Search PubMed⌕ Search

Biomedical subjects

M Klingenberg

Publications and source records attributed to M Klingenberg.

At least 145 records · Page 8Linked to original sources
PubMed1976

The state of ADP or ATP fixed to the mitochondria by bongkrekate.

The reported studies are intended to clarify the binding state of ADP fixed to mitochondria under the influence of bongkrekate, and thus to discern between the affinity increase and reorientation mechanism proposed for the bongkrekate effect. (a) The composition of the intramitochondrial adenine nucleotide pool is not changed under the influence of bongkrekate with and without added nucleotides. (b) The added ADP and ATP fixed by bongkrekate can be identified as AMP, ADP and ATP in the same proportions as in the endogenous pool. (c) The bound nucleotides respond to oxidative phosphorylation or uncoupler stimulated dephosphorylation similar as endogenous nucleotides. It can be concluded that the ADP or ATP fixed under the influence of bongkrekate to the mitochondria are equilibrated with the intramitochondrial adenine nucleotide pool and are active in intramitochondrial phosphate transfer reactions. The results disagree with the affinity increase mechanism but support the reorientation mechanism which postulates that ADP and ATP are trapped in the mitochondria under the influence of bongkrekate in the same amount as there are carrier sites available outside before bongkrekate addition.

Adenosine Diphosphate↗
PubMed1976

Antibody evidence for different conformational states of ADP, ATP translocator protein isolated from mitochondria.

Consistent with the previously proposed reorientation mechanism for the ADP,ATP translocator protein of mitochondria, evidence has now been obtained for the existence of two distinct conformational states of the isolated translocator protein. Previous studies indicated that when the mitochondrial translocator protein is in the c-state(i.e., when its binding site faces the cytosol side) the protein binds primarily the ligand carboxyatractylate (CAT), and when the translocator protein is in the m-state(i.e., when its binding site faces the mitochondrial matrix) the translocator protein binds primarily bongkrekate. Direct evidence for this formulation has now come from the application of antibodies to the isolated translocator protein-ligand complex. Two antibodies were produced against the ADP,ATP translocator protein isolated from beef heart mitochondria. One antibody, which was produced against the protein isolated as the CAT-binding protein complex, was found to be highly specific for that complex and did not react with the protein in the conformation state conferred by the bongkrekate ligand. This antibody did not cover the CAT-binding site, as evidenced by the exchange of unlabeled CAT with [35S]CAT bound to the translocator protein. However, the same antibody inhibited a transition of the protein from the c-state to the m-state, as evidenced by an inhibition of the displacement of[35S]CAT by bongkrekate (added jointly with ADP). It appears, therefore, that the antibody immobilized the translocator protein in the c-state. The second antibody produced against the (somewhat less pure) ADP,ATP translocator protein, isolated as the bongkrekate-binding protein complex, did not react with the CAT-binding protein. Thus, the second antibody appeared to be specific for the translocator protein in the m-state. Neither antibody inhibited mitochondrial ADP,ATP transport.

Adenosine Diphosphate↗
PubMed1975

The binding of atractylate and carboxy-atractylate to mitochondria.

35S-labelled atractylate and carboxy-atractylate are produced biosynthetically and used for studying the binding of these specific ligands to the ADP, ATP carrier in beef heart mitochondria. The following results are obtained. 1. Inhibition of translocation activity goes parallel to the increase of binding by [35S]atractylate. No additional binding is observed after full inhibition of translocation is reached giving evidence that atractylate binds exclusively to the carrier. 2. The maximum number of binding sites of both atractylates is about 1.6 mumol/g protein in beef heart mitochondria and decreases on treatment of the membrane by Pi, freezing, ageing, etc. The dissociation constants of the binding are approximately for atractylate Kd = 5-10(-8) M and for carboxy-atractylate Kd = 10(-8) M. The mass action plots of the concentration dependence for the binding are nonlinear-convex in particular with carboxy-atractylate and more linear with atractylate. Nonlinearity appears to be caused by some retardation of equilibration in the case of very high affinity binding. 3. The binding of atractylate and carboxy-atractylate is relatively fast in intact mitochondria and slower in aged membranes. There is a slower and a faster binding portion. 4. The atractylates remove ADP in a nearly 1:1 stoichiometry from untreated mitochondria. In aged and Pi-treated membranes the ratio deltaADP/deltaatractylate approaches 0. Obviously binding of carrier sites to ADP is more sensitive to alterations than that of the atractylates. The assumption is maintained that the binding site for atractylate is identical with that for ADP and ATP. 5. Bongkrekate prevents binding of both atractylates. However, when added after, it only removes atractylate but not the carboxy compound because of its different tight binding. The removal of atractylate depends on the synergistic effect of bongkrekate with ADP. 6. The binding studies with [35S]atractylate and in particular the interaction with bongkrekate support the reorienting carrier model in which atractylate as an impermeable ligand fixes the binding site of the carrier outside while with bongkrekate the carrier site is turned to the inside.

