATP)/AAC content] in oxidative phosphorylation is reduced to 10% or 20% except for the two intrahelical mutants R96H and R294A. In the three Arg triplet mutants, it is nearly zero. In conclusion, the first two intrahelical arginines R96 and R204, are essential for expression but probably also for the activity of AAC. R294A still retains good transport activity and remarkably high expression of AAC. All arginines in the triplet 252, 253, 254 are essential. Extrapolation of the in vitro phosphorylation rates to the cellular level by the cytochrome c factor reveals a large discrepancy to the in vivo rates in particular for R294A. This indicates that these mutations render the AAC more sensitive to the regulatory intracellular ATP/ADP ratio than the wt AAC.

Amino Acid Sequence↗
PubMed1996

Probing the role of positive residues in the ADP/ATP carrier from yeast. The effect of six arginine mutations on transport and the four ATP versus ADP exchange modes.

Mutagenesis of three intrahelical arginines, R96, R204, or R294, and of each member of the arginine triplet R252, R253, R254 into neutral residues had resulted in a strong suppression of oxidative phosphorylation in cells and isolated mitochondria [Müller, V., Basset, G., Nelson, D. R., & Klingenberg, M. (1996) Biochemistry 35, 16132-16143]. Here we determine the transport activity of wild-type and mutant AAC in reconstituted proteoliposomes using a new rapid removal-stop method without relying on the inhibitor stop which can be compromised by mutations. The basic electroneutral ADP/ADP exchange activity is strongly or totally suppressed in six out of seven of these mutations, with the exception of R294A, which retains nearly wild-type activity. Carboxyatractylate (CAT) inhibits the ADP/ATP exchange rate only to 3-10% in wild type and R294A and up to 40% in other mutants, whereas bongkrekic acid (BKA) inhibits 50% (wild type and R294A) and 90% (other mutants). Consequently, AAC is preferentially reconstituted with the matrix surface outside. All these mutations drastically change activity distribution among the four exchange modes ADP/ADP, ADP/ATP, ATP/ADP, and ATP/ATP. Whereas in wild-type AAC the homo ATP/ATP exchange is twice as high as the ADP/ADP exchange, in mutants it is 10 to 15 times lower. Similarly, the hetero ATP/ADP exchange in wild-type AAC is higher than the ADP/ ATP exchange, but in mutants it is several times lower. Thus, these mutations afflict the ATP-linked modes, in particular those linked to external ATP. The inhibition of oxidative phosphorylation is thus explained by the suppression of ATP export versus ADP import mode. The "extra"-inhibition of oxidative phosphorylation in mutant cells is explained by the extreme shift in mutants in favour of ATP import versus ADP export. Besides structural changes, the mutant effects indicate electrostatic interactions of these arginines with the anionic substrates. The loss of one positive charge raises the translocation barrier the more negative the substrate, i.e. more for ATP4- than for ADP3-. In none of these arginine mutants was the binding of CAT or BKA abolished.

Arginine↗
PubMed1996

Mitochondrial ADP/ATP carrier can be reversibly converted into a large channel by Ca2+.

Single-channel current measurements of excised patches with reconstituted purified mitochondrial ADP/ATP carrier (AAC) indicates the presence of a large low cation selective (PK+/PCl- = 4.3 +/- 0.6) channel. The channel conductance has multiple sublevels and varies from 300 to 600 pS. It has low probability of current fluctuations at Vhold up to 80-100 mV of both signs and is reversibly gated at Vhold > 150 mV. The opening of the channel is Ca(2+)-dependent (1 mM Ca2+) and can be reversibly closed on removal of Ca2+. It is strongly pH dependent and closes completely at pHex 5.2. The AAC-specific inhibitor bongkrekate inhibits the channel partially and completely in combination with ADP, whereas carboxyatractylate did not affect the conductance. The effects of these AAC-specific ligands prove that the channel activity belongs to AAC. The AAC-linked conductance can clearly be differentiated from the porin channel, rarely detected in our preparations. The properties of the AAC-linked channel coincide with the mitochondrial permeability transition pore (MTP), which is also affected by the AAC ligands [Hunter, D. R., & Haworth, R. A. (1979) Arch. Biochem. Biophys. 195, 453-459] and resembles the mitochondrial "multiconductance channel" [Kinnally, K. W., Campo, M. L., & Tedeschi, H. T. (1989) J. Bioenerg. Biomembr. 21, 497-506] or "megachannel" [Petronilli, V., Szabo, I., & Zoratti, M. (1989) FEBS Lett. 259, 137-143]. Therefore we conclude that the AAC, when converted into a large unselective channel, is a key component in the MTP and thus is involved in the ischemia-reperfusion damage and cytosolic Ca2+ oscillations. The channel opening in AAC is proposed to be caused by binding of Ca2+ to the cardiolipin, tightly bound to AAC, thus releasing positive charges within the AAC which open the gate.

