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M Klingenberg

Publications and source records attributed to M Klingenberg.

At least 127 records · Page 7Linked to original sources
PubMed1978

Isolation of the ADP, ATP carrier as the carboxyatractylate . protein complex from mitochondria.

The procedure for the isolation from mitochondria of the undenatured ADP, ATP carrier is described. The condition of retaining the nativity are elaborated. 1. As indicator for the ADP, ATP carrier (35S)- or (3H) carboxyatractylate were used. By preloading the mitochondria with carboxyatractylate, a stable carboxyatractylate . protein complex could be retained after solubilization with Triton X-100. Among the polyoxyethylene detergents emulphogen is also solubilizing, whereas Brij and Lubrol fail to solubilize. 2. When unloaded mitochondria are solubilized the capacity for binding carboxyatractylate disappears rapidly, particularly at 20 degrees C. 3. When mitochondria are preloaded with atractylate, the binding after solubilization with Triton X-100 is considerably lower than with carboxyatractylate, indicating that the high affinity of carboxyatractylate is required for effectively protecting the protein. 4. For purification hydroxyapatite is most effective. The carboxyatractylate-protein complex appears in the pass-through whereas the bulk of other mitochondrial proteins are retained such that a 7-fold purification is obtained. The nonadsorptivity to hydroxyapatite is dependent on the undenatured state maintained in the carboxyatractylate . protein complex. 5. Subsequent gel filtration on Sepharose results in a 1.5-fold further enrichment of specific carboxyatractylate binding up to 17 mumol/g protein, corresponding to a 10-fold purification from mitochondria. This value cannot be increased with further measures. 6. At the last purification step, in sodium dodecyl sulfate polyacrylamide gel electrophoresis virtually a single band of 30 000 molecular weight is found, confirming the purity at this stage. A molecular weight of 60 000 is calculated from the carboxyatractylate binding, indicating that the carboxyatractylate protein complex consists of two 30 000 subunits. From this the protein share of the ADP, ATP carrier in beef heart mitochondria can be calculated to amount to 9.5%9 7. The intact carboxyatractylate . protein complex is protected against proteolytic degradation. The release of carboxyatractylate ensues a conformational change of protein as assayed by conformation specific antibodies, concomitant with unmasking of proteolytic site as assayed by tryptic digestion. 8. The amino acid composition indicates hydrophobicity (39% polarity) and a high content of basic amino acid such as lysine and arginine. There is 1.5 mol percent cysteine and a blocked N-terminal. 9. From the solubilized complex (35S) carboxyatractylate can be removed by carboxyatractylate, ADP and ATP but not by ITP, etc., indicating the presence of recognizing sites specific fof ADP, ATP and therefore, identity with the ADP, ATP carrier. 10. Other reported procedures for isolating the ADP, ATP carrier are shown to either fail or have lower yield than the present, original procedure.

Adenosine Diphosphate↗
PubMed1978

The biosynthesis of the mitochondrial ADP, ATP translocator.

The biosynthesis of the ADP,ATP carrier was studied in mitochondria of Neurospora crassa. The carrier was isolated as the carboxyatractylate-protein complex and characterized in dodecylsulphate/polyacrylamide gel electrophoresis to have a Mr = 33 000. Applying the inhibitors chloramphenicol for the intramitochondrial translation and cycloheximide for extramitochondrial translation, the site of synthesis of this polypeptide was found to be extramitochondrially located.

Adenosine Diphosphate↗
PubMed1978

Kinetics of ADP, ATP transport in mitochondria as studied by the quench-flow method.

