Structural and functional asymmetry of the ADP/ATP carrier from mitochondria.
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Anions↗
Biomedical subjects
M Klingenberg
Publications and source records attributed to M Klingenberg.
The antigenic characteristics and the significance of the adenine nucleotide translocator as a major autoantigen to antimitochondrial antibodies in dilated cardiomyopathy.
To obtain further knowledge of the antigen-antibody system, immunochemical characterization of the adenine nucleotide translocator was achieved by crossed immunoelectrophoresis, immunoreplica technique, radioimmunoassay, immunoabsorption studies, and nucleotide-transport measurements. Sera of 18 patients with proven congestive cardiomyopathy (CCM) were tested. On the adenine nucleotide translocator (ANT) from heart, kidney, and liver, organ-specific antigenic determinants were demonstrable, although a partial cross-reactivity existed. Conformation specificity was also confirmed by experimental studies. Of the patients studied, 17 of 18 with CCM showed a significant binding to the heart ANT, while no or a lower binding was seen on the kidney/liver ANT. In CCM, a correlation exists between the ejection fraction and the anti-ANT titer. These results give new evidence for autoimmunological events in MC and CCM and indicate a possible causal relationship between these two diseases.
Adult↗
Immunochemical characterization of the adenine nucleotide translocator. Organ specificity and conformation specificity.
Antibodies have been prepared against purified preparations of the heart and kidney nucleotide translocator in the 'c'-conformation. The results show organ-specific antigenic determinants on the translocator proteins isolated from heart, kidney and liver although a partial cross-reactivity between these three proteins was demonstrable. The organ specificity was observed both with the solubilized and with the membrane-bound translocator protein indicating organ-specific determinants on exposed regions of the carrier. An organ-specific inhibition of the nucleotide transport in heart mitochondria by the heart carboxyatractylate-protein antiserum leads to the conclusion that the organ specificity is at least partially conditioned by the binding site for the substrate and/or the closely linked gate of the carrier protein. Apart from the organ specificity the results also demonstrate a specificity of the antibodies for the translocational conformations of the carrier: the 'c'-conformation stabilized in the carboxyatractylate-protein complex and the 'm'-conformation present in the bongkrekate-protein complex. However, after denaturation of the carboxytraktylate-protein and bongkrekate-protein complexes the binding of the anti-(carboxyatractylate-protein) antiserum to both inhibitor-protein complexes was nearly identical. The conformation specificity was also expressed by the inhibition of the conformation transition from the 'c'- to the 'm'- state. This side-specific inhibition of the nucleotide transport and the identical binding activity of the carboxyatractylate-protein antiserum against the denatured carboxyatractylate-protein and bongkrekate-protein complexes suggested that the conformation-specific antigenic determinants are topographic surface regions which are determined by the chain folding.
Animals↗
Interaction of fluorescent adenine nucleotide derivatives with the ADP/ATP carrier in mitochondria. 1. Comparison of various 3'-O-ester adenine nucleotide derivatives.
Fluorescent 3'-O-acyl-substituted adenine nucleotide (dimethylamino)naphthoyl and trinitrophenyl groups were studied for binding to the ADP/ATP carrier in mitochondria and submitochondrial particles. The changes in fluorescence intensity and emission maximum are for the most part similar to those observed in nonaqueous solvents. The (dimethylamino)naphthoyl derivatives from a largely quenched aqueous state have a shortwave shift up to 85 nm and increase up to 90-fold (1,5 derivative), whereas the little quenched naphthoyl derivatives show a fluorescence decrease and the weakly fluorescent trinitrophenyl derivative shows only a small increase on binding. All derivatives are good inhibitors (K1 = 1-10 microM) of nucleotide transport. The fluorescence titrations have an apparent K1/2 = 2-7 microM. The fluorescence of the 1,5-DAN nucleotide is fully suppressed by bongkrekate but only partially suppressed by carboxyatractylate. The fluorescence response is much stronger in submitochondrial particles than in mitochondria. Both facts suggest fluorescent binding to the "m" state of the carrier site at the inner face of the membrane. 1,5-DAN-AMP shows a slightly more efficient binding than DAN-ADP or DAN-ATP.
