PubMed Health⌕ Search

Biomedical subjects

R Bohnensack

Publications and source records attributed to R Bohnensack.

At least 55 records · Page 3Linked to original sources
PubMed1984

Rate law of mitochondrial respiration versus extramitochondrial ATP/ADP ratio.

Rate equations describing the dependence of mitochondrial respiration or ATP formation on the extramitochondrial ATP/ADP ratio were derived from a simplified reaction scheme of oxidative phosphorylation. The equations include maximum velocities of (1) respiration, (2) ATP formation, and (3) other energy utilizing side reactions; apparent Michaelis constants for ATP and ADP; an apparent cooperativity coefficient; and in addition an apparent equilibrium constant which is equal to the ATP/ADP ratio at zero net formation of ATP in the resting state of mitochondria. The control characteristics described by the rate equation are in close agreement with curves that can be obtained from a much more complex mathematical model of phosphorylating mitochondria. The kinetic parameters of the equation can be estimated from experimental data by a nonlinear regression procedure. The results demonstrate a weak apparent cooperativity and a strong shift of the apparent affinity ratio for ATP and ADP towards ADP.

Adenosine Diphosphate↗
PubMed1983

Control of mitochondrial respiration. The contribution of the adenine nucleotide translocator depends on the ATP- and ADP-consuming enzymes.

The consequence of the complexity of the metabolic network on the amount of control strength of adenine nucleotide translocator was investigated with isolated rat liver mitochondria. Two experimental systems were compared: (i) mitochondria in the presence of yeast hexokinase (hexokinase system) and (ii) the same system plus additional pyruvate kinase (pyruvate kinase system). In both systems the control strength was analysed for the adenine nucleotide translocator by inhibitor titration studies with carboxyatractyloside and for the hexokinase or pyruvate kinase by changing their relative activities. Experimental results were compared with computer simulation of these systems and that of a third one, where the extramitochondrial ATP/ADP ratio was held constant by perifusion (perifusion system). The results demonstrate quite different flux-dependent control strength of the translocator in the three systems. In the hexokinase system the control strength of the translocator on mitochondrial respiration was zero up to respiration rates of about 60 nmol O2/mg protein per min. For higher rates, the control strength increased until the maximum value (0.45) was reached in the fully active state. Here, the same value was also found in the pyruvate kinase system. In all other states of respiration the translocator exerts a higher control strength in the pyruvate kinase system than in the hexokinase system. This different behaviour was attributed to the various changes in the adenine nucleotide pattern caused by partial inhibition of the translocator in the hexokinase and pyruvate kinase system. The data clearly show that the sharing of control strength depends not only on the respiration rate but also on the complexity of the metabolic system.

Adenosine Diphosphate↗
PubMed1983

Control of mitochondrial respiration.

The control theory of Kacser and Burns [in: Rate Control of Biological Processes (Davies, D.D. ed) pp. 65-104, Cambridge University Press, London, 1973] and Heinrich and Rapoport [Eur. J. Biochem. (1974) 42, 97-105] has been used to quantify the amount of control exerted by different steps on mitochondrial oxidative phosphorylation in rat-liver mitochondria. Inhibitors were used to manipulate the amount of active enzyme. The control strength of the adenine nucleotide translocator was measured by carrying out titrations with carboxyatractyloside. In state 4, the control strength of the translocator was found to be zero. As the rate of respiration was increased by adding hexokinase, the control strength of the translocator increased to a maximum value of approximately 30% at approximately 80% of state 3 respiration. In state 3, control of respiration is distributed between a number of steps, including the adenine nucleotide translocator, the dicarboxylate carrier and cytochrome c oxidase. The measured values for the distribution of control agree very well with those calculated with the aid of a model for mitochondrial oxidative phosphorylation developed by Bohnensack et al. [Biochim. Biophys. Acta (1982) 680, 271-280].

Adenosine Diphosphate↗
PubMed1983

Influence of the beta-hydroxybutyrate/acetoacetate ratio on the redox states of mitochondrial NAD(P) and cytochrome c systems, extramitochondrial ATP/ADP ratio and the respiration of isolated liver mitochondria in the resting state.