Animals↗
PubMed1996

Two-stage nucleotide binding mechanism and its implications to H+ transport inhibition of the uncoupling protein from brown adipose tissue mitochondria.

The uncoupling protein (UCP) from brown adipose tissue mitochondria is the simplest H+ translocator known. H+ transport is regulated by fatty acids as activators and by pruine nucleotides as inhibitors. Nucleotide binding again is strongly influenced by the pH [Klingenberg, M. (1988) Biochemistry 27, 781-791]. Previously, by using fluorescent 2'-O-dansyl (DANS) derivatives of purine nucleotides, a two-stage binding mechanism was unraveled with a slow transition from a loose into a tight conformational state in the isolated UCP [Huang, S.-G., & Klingenberg, M. (1995) Biochemistry 34, 349-360]. Whereas with the unsubstituted nucleotides the transition to the tight state is nearly complete, various DANS and DAN (dimethylaminonaphthoyl) nucleotides bind more to the loose state. Here we investigated the relationships between the two-stage nucleotide binding and the inhibition of the H+ transport activity in reconstituted proteoliposomes. Further, limited tryptic digestion was used as an indicator of conformational change induced by the nucleotide binding in the isolated protein. The inhibition of H+ transport activity in reconstituted UCP proteoliposomes correlated only with the fraction of tight state of nucleotide binding. Unsubstituted nucleotides (ATP, GTP, and ADP) as well as DANSGTP inhibit fully the H+ transport, whereas DANSATP and DANSADP inhibit only to about 50%, and DANSAMP is nearly ineffective. Even for the loose conformational state the nucleotide derivatives exhibit considerable affinity. This allows DANSAMP to replace prebound ATP from UCP and relieve the inhibition of H+ transport by reversing the distribution of UCP from the tight into the loose conformational state. The pH dependence of the fraction of nucleotide binding in the tight state correlates closely with the pH dependence of the degree of H+ transport inhibition. Titration with DANS nucleotides of UCP incorporated into phospholipid vesicles revealed that over 70% of binding sites had an affinity comparable with that for the isolated UCP while the remaining sites displayed substantially lower affinity, due to nonhomogeneity of the reconstituted system. The sensitivity against trypsin digestion is inversely correlated with the fraction of nucleotide binding in the tight state. Whereas unsubstituted nucleotides and DANSGTP protect strongly against trypsinolysis, DANSATP and DANSADP do only partially, and DANSAMP does not at all. The counteracting influences of the DANS substitution are shown with DANSAMP, which has an affinity comparable to that of DANSATP or DANSADP but cannot form the tight inhibited complex. These data show that nucleotide binding only in the tight state is associated with a strong conformational change, which further causes an inhibition of H+ transport. In conclusion, UCP can exist in a loose noninhibited and a tight inhibited conformational state. The equilibrium between these two conformations is shifted to the tight state with unsubstituted nucleotides but remains to variable degrees in the loose state with DANS and DAN derivatives. The DANS group hinders progressively the transition to the tight state as the binding affinity of the underlying nucleotide decreases.

Adipose Tissue, Brown↗
PubMed1996

Electrical currents associated with nucleotide transport by the reconstituted mitochondrial ADP/ATP carrier.

The electrophoretic export of ATP against the import of ADP in mitochondria bridges the intra- versus extramitochondrial ATP potential gap. Here we report that the electrical nature of the ADP/ATP exchange by the mitochondrial ADP/ATP carrier (AAC) can be directly studied by measuring the electrical currents via capacitive coupling of AAC-containing vesicles on a planar lipid membrane. The currents were induced by the rapid liberation of ATP or ADP with UV flash photolysis from caged nucleotides. Six different transport modes of the AAC were studied: heteroexchange with either ADP or ATP inside the vesicles, initiated by photolysis of caged ATP or ADP; homoexchange with ADPex/ADPin or ATPex/ATPin; and caged ADP or ATP with unloaded vesicles. The heteroexchange produced the largest currents with the longest duration in line with the electrical charge difference ATP4- versus ADP3-. Surprisingly, also in the homoexchange and with unloaded vesicles, small currents were measured with shorter duration. In all three modes with caged ATP, a negative charge moved into the vesicles and with caged ADP it moved out of the vesicles. All currents were completely inhibited by a mixture of the inhibitors of the AAC, carboxyatractyloside and hongkrekate, which proves that the currents are exclusively due to AAC function. The observed charge movements in the heteroexchange system agree with the prediction from transport studies in mitochondria and reconstituted vesicles. The unexpected charge movements in the homoexchange or unloaded systems are interpreted to reveal transmembrane rearrangements of charged sites in the AAC when occupied with ADP or ATP. The results also indicate that not only ATP4- but also ADP3- contribute, albeit in opposite direction, to the electrical nature of the ADP/ATP exchange, which is at variance with former conclusions from biochemical transport studies. These measurements open up new avenues of studying the electrical interactions of ADP and ATP with the AAC.