Adenine Nucleotides↗
Interaction of fluorescent adenine nucleotide derivatives with the ADP/ATP carrier in mitochondria. 2. [5-(Dimethylamino)-1-naphthoyl]adenine nucleotides as probes for the transition between c and m states of the ADP/ATP carrier.
The binding to the ADP/ATP carrier in mitochondrial membranes of the 3'-O-(dimethylamino)naphthoyl (DAN) derivatives of AMP, ADP, and ATP was quantitatively analyzed. The sidedness of the fluorescent type binding to the "m" side only was shown comparing the mitochondrial membranes in various stages of integrity and surface orientation. In particles displacement by bongkrekate (BKA) is direct, whereas in the case of carboxyatractylate (CAT) the requirement for ADP and ATP demonstrates the transition from the "m" to the "c" state. Quantitatively the "physical" binding of [3H]DAN-AMP and fluorescence are well correlated, allowing for a little nonfluorescent binding to the c side. For DAN-AMP KD is 1.6 microM, for DAN-ADP KD is 0.8 microM, and in the Hill plot a straight line with n = 1.25 is obtained. The maximum number of binding sites for [3H]DAN-AMP (1.5 mumol/g of protein) is about equal to the sites found for [3H]BKA if the unspecific binding of both ligands is differentiated by blocking carrier sites with CAT. [3H]CAT binding is somewhat lower in accordance with the limited access of CAT to inverted vesicles. ADP is able to decrease fluorescence only by about 35% at high concentrations (10 mM) whereas GDP has virtually no effect. With ADP, DAN-AMP binding decreases by 30% of the total binding sensitive to BKA. Binding to ATPase is low because of the absence of Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenine Nucleotides↗
A detergent-induced charge shift as a prerequisite for the electrochemical analysis of the adenine nucleotide translocator, a basic membrane protein.
The adenine nucleotide translocator is a hydrophobic, basic protein of the inner mitochondrial membrane which is solubilized by the non-ionic detergent Triton X-100. For immunochemical characterization of this membrane-protein by crossed immunoelectrophoresis a charge shift of the protein-Triton X-100 micelle by the introduction of an ionic detergent (deoxycholate) was necessary as a prerequisite to avoid unspecific precipitation of the protein. Beside the charge shift of the protein-detergent micelle, the selection, concentration and ratio of the detergents used and the choice of the agarose with different degrees of electroendosmosis should be carefully considered. The principle derived from these results provides a new methodological possibility for the immunochemical characterization of hydrophobic, basic membrane proteins.
Animals↗
Characteristics of the uncoupling protein from brown-fat mitochondria.
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Adenosine Diphosphate↗
Inhibition of the adenine nucleotide translocator by organ specific autoantibodies in primary biliary cirrhosis.
Sera from 13 patients with proven primary biliary cirrhosis (PBC) were studied for the capacity to bind to the adenine nucleotide translocator (ANT) isolated from heart, kidney and liver mitochondria. Antibodies against the ANT from liver were detected in the serum of all PBC patients, while 10 of 13 sera were negative when tested with the ANT from heart. None of the sera showed a significant binding to the ANT from kidney. The specific binding and the organ specificity of the autoantibodies against the ANT from liver were also confirmed by immunoabsorption studies on the isolated proteins. To distinguish between antibody titre and antibody activity, we measured the ability of the antisera to inhibit the adenine nucleotide transport across inner mitochondrial membrane using isolated mitochondria from heart, kidney and liver. Six of 13 patient sera tested inhibited the adenine nucleotide transport from liver mitochondria, however, none of the sera inhibited the transport from heart or kidney mitochondria again indicating the organ specificity of the antigen and the autoantibodies.
Adult↗
Binding of spin-labeled carboxyatractylate to mitochondrial adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier as studied by electron spin resonance.