The effect of changes in hydrogen supply on the rate of energy transformation (measured as respiration rate), on the redox state of the NAD(P) and cytochrome c couples and on the extramitochondrial ATP/ADP was investigated with isolated rat liver mitochondria. Hydrogen supply was changed by variation of the beta-hydroxybutyrate/acetoacetate ratio in the incubation medium. In the active state respiration rate was strongly diminished by lowering the beta-hydroxybutyrate/acetoacetate ratio, whereas in the resting state a minor effect was observed. This resulted in a decrease in the respiratory control index (RCI). Even under a metabolic situation of strongly diminished hydrogen supply corresponding to a beta-hydroxybutyrate/acetoacetate ratio equal to 0.5 added ADP was phosphorylated. However, the ATP/ADP ratio generated extramitochondrially amounted to half that obtained at a beta-hydroxybutyrate/acetoacetate ratio of more than 100. Nevertheless at a beta-hydroxybutyrate/acetoacetate ratio of 0.5 the generated extramitochondrial ATP/ADP ratio is near to 100. A comparison of the changes in the available free redox energy with those in the extramitochondrial phosphorylation potential points to a disequilibrium between them in the resting state, too. The experimental results are discussed in the light of the metabolic conditions in vivo.

3-Hydroxybutyric Acid↗
PubMed1982

Rate-controlling steps of oxidative phosphorylation in rat liver mitochondria. A synoptic approach of model and experiment.

The contribution of different steps to the control of oxidative phosphorylation in isolated rat liver mitochondria was investigated by a combination of experiments and computer simulations. The parameters of the mathematical model of phosphorylating mitochondria were derived from experimental data. The model correctly described the competition between ATP utilization inside and outside mitochondria for the ATP generated in mitochondria. On the basis of the good agreement between experiments and simulations, the contribution of different steps to the control of respiration was estimated by computing their control strengths, i.e., the influence of their activities on the rate of respiration. The rate-controlling influences vary depending on the load of oxidative phosphorylation. The predominant steps are: in the fully active state (State 3)--the hydrogen supply to the respiratory chain; in the resting state (State 4)--the proton leak of the mitochondrial inner membrane; in states of non-maximum ATP export--the adenine nucleotide translocator. Titrations of respiration with phenylsuccinate, antimycin, oligomycin and carboxyatractyloside completely support these conclusions.

Adenosine Diphosphate↗
PubMed1982

The role of the adenine nucleotide translocator in oxidative phosphorylation. A theoretical investigation on the basis of a comprehensive rate law of the translocator.

A minimum model of adenine nucleotide exchange through the inner membrane of mitochondria is presented. The model is based on a sequential mechanism, which presumes ternary complexes formed by binding of metabolites from both sides of the membrane. The model explains the asymmetric kinetics of ADP-ATP exchange as a consequence of its electrogenic character. In energized mitochondria, a part of the membrane potential suppresses the binding of extramitochondrial ATP in competition with ADP. The remaining part of the potential difference inhibits the back exchange of internal ADP for external ATP. The assumption of particular energy-dependent conformational states of the translocator is not necessary. The model is not only compatible with the kinetic properties reported in the literature about the adenine nucleotide exchange, but it also correctly describes the response of mitochondrial respiration to the extramitochondrial ATP/ADP ratio under different conditions. The model computations reveal that the translocation step requires some loss of free energy as driving force. The size of the driving force depends depends on the flux rate as well as on the extra- and intramitochondrial ATP/ADP quotients. By both quotients the translocator control the export of ATP formed by oxidative phosphorylation in mitochondria.

Adenosine Diphosphate↗
PubMed1982

Interrelationships between hydrogen-supplying reactions, respiration rate and extramitochondrial adenine nucleotide pattern.

1. The influence of a diminished hydrogen supply on the regulation of oxidative phosphorylation of isolated rat liver mitochondria in dependence on the extramitochondrial (ATP)/(ADP) ratio was investigated. 2. The hydrogen supply was diminished by using various (beta-hydroxybutyrate)/(acetoacetate) ratios as a redox buffer and the results were compared with those of experiments using perifusion of immobilized mitochondria with non-saturating substrate concentrations. 3. In both experimental approaches the influence of a diminished hydrogen pressure on the maximum (ATP)/(ADP) ratio at minimum flux was low. An extreme decrease in the (beta-hydroxybutyrate)/(acetoacetate) ratio by more than two orders of magnetitude causes the (APT)/(ADP) ratio to decrease by about 50%. 4. The load capacity of oxidative phosphorylation (maximum flux) is considerably decreased by diminished hydrogen pressure. 5. The borderline cases of purely kinetic and thermodynamic limitations of hydrogen supply were calculated by computer simulation with respect to the regulating behaviour of oxidative phosphorylation and changes in the control strength of adenine nucleotide translocator and hydrogen supply in the overall reaction. 6. A prevalent thermodynamic influence of hydrogen supply on oxidative energy transformation in the cell is discussed in the light of experimental data.