Adenine Nucleotides↗
PubMed1995

Nature of the masking of nucleotide-binding sites in brown adipose tissue mitochondria. Involvement of endogenous adenosine triphosphate.

Binding of the fluorescent nucleotide derivative 2'-O-dansyl GTP and purine nucleotides to brown adipose tissue mitochondria from hamster was studied. 2'-O-Dansyl GTP binds with enhanced fluorescence to the uncoupling protein (UCP) in the mitochondria, similar to the isolated protein. The fluorescence signal showed biphasic fast and slow increases. Treatment of the mitochondria with an anion exchanger (Dowex) increased the total fluorescence but decreased the slower phase. The biphasic fluorescence response was restored by incubation with only 1 microM ATP, indicating that residual bound ATP may be responsible for the observed slow phase. The binding of [14C]GTP and GDP also increased after Dowex treatment. The dissociation of bound [14C]ATP but not of bound [14C]ADP was slow and apparently limited the binding assays. Short incubation (5 min) resulted in a curvature of the Scatchard plot, where the 'high-affinity sites' correspond to the free UCP sites; GDP had apparently higher affinity than GTP. Dowex treatment and incubation for 60 min produced a more linear Scatchard plot. Under such conditions, one measures the maximal UCP-binding sites (1.2 mumol/g protein); GTP exhibited higher affinity (Kd = 0.64 microM) than GDP (Kd = 3.1 microM). Acute cold adaptation (40 min at 4 degrees C) of hamsters caused an increase by over 40% of [14C]GTP binding, as compared to the control warm-(28 degrees C)-adapted animals. Dowex treatment completely abolishes this unmasking/masking effect, where both mitochondria had identical binding capacity and affinity for GTP. The inhibition by purine nucleotides of H+ transport as measured by potassium-acetate-induced mitochondrial swelling was dependent on the incubation time. Diphosphates inhibited faster and triphosphates required longer incubation (10 min) but inhibited more strongly. A linear correlation between the mitochondrial swelling rate and GDP binding was observed for mitochondria with depleted endogenous ATP or with added ATP. These data indicate that residual bound ATP from the tissue is responsible for the masking phenomenon.

Adaptation, Physiological↗
PubMed1995

Fluorescent nucleotide derivatives as specific probes for the uncoupling protein: thermodynamics and kinetics of binding and the control by pH.

Fluorescent 2'-O-dansylated (DANS) purine nucleotides were synthesized. The fluorescence of the nucleotide derivatives is quenched in aqueous solutions but strongly enhanced on binding to the uncoupling protein (UCP) from brown adipose tissue mitochondria. The fluorescence enhancement was 30-, 10-, and 10-fold for DANSGTP, DANSATP, and DANSADP. One mole of DANS nucleotide binds to 1 mol of dimeric UCP. The binding affinity ranges from 10(5) to 10(8) M-1, similar to that of the unsubstituted nucleotides, while dansylation of AMP increases the affinity 50-fold. The pH dependence in the pKD/pH plots for the DANS nucleotides is basically similar to that for the unsubstituted nucleotides, i.e., for nucleoside diphosphates the slope delta pKD/delta pH < -1 at pH 5-6.5, = -1 at pH > 6.8, and only for triphosphates = -2 at pH > 7.2. Two different protonation sites with a pKH approximately 4 (Asp/Glu) and pKH approximately 7.2 (His), only for nucleoside triphosphates, are suggested to be involved in binding. The higher affinity of DANSGTP indicates additional participation in binding of the C-6 oxygen on the guanine. The binding as measured with the anion exchange method agrees with the fluorescence measurement for DANSGTP, whereas for the more loosely binding DANSATP it is 40% lower. This is interpreted in terms of tight/loose UCP-nucleotide complexes, 100% tight complex for DANSGTP (as well GTP or ATP) but 40% loose complex for DANSATP. By measuring the rapid kinetics using the fluorescence signal, the binding rate is found to be fast and fairly constant for the various nucleotides, whereas the dissociation is slow and strongly nucleotide dependent. The rates are pH dependent with delta pkon/delta pH = 1 for all the nucleotides and delta pkoff/delta pH = -1 for DANSNTP but more weakly with delta pkoff/delta pH < -0.5 for DANSADP and DAN-ATP. The pH dependence of the binding rate corresponds to a protonation at the carboxylate group (Glu/Asp). The high pH dependence of the dissociation rate only for DANSNTP is explained by deprotonation at the HisH+ which is involved only in nucleoside triphosphate binding. This is in line with the very strong pH dependence of nucleoside triphosphate affinity above pH 7 with a delta pKD/delta pH = -2 as an important regulatory mechanism for the H+ transport activity of UCP. The differences of the DANS nucleotides versus the DAN and unsubstituted nucleotides as well as the nucleoside tri- versus diphosphate are rationalized in a specific H+ dependent regulatory mechanism at the binding site.