The spin-label 2,2,5,5-tetramethyl-1-oxy-3-pyrroline-3-carboxylic acid was attached to the inhibitor carboxyatractylate of the mitochondrial ADP/ATP carrier. Being closely linked to the inhibitor, the spin-label should reflect the mobility of the carboxyatractylate. When bound to the carrier in mitochondria, spin-labeled carboxyatractylate reveals a most unusual hyperfine splitting of 72 G. A second spectral component with a hyperfine splitting of 62 G is also mainly due to carrier-bound inhibitor. A similar spectrum with somewhat reduced hyperfine splitting was observed with the detergent-solubilized protein, whereas reincorporation into phospholipid membranes yielded almost the same spectra as in mitochondria. The carrier-bound spin-label is concluded to be highly immobilized. The less immobilized spectral component is discussed in terms of strongly anisotropic label motion. In addition, the unusual splitting is interpreted to indicate the highly polar environment of the nitroxide. The interpretations are supported by the temperature dependence, which indicates a reversible progressive spin-label mobilization up to 50 degrees C. Membrane-impermeable reducing agents showed that the spin-label is easily accessible from the aqueous phase.
Animals↗
The binding of bongkrekate to mitochondria.
The binding of bongkrekate to mitochondrial membrane was investigated using [3H]bongkrekate. These measurements were designed to examine the previously derived reorienting site mechanism which implies that bongkrekate binds to the single carrier site only from the inner face of the mitochondrial membrane. The binding studies confirm pH-dependent accumulation of [3H]bongkrekate inside the mitochondria which superimposes on to binding of carrier sites. By breaking the membrane with Lubrol or sonication, binding to the carrier sites can be titrated and Kd approximately equal to 5 X 10(-8) M is determined. ADP or ATP increases the amount of binding but does not change the Kd. Reciprocally bongkrekate increases ADP binding in those sections of a titration curve where bongkrekate binding is increased by ADP. [35S]Atractylate is displaced by [3H]bongkrekate at a 1:1 molar ratio. This displacement is dependent on ADP concentration with the Km = 0.5 X 10(-6) M. The earlier described isomer, isobongkrekate, also binds specifically to the carrier sites. From competition with bongkrekate a ratio KisoBKAd/KBKAd = 0.10 is determined. [35S]Carboxyatractylate displaces most of [3H]isobongkrekate but only little of [3H]bongkrekate. The rate of displacement is more than 10-times faster for isobongkrekate than for bongkrekate. The displacement is dependent on ADP with a Km = 5 X 10(-6) M. All these results are fully consistent with the single site reorienting mechanism. In no instant do bongkrekate and atractylate as well as ADP or ATP bind simultaneously to the carrier.
Adenosine Diphosphate↗
Reincorporation of adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier into phospholipid membranes. Phospholipid-protein interaction as studied by phosphorus-31 nuclear magnetic resonance and electron microscopy.
Combined phosphorus-31 nuclear magnetic resonance (31P NMR) and electron microscopic studies were performed on the ADP/ATP carrier protein from beef heart mitochondria. The protein was incorporated into phospholipids by addition of Triton-protein micelles to a lipid suspension or to the dry lipid. All of the phospholipid (egg phosphatidylcholine or mixtures of egg phosphatidylcholine and egg phosphatidylethanolamine) that contributed to the observed 31P NMR signal under these conditions appeared to be in a bilayer configuration. Freeze-fracturing and negative-staining electron microscopy showed unilamellar vesicles and multilayers. An isotropic signal could be attributed to vesicle rotation, judging from its sensitivity to increasing viscosity. The presence of small vesicles was also noticeable in the 31P NMR spectra of planar oriented membranes. In the presence of phosphatidylethanolamine, aggregation of protein particles was observed. Gel chromatography of the protein-Triton-phospholipid mixture revealed that, before Triton removal, large amounts of protein are associated with multibilayers. Separation of loaded and unloaded membranes by centrifugation in D2O showed that, upon stepwise addition, protein incorporates preferentially into unloaded liposomes. From these findings a mechanism of protein reincorporation was deduced.
Animals↗
The mitochondrial adenine nucleotide translocator is an antigen in primary biliary cirrhosis.
Circulating antibodies reacting specifically with the adenine nucleotide translocator from liver mitochondria were detected in sera from 12 patients with proven primary biliary cirrhosis (PBC) by a solid phase double antibody immunoradiometric assay (IRMA). Furthermore these antibodies were absorbed with the isolated adenine nucleotide translocator from liver mitochondria. None of the sera from 20 normal individuals, four patients with anti-mitochondrial positive pseudolupus syndrome (PLE) sera (M-3) and three patients with syphilis (anti-M-l) had antibodies directed against this protein from inner mitochondrial membrane. The adenine nucleotide translocator as antigen in PBC could clearly be distinguished from the ATPase associated PBC specific M-2 antigen. With the present study, for the first time, a well characterized protein from inner mitochondrial membrane has been clearly defined as an autoantigen in primary biliary cirrhosis.