3-Hydroxybutyric Acid↗
PubMed1982

Interrelationship between oxidative energy transformation and energy consumption at mitochondrial and cellular levels.

The adaptation of oxidative energy transformation in mitochondria to the energy demand of cellular metabolism was investigated in experiments with isolated mitochondria and liver cells and by computer simulation in terms of a mathematical model. Separate draining of different energy pools allowed the determination of the relation between these pools and the elucidation of the importance of the connecting enzyme reactions to the regulation of the whole process. The following conclusions can be drawn from the results: 1. The intramitochondrial adenine nucleotide pool exhibits a homogeneous behaviour, and its changes are the signal for ATP synthesis. 2. The proton-motive force which is in near-equilibrium with the intramitochondrial phosphorylation potential is the immediate signal for the respiratory chain. 3. The intramitochondrial phosphorylation potential is transformed into the external one by a flux-dependent non-equilibrium reaction of the translocator. 4. The rate of respiration-linked ATP formation is regulated by more than one reaction step with varying control strength. 5. In both isolated mitochondria and hepatocytes an activation of respiration is provoked by a decrease in the mitochondrial energy state caused by cellular energy utilization.

Adenosine Triphosphate↗
PubMed1982

A quick test for the simultaneous determination of intactness and concentration of spermatozoa.

The fluorescence of ethidium bromide bound to the DNA of bull spermatozoa can be used for the estimation of intactness and concentration of spermatozoa of washed sperm samples. For the full permeability of the spermatozoal membrane for ethidium bromide digitonin is used in a concentration of 50 micrograms/ml. The fluorescence signals before and after digitonin treatment reflect the portion of cells with intact cellular membrane in the sample. The signal after addition of digitonin correlates to a high degree with the sperm concentration. The method exhibits a sufficient accuracy for the estimation of sperm quality. That applies for the reproducibility with individual ejaculates as well as for the correlation to other methods determining the intactness and sperm concentration. This simple single-step technique requires only about 2 X 10(7) spermatozoa corresponding to 10 to 20 microliters of an average bull ejaculate.

Animals↗
PubMed1982

[Effect of temperature decrease on the intactness of the cell membrane of ejaculated bull spermatozoa].

Respiration of washed ejaculated bull spermatozoa with exogenous succinate is drastically increased after a sudden cooling of spermatozoa (cold shock). Loss of intactness of the cellular membrane occurs in a small temperature range between about 16 degrees C and 8 degrees C. A low cooling rate in critical temperature range connected with a sufficient equilibration time at this temperatures strongly restricts the damage of the spermatozoal membrane.

Animals↗
PubMed1981

Control of energy transformation of mitochondria. Analysis by a quantitative model.

A mathematical model of control of energy transformation in mitochondria is presented. The considered processes are: the proton translocation by the respiratory chain, the production of ATP by ATPase, the translocation of adenine nucleotides and of phosphate by their translocators, and a passive backflow of protons through the mitochondrial membrane. The mathematical equations expressing the steady-state kinetics of these processes and the relations between them were derived on the basis of current experimental data. The model predicts fairly well the values of the proton electrochemical gradient, of the ATP/ADP ratios within and outside mitochondria and of the distribution of phosphate between both compartments in different metabolic states of mitochondria. From the general agreement of model computations with experimental data, it is suggested that the electron flux through the respiratory chain is immediately controlled by the energy back-pressure of the proton electrochemical gradient, that the ATPase reaction is near equilibrium in phosphorylating mitochondria but that the adenine nucleotide exchange across the mitochondrial membrane requires some loss of energy. The latter is caused by an inhibition of the translocator by ATP from the outer side or by ADP from the inner side depending on the actual ATP/ADP in both compartments. It explains that no fixed relation exists between the rate of respiration and the phosphorylation state of extramitochondrial adenine nucleotides. The relation is modified by the concentration of phosphate and by intramitochondrial energy utilization.

Adenine Nucleotides↗
PubMed1980

[Relation between intactness and adenine nucleotide pattern of ejaculated bull spermatozoa].