Animals↗
PubMed1975

Energy transfer in mitochondrial synthesis of ATP; a survey.

The energy transduction in mitochondria, with its principal agent ATP, still represents a major challenge for biological research. In general, the energy transduction process is divided into three sections: (1) the redox processes; (2) a conservation of intermediary energy forms; (3) synthesis of ATP. All three processes are linked to the membrane and are, therefore, as difficult to resolve as are processes linked to other biomembranes. It is probable that the electron transport system is constructed in such a way as to provide energy for synthesis of ATP and related processes. Important for this function is the transversal distribution of these components across the membrane, facilitating generation of membrane potential by electron or proton transfer. The exact composition of the respiratory chain is not yet known, in particular with respect to iron-sulphur proteins. Progress is achieved by defining single species of the respiratory chain, subunit composition, amino acid sequences and genetic derivation from intra- or extra-mitochondrial translation. Energy generated by oxidation can be trapped before ATP is formed by a number of reactions, in particular reversed electron transport, energy-dependent transhydrogenation and uptake of anions or cations into the mitochondria. The latter reaction is of major importance for understanding the intermediate energy form, as it appears to use energy most directly and be driven mainly by membrane potential or proton gradient across the membrane. The formation of ATP is a major problem hindering elucidation of the mechanism of oxidative phosphorylation. The mechanism of this enzymic process is not yet understood although the enzymes have been isolated and the subunits have been defined. Most probably, a concerted reaction between ADP and phosphate, driven by some conformational transition of the complex, leads to the formation of ATP. Release of ATP from a hydrophobic to hydrophilic environment may consume most of the energy.

Adenosine Triphosphate↗
PubMed1975

Energetic aspects of transport of ADP and ATP through the mitochondrial membrane.

Evidence from various sources demonstrates that the release of ATP in exchange for the entry of ADP across the mitochondrial membrane is an active process requiring energy. The necessary energy may be derived from the same source of energy as that used for oxidative phosphorylation. The following results will be discussed:--(1) The exchange is asymmetric with respect to the specificity of ADP and ATP in 'energized' mitochondria. From the outside ADP is much preferred to ATP, but from the inside both exchange with equal specificity. This asymmetry is abolished by de-energization of the membrane. (2) The ADP-ATP exchange is about 50% electrogenic: about half the ATP released against ADP is protonated. The excess of negatively charged ATP might prevent ADP from entering mitochondria against a membrane potential. (3) The ratio of ATP to ADP across the inner mitochondrial membrane is higher outside than inside only in the energized state. Variation of the ATP/ADP ratio maintains this difference. (4) The ADP/ATP ratio apparently varies with changes in the membrane potential as measured by Rb+-distribution. The correlation factor between deltaE changes and this ratio is 0.5, in agreement with predictions from proton-stoichiometry measurements. The deltapH does not significantly change the distribution ratio. (5) By following the P/O ratio, one can show that energy derived from expelling ATP against the ADP/ATP gradient lowers the amount of ATP synthesis. The P/O ratio is lowered as the imbalance of ATP to ADP increases. (6) The energy difference of the phosphorylation potential of ATP is calculated by various methods to be about 8-12 kJ, depending on the conditions. This free energy is the result of the 'active' transport which corresponds to the release of ATP outside the mitochondria. (7) In vivo studies on the distribution of ADP and ATP inside and outside the mitochondria in liver show a corresponding ratio difference of about 15 as predicted from the in vitro studies.

Adenosine Diphosphate↗