The kinetics in the millisecond range of ADP, ATP counterexchange in rat liver mitochondria were investigated using a quench-flow apparatus. The exchange was stopped with atractylate and the mitochondria were separated by centrifugation. 1. After correcting for leakage due to flow stress, apparent biphasic exchange kinetics were observed, with a more rapid phase within 100--200 ms, which extends to 10% of the total exchange. In general, the extent of the rapid phase increased when the translocation rate was changed under various conditions, in agreement with the model of the quench mechanism by atractylate. 2. The nature of the "rapid" phase was analyzed in the "steady" and "transient" state of the translocation and was shown to be caused by a delayed binding of atractylate due to competition with ADP. This quench delay results in a residual exchange which could explain the rapid part of the kinetics. 3. Deenergization of mitochondria by valinomycin or by uncoupler largely abolishes the rapid kinetic phase. This is explained by an increased availability of carrier sites at the outer face of the membrane to atractylate in the deenergized state, resulting in a more rapid quench. 4. The interpretation of the rapid exchange phase as a function of carrier sites accessible to atractylate quenching at the outer membrane face was simulated by a computer program based on the reorientating carrier model. With a set of rate constants, an approximate fit for the extent of quench delay with the experimental data is obtained.

Adenosine Diphosphate↗
PubMed1978

Interaction of an amine oxide detergent with lecithin vesicles as studied by nuclear magnetic resonance.

The interaction of an amine oxide detergent with single bilayer lecithin vesicles was investigated with proton and phosphorus magnetic resonance. The addition of the detergent micelles to vesicles suspensions leads to rapid detergent incorporation into the vesicle bilayer, resulting in a heterogenous vesicle population. Initially, some vesicles take up the equivalent of one detergent micelle, whereas others contain no detergent. Subsequently, the detergent is distributed between the vesicles by vesicle-vesicle collisions. This can be followed by the change in the Pr3+-shifted spectral positions of the detergent and lecithin head groups with time. From the intensity of the head-group signals, it can be concluded that after about 20 h the detergent is almost equally distributed between the outer and inner vesicle membrane monolayers. Vesicles obtained by cosonication of the detergent and lecithin take up metal ions. This ion permeability depends on the vesicle concentration and can be attributed to vesicle-vesicle or vesicle-mixed micelle collisions. Egg lecithin vesicles are stable against the detergent up to molar ratios of detergent to lecithin of 0.2--0.3. At larger ratios mixed micells and multibilayers are formed. Measurements of proton spin-lattice relaxation times confirmed that the internal architecture of the vesicle bilayer is almost unaffected by the incorporated detergent.

Detergents↗
PubMed1978

Isolation of the ADP/ATP translocator from beef heart mitochondria as the bongkrekate-protein complex.

1. The isolation of the ADP/ATP translocator from beef heart mitochondria as the bongkrekateprotein complex is described, using hydroxyapatite chromatography and gel filtration in Triton X-100 solution. 2. The inhibitor is bound to the protein prior to solubilization with detergent for protection against denaturation. Only the intact bongkrekate-protein passes easily through the hydroxyapatite column. Bongkrekate shileds the protein in contrast to carboxyatractylate only partially against proteinases present in the crude extract. 3. The isolated bongkrekate protein shows the same molecular weights in dodecylsulfate and Triton X-100, the same amino acid composition and the same isoelectric point as the earlier isolated carboxyatractylate-protein complex. It differs by its higher sensitivity against trypsin and thermolysin. 4. The identity of both proteins is demonstrated by interconversion of the bongkrekate-protein into the carboxyatractylate-protein. The process requires the catalysis by ADP or ATP, the natural substrates of the protein. 5. The formation of the extractable [3H]bongkrekate-protein complex in mitochondria requires the presence of ADP or ATP. 6. These data, the immunological studies presented earlier, and the differences in the reactivity of -SH groups of the isolated bongkrekate and carboxyatractylate complexes (to be published) indicate that both proteins represent different conformational states of the translocator protein (m-state and c-state).

Adenosine Diphosphate↗
PubMed1978

[Transport catalysis in biomembranes elucidated by the interactions of ADP, ATP-carriers in mitochondria].

A basic issue of biomembranes is their ability to facilitate specific transport of selected molecules. This transport is catalyzed by carriers which are membrane proteins and form, analogous to enzymes, carrier-substrate complexes. The ADP, ATP carrier of mitochondria is highly suitable for elucidating the mechanism of this catalysis due to its unique qualities such as great abundance in higher cells, easy isolation in native state by detergents, existence of inhibitors specific for either the in- or outward looking binding site and direct observation of a carrier-substrate complex. As central catalytic steps, the reorientation of the substrate-binding site at the carrier during translocation across the membrane could be demonstrated at the intact membrane and the isolated protein. The results are interpreted by the gated-pore mechanism where two subunits form a gate with a central binding site which radically change conformation and specificity on transition from one to the other side of the membrane.