The intactness of bull spermatozoa was determined by exclusion of external succinate. This was oxygraphically measured in the presence of cytochrome c after inhibition of the cell respiration with rotenone. An addition of cytochrome c was necessary for a reproducible respiration of damaged cells. The portion of intact cells determined in this way correlated with that obtained by the fluorescent dye primuline. The content of adenine nucleotides and the percentage of ATP of washed fresh spermatozoa depended on the intactness of the sperm sample. The adenine nucleotide pattern of spermatozoa after deep freezing corresponded to that of washed fresh ejaculates with a high percentage of damaged cells. The major part of ejaculates tested showed only a small increase of damaged cells during substrate-free incubations at 38 degrees C up to 4 h under aerobic and anaerobic conditions. Ejaculates which had shown a drastic increase of damaged cells were not recognizable by the initial portion of damaged cells. Also a lowered content of ATP in the cells was no indicator of an increased fragility of the cell membrane. A remarkable loss of total adenine nucleotides was observed under conditions of cold shock. It indicates that the changes in the sperm membrane induced by cold shock were qualitatively different in comparison with changes caused by storage and by a slow decrease in temperature.

Adenine Nucleotides↗
PubMed1979

The stimulation of the mitochondrial respiration by citrulline synthesis.

1. The influence of ammonia and ornithine on the oxygen uptake and the formation of citrulline was investigated with isolated rat liver mitochondria. The experiments were performed in a cytosol-like saline medium at 38 degrees C. 2. Under these conditions an increase of the respiration rate by ammonia and ornithine was observed, but a small response to external ADP, only. The missing stimulation by ADP was due to a partial inhibition of the respiratory chain by traces of zinc (approximately 1 microM) present in the medium. This inhibition was only detected at low concentrations of mitochondria. 3. For activation of respiration by ammonia plus ornithine two different processes were responsible: (i) chelation of the inhibiting zinc by ornithine, which could be prevented by EDTA; (ii) ADP production in the matrix space during formation of carbamoyl phosphate, which could be prevented by oligomycin but not by carboxyatractyloside. 4. This stimulus of the carbamoyl phosphate formation and of the equivalent citrulline synthesis on the mitochondrial respiration ran to 12% of that increase caused by phosphorylation of external ADP. The maximum rate of citrulline formation was limited by the activity of carbamoyl phosphate synthetase. 5. Added ADP suppresses the production of citrulline probably by the exchange of extramitochondrial ADP versus intramitochondrial ATP. The data suggest a common adenine nucleotide pool delivering ATP to the adenine nucleotide translocase as well as to the carbamoyl phosphate synthetase.

Animals↗
PubMed1978

Mathematical model of regulation of oxidative phosphorylation in intact mitochondria.

1. A mathematical model of the regulation of mitochondrial ATP synthesis by the extramitochondrial ATP/ADP ratio is presented taking into account the transport processes of phosphate and of adenine nucleotides by their specific translocators. 2. In agreement with known experimental data the model describes the control of respiration by the extramitochondrial ATP/ADP ratio as well as the distribution of adenine nucleotides and of inorganic phosphate between the extramitochondrial and the intramitochondrial compartment. 3. In the extramitochondrial compartment the phosphorylation potential is predicted by the model to be higher than in the matrix space. 4. Despite the differences in the phosphorylation potentials no particular translocation energy is necessary. This has been achieved by postulating a charge compensation between the movement of adenine nucleotides and the uptake of phosphate during ATP synthesis. 5. The proton stoichiometry of the proton pump must be higher than stated by the chemiosmotic coupling hypothesis in its present form, otherwise sufficient results could not be obtained. 6. With increasing activities of non-phosphorylating energy requiring side reactions (as uncoupling) at first the difference of the respiration rates between the phosphorylating and the non-phosphorylating state disappears, at higher activities the ADP phosphorylation stops, but the membrane potential collapses at very high activities only.

Adenosine Diphosphate↗
PubMed1976

Control of oxidative phosphorylation by the extra-mitochondrial ATP/ADP ratio.

The control of mitochondrial ATP synthesis by the extramitochondrial adenine nucleotide pattern was investigated with rat liver mitochondria. It is demonstrated that any stationary state between the two limit states of maximum activity (state 3) and of resting activity (state 4) can be obtained by a hexokinase-glucose trap as an ADP-regenerating system. These intermediate states are characterized by stationary respiratory rates, stationary redox levels of the cytochromes b and c and stationary levels of extramitochondrial ATP and ADP between the rates and levels of the limit states. At a constant concentration of inorganic phosphate the activity of mitochondria between the limit states is controlled by the extramitochondrial ATP/ADP ratio independent of the total concentration of adenine nucleotides present. The control range was found to be between ratios of about 5 and 100 at 10 mM phosphate. At lower ratios the mitochondria are in their maximum phosphorylating state. With succinate+rotenone and glutamate+malate the same control range was observed, indicating that it is independent of the nature of substrate oxidized. The results suggest that in the control range the mitochondrial activity is limited by the competition of ADP and ATP for the adenine nucleotide translocator.

Adenosine Diphosphate↗