Adenosine Triphosphatases↗
Characteristics of the isolated purine nucleotide binding protein from brown fat mitochondria.
The isolation of the purine nucleotide binding protein (NbP), the putative uncoupling protein, from hamster brown adipose tissue mitochondria and some of its functional characteristics are described. (1) Among various detergents tested, Triton is the most suitable; the total GDP binding capacity can be recovered after solubilization by Triton and is rather stable in this extract. (2) For separation of NbP from the ADP/ATP carrier, differences in the solubilizing conditions and the stability at room temperature between both proteins are exploited. The preparation is substantially free of ADP/ATP carrier. (3) The purified NbP has a binding capacity for 16 mumol of GDP/g of protein, corresponding to a 16-fold purification from mitochondria. (4) In sodium dodecyl sulfate-polyacrylamide gel electrophoresis in single band of Mr 32 000 is found. A dimer structure is suggested from chemical cross-linking, from the binding capacity for GDP, and from the previously reported centrifugation equilibrium. (5) The isolated NbP preparation consists of Triton-protein-phospholipid mixed micelles with a Stokes radius of 60.5 A as determined by gel filtration. The Triton binding is 1.9 g/g of protein, and the phospholipid binding is 0.2 g/g of protein. (6). The amino acid composition has a polarity index of 43.5%. The N-terminal peptide has the sequence Val-Asp-Pro-Thr-Thr-Ser-Glu-Val. (7) The affinity of NbP for different purine nucleotides decreases in the order GTP greater than GDP greater than ATP greater than ITP greater than ADP greater than IDP. The affinity for the monophosphates is 100 time lower. (8) Photooxidation and the lysine reagent 2,4,6-trinitrobenzenesulfonic acid decrease the binding capacity without influencing the affinity of the unaffected sites. GDP protects against photooxidation.
Adipose Tissue, Brown↗
Isolation of the GDP binding protein from brown adipose tissue mitochondria of several animals and amino acid composition study in rat.
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Adipose Tissue, Brown↗
Electrophoretic control of reconstituted adenine nucleotide translocation.
The initial velocity of adenine nucleotide exchange catalyzed by the reconstituted ADP-ATP carrier from beef heart mitochondria was measured under the influence of membrane potential and with different nucleotide distributions between the internal liposomal and the external buffer volume. Both Vmax and Km of adenine nucleotide uptake not only changed due to the applied potential but also depended on the respective nucleotide distribution. The rate equations for the ADP-ATP exchange under the various conditions were derived. These equations were simplified by assuming two alternative situations; either (a) af affinity type model, where the membrane potential influences only the affinity of the adenine nucleotide carrier toward ATP and ADP, or (b) a velocity type or distribution model, where the membrane potential modulates the rate constants of the ADP-ATP exchange. On the basis of several simplifications in the reconstituted system, the rate equations could be solved and the rate constants and dissociation constants of the exchange in the "energized" and in the "deenergized" state could be calculated. These values were used to derive prediction tables for normalized exchange rates under different nucleotide distributions, which were then compared with the experimental data. Only the exchange rates predicted by the velocity-type model agreed with the measured values. On the basis of this model a definite asymmetry caused by the membrane potential could be seen. Whereas this asymmetry is not very pronounced in the case of carrier-ADP complexes, about 40 times more ATP-loaded binding sites face the outside of the vesicles in the energized state.
Adenosine Diphosphate↗
Incorporation of N-ethylmaleimide into the membrane-bound ADP/ATP translocator. Isolation of the protein labeled with N-[3H]ethylmaleimide.