Adenosine Diphosphate↗
PubMed1977

Isolation of the unliganded adenosine 5'-diphosphate, adenosine 5'-triphosphate carrier-linked binding protein and incorporation into the membranes of liposomes.

The ADP,ATP carrier-linked binding protein of beef heart mitochondria was isolated free of ligands, using the detergent 3-lauramido-N,N-dimethylpropylamine oxide. Unlike the preparation of the carboxyatractylate (CAT)-protein complex described earlier, this protein enables direct binding studies to be made with the inhibitor ligands. The protein was characterized with respect to its polypeptide composition, stability against degradation, and immunological properties; the identity of the binding protein with the previously isolated CAT-protein complex was thereby shown. As a step toward reconstitution studies, the isolated binding protein was incorporated into liposomes by a simple rapid mixing process. The complete insertion into the vesicular membrane was demonstrated by chromatography on Sepharose 6B and by immunoprecipitation reactions.

Adenosine Diphosphate↗
PubMed1977

Reconstitution of inhibitor binding properties of the isolated adenosine 5'-diphosphate,adenosine 5'-triphosphate carrier-linked binding protein.

We studied the binding of carboxyatractylate (CAT) and bongkrekate (BKA) to the solubilized ATP,ADP carrier-linked binding protein, which had been incorporated into liposomes. After solubilization with 3-lauramido-N,N-dimethylpropylamine oxide the binding protein had largely lost it affinity and binding capacity for both CAT and BKA. On incorporation into phospholipid vesicles, CAT binding was restored to a considerable extent (3.5 mumol of CAT/g of protein), reaching the original affinity as observed in mitochondria (Kd = 10(-8) M). With high amounts of CAT and under the influence of ADP the binding can be increased to 6.8 mumol of CAT/g of protein, indicating a movement of binding sites in the liposomal membrane. The binding of BKA was also reconstituted with high affinity (Kd = 8 X 10(-8) M) and to the same extent (6.4 mumol of BKA/g of protein). As in the case of intact mitochondria, this reconstituted binding depends on the presence of ADP. This dependence on ADP has an apparent Km = 7 muM, similar to the carrier affinity for ADP in intact mitochondria. The reorientation model of Klingenberg for the ADP,ATP carrier implicating an ADP-catalyzed transition between the CAT binding form (c state) and BKA binding form (m state) in the inner mitochondrial membrane has been confirmed in this reconstituted system.

Adenosine Diphosphate↗
PubMed1977

Relation between the gradient of the ATP/ADP ratio and the membrane potential across the mitochondrial membrane.

The relation between the intramitochondrial and extramitochondrial ratio ATP/ADP, the transmembrane potential and pH gradient is investigated in the present communication. For this purpose mitochondria are equilibrated with added [14C]ATP in the presence of substrate and oligomycin for eliminating phosphate transfer by ATPase. The membrane potential was measured by the distribution of 86Rb+ in the presence of valinomycin, the deltapH by the distribution of [14C]acetate. In the energized state by varying deltapsi between 60 and 160 mV, the internal (ATP/ADP)i is decreased 30-fold, the external (ATP/ADP)e remains largely constant. As a result, the deltalog (ATP/ADP)e/(ATP/ADP)i = deltalogphi is increased linerly with deltapsi according to the following relation: deltalogphi = 0.85 deltapsi - 0.35. The deltapH was changed between 0.1 and 0.8 by increasing the Pi concentration causing only a minor decrease of deltalogphi would be expected if the ATP-ADP exchange has a significant electroneutral portion. Also in the uncoupled and respiration-inhibited state the same function between deltalogphi and deltapsi is found as in the energized states. It is concluded that under these conditions the ATP-ADP exchange is largely electrical.

Adenosine Diphosphate↗