Animals↗
PubMed1994

The reconstituted ADP/ATP carrier can mediate H+ transport by free fatty acids, which is further stimulated by mersalyl.

In a reconstituted system, the participation of the ATP/ADP carrier (AAC) in the free fatty acid (FFA)-induced proton transport was demonstrated (i) by direct measuring of the proton transport through the membranes of AAC proteoliposomes and (ii) by monitoring of the transmembrane potential delta psi in AAC-cytochrome-c oxidase (COX)-coreconstituted proteoliposomes. FFA increased the initial rate of proton transport in AAC proteoliposomes and decreased delta psi in AAC-COX proteoliposomes. Inhibitors of AAC suppressed the effects of FFA. Without AAC or with inactive AAC, FFA cannot maintain proton leakage through the membrane. In these cases, even a small increase of delta psi was induced by FFA. These results demonstrate for the first time with purified components a participation of AAC in FFA-induced proton transport supporting an earlier suggestion (Skulachev, V.P. (1991) FEBS Lett. 294, 158-162). Mersalyl treatment of the AAC-COX proteoliposomes resulted in an increase of the AAC-mediated protonophoric action of FFA. Mersalyl also sensitized the protonophoric action of the FFA against nucleotides so that even guanine nucleotides, which are inactive in transport, become inhibitory. The effect of mersalyl is rationalized in terms of a specific interaction with cysteine 159 being attracted as anion by surrounding positive charges. This might open a gate similarly as suggested for eosin 5-maleimide interaction (Majima, E., Koike, H., Hong, Y.-M., Shinohara, Y., and Terada, H. (1993) J. Biol. Chem. 268, 22181-22187) and, thus, transform the AAC into undirectional transport mode.

Adenosine Triphosphate↗
PubMed1994

Effect of fatty acids on H+ transport activity of the reconstituted uncoupling protein.

A detailed study on the activation of H+ transport by reconstituted uncoupling proteins from brown adipose tissue is given, including the influence of chain lengths and of other structural modifications, concentration dependence, and the influence of nucleotides. Uncoupling protein was reconstituted with phosphatidylcholine in such a way as to keep H+ transport with endogenous fatty acids at a minimum. Using excess of polystyrene beads on reconstitution avoided the complications arising from the use of albumin. Both delta psi-driven H+ uptake and H+ efflux systems are used by changing the polarity. Fatty acids stimulate H+ uptake up to 6-fold and H+ efflux more than 10-fold. There is no competition between the inhibition by nucleotides (GTP) and fatty acids. Also, the binding of GTP and ATP is not affected. Only fatty acids starting from C8 activate, reaching a maximum at C14. However, unsaturated homologous of C18 (oleic, linoleic, etc.) are fully active. Hydrophilic substitutions by hydroxyl, CO2H, bromo, doxyl groups also permit good activation, probably due to improved uptake into the lipid phase. The hydrophobic moiety exhibits a low specificity. Blockage of carboxyl by esterification abolishes the activation. Maximum activation requires high total concentrations of 200-300 microM. The distribution of fatty acids between proteoliposomes and solution was determined. The activation mode of fatty acids is discussed either as regulatory activators or as cofactors in H+ translocation involving their carboxyl groups. Two alternatives are considered, namely that the fatty acids carboxyl group is at the translocation center or in the channel facilitating H+ transfer to the constituent H(+)-translocating carboxyl groups.

Adenosine Triphosphate↗