The incorporation of N-ethylmaleimide into the 30,000-Mr component of beef-heart mitochondria has been studied as a function of various ligands to the ADP/ATP carrier and the isolation of the N-ethylmaleimide-labeled protein is reported. 1. The incorporation of N-ethylmaleimide into the 30,000-Mr component is specifically stimulated by ADP and ATP. Thus by differential incorporation of N-ethylmaleimide, the 30,000-Mr component is preferentially labeled. 2. Addition of carboxyatractylate inhibits, whereas bongkrekate tolerates, the incorporation of N-ethylmaleimide. 3. After solubilization by Triton the purification of N-ethylmaleimide-labeled protein is facilitated in the presence of bongkrekate but not of carboxyatractylate, in agreement with the postulated existence of only a bongkrekate-N-ethylmaleimide-protein complex. The labeled protein was purified to homogeneity on hydroxyapatite in Triton and subsequently, after denaturation in dodecylsulfate, on Sepharose 6B. 4. The identify of the isolated labeled protein with the formerly isolated bongkrekate-protein or carboxyatractylate-protein complexes is confirmed by the isoelectric point and amino acid composition. 5. Two moles of N-ethylmaleimide must be incorporated into the 30,000-Mr component in order to inhibit fully the binding of one mole carboxyatractylate. This corresponds to one -SH group per unit.
Animals↗
The reactivity of -SH groups in the ADP/ATP carrier isolated from beef heart mitochondria.
The emergence of the reactivity of -SH groups associated with conformation changes has been studied on the ADP/ATP carrier, is isolated in three different inhibitor-protein complexes. 1. The bongkrekate-protein complex incorporates approximately one molecular more of N-ethylmaleimide than the carboxyatractylate-protein complex. After extensive denaturation by dodecylsulfate in urea, both inhibitor complexes exhibit four reactive -SH groups per subunit. Thus one of four -SH groups per subunit has been unmasked in the bongkrekate-protein complex. 2. The interconversion from the bongkrekate-protein complex to the carboxyatractylate-protein complex is inhibited after the -SH groups have been blocked. 3. The protein complex isolated with the more easily dissociable atractylate, is used to demonstrate, by the emergence of the -SH groups, the transition into the m-state. This transition is specifically catalyzed by ADP and ATP. 4. Using 2,2'-dinitro-5,5'-dithiodibenzoate, the appearance of the -SH groups on transition from the c-state to the m-state can be followed spectrophotometrically. The specificity for the catalyzing nucleotides is identical with that for the transport. The Km for ADP and ATP is in the range of 1 microM. In conclusion, the thiol groups of the isolated ADP/ATP carrier behave as in the mitochondrial membrane. The unmasking of -SH groups is in full accordance with the concept of two conformational states (c and m).
Adenosine Diphosphate↗
Temperature dependence of ADP/ATP translocation in mitochondria.
The temperature dependence of the adenine nucleotide exchange in mitochondria has been determined by employing a rapid mixing, quenching and sampling apparatus and the inhibitor quench-back exchange method. Thus the exchange is resolved down to 0.1 s. Rates are evaluated from accumulating the time-dependent progress at about 10 points. The exchange rate in liver mitochondria was determined from -10 degrees C to + 10 degrees C in the presence of 20% glycol, from 0 degrees C to 25 degrees C, and from 20 degrees C to 40 degrees C under partial inhibition by carboxyatractylate. The total range between -10 degrees C to + 40 degrees C has only one temperature break at 13 degrees C. From the Arrhenius plot between -10 degrees C to + 13 degrees C, EA approximately equal to 140 kJ and above 13 degrees C, EA approximately equal to 56 kJ is evaluated, corresponding to a Q10 of 8 and 2 respectively. In beef heart mitochondria the exchange rate was measured between 0 degrees C and 20 degrees C, and between 15 degrees C and 30 degrees C under partial inhibition with carboxyatractylate. There is a temperature break around 14 degrees C with EA approximately equal to 143 kJ between 0 degrees C and 14 degrees C and EA approximately equal to 60 kJ from 15 degrees C to 30 degrees C. The extrapolated translocation rates at 37 degrees C are 500 and 1800 mumol min-1 (g protein)-1 for rat liver and for beef heart mitochondria respectively. The temperature break is suggested to reflect a conformation change since there is no reversed break at low temperature, the temperature break changes in sonic particles and no lipid phase transition at 14 degrees C in mitochondria has been reported.
Adenosine Diphosphate↗