Peter J. A. M. Plomp

On the origin of the limited control of mitochondrial respiration by the adenine nucleotide translocator

A thermodynamic control theory previously developed has been applied to mitochondrial oxidative phosphorylation with emphasis on the role of delta microH and coupling and within the paradigm of delocalized chemiosmotic coupling. The basis for the observed distribution of flux control over the participating enzymes is shown to lie in the relative magnitudes of so-called delta microH elasticity coefficients, i.e., the delta microH dependencies of the different mitochondrial processes. In particular the relatively strong delta microH dependence of mitochondrial respiration is responsible for the significant role of the adenine nucleotide translocator in the control of oxidative phosphorylation. Uncoupling decreases the control exerted by this translocator on respiration but increases that exerted on phosphorylation.

Control of proteolysis in perifused rat hepatocytes

The mechanism by means of which amino acids inhibit intrahepatic protein degradation has been studied in perifused rat hepatocytes. Proteolysis was extremely sensitive to inhibition by low concentrations of amino acids. A mixture of 0.5 mM leucine and 1–2 mM alanine, concentrations found in the portal vein of the rat after feeding, inhibited proteolysis to the same extent as a complete physiological mixture of amino acids. Inhibition by these two amino acids was accompanied by a rise in the intracellular concentrations of glutamate and aspartate, and was largely prevented by addition of glucagon, by addition of the transaminase inhibitor aminooxyacetate, or by omission of K+. Acceleration of proteolysis by K+ depletion was accompanied by a fall in intracellular glutamate caused by an increased rate of transport of this amino acid to the extracellular fluid. It is concluded that intracellular leucine, glutamate and aspartate are important elements in the control of hepatic protein degradation.

Mechanism of the stimulation of respiration by fatty acids in rat liver

The mechanism of stimulation of hepatic respiration by fatty acids was studied in isolated rat hepatocytes. Stimulation of respiration by fatty acids varied from about 35% to about 105% depending on chain length. The stimulatory effect of octanoate (1 mM) or oleate (0.5 mM) was prevented by oligomycin (2 ). With carboxyatractyloside (100 μM) and ouabain (2 mM) the stimulation of respiration was partially inhibited (by 50–70 and 50–60%, respectively). From these results it can be concluded that the increased rate of respiration after addition of fatty acids is coupled to ATP synthesis. A large part (50–60%) of this ATP is utilized by the (Na+ + K+)‐ATPase

Hepatic autophagy and intracellular ATP. A morphometric study.

In order to estimate the sensitivity of macroautophagy in liver toward changes in ATP we have analyzed the volume density of the autophagic/lysosomal system in isolated rat hepatocytes, incubated under conditions where intracellular ATP was partially depleted. (a) It appeared that reduction of the intracellular ATP concentration by 30-50% decreased the volume density of autophagic vacuoles by 70%. (b) Partial ATP depletion did not involve significant changes in the volume density of dense bodies. Together with studies showing that the rate of overall proteolysis via macroautophagy decreases with decreasing ATP concentration (P.J.A.M. Plomp, E.J. Wolvetang, A.K. Groen, A.J. Meijer, P.B. Gordon, and P.O. Seglen (1987) Eur. J. Biochem. 164, 197-203) our data indicate that changes in intracellular ATP primarily affect early steps in the autophagic/proteolytic pathway.

Thermodynamics of the control of metabolism

A theory is presented, describing the control analysis of metabolic systems in terms of Gibbs free energies, extending earlier work of Kacser and Burns (25), and Heinrich and Rapoport (29). It is shown that relationships exist between flux control coefficients (the degree to which enzymes control steady-state fluxes) and free-energy elasticity coefficients, defined as the fractional change in the rate of a reaction induced by a standard change in one free-energy difference, while all the other free-energy differences are kept constant.Application of this extended control analysis to some biochemical reactions, including proton translocation, demonstrates that1.Problems arising in the control analysis because of conservation (sum concentration of substrate and product constant) can be circumvented.2.Although free-energy elasticity coefficients are maximal when the reaction is close to equilibrium, they can also be significant when the reaction is not close to equilibrium.3.Problems in the control analysis caused by compartmentation can be resolved by defining control parameters that refer to the organelle as a whole.4.These latter control parameters obey the above-mentioned relationships.

Cytochemical and morphometric analysis of autophagy in energy depleted rat hepatocytes

The energy dependence of lysosomal enzyme acquisition by autophagosomes was studied in isolated rat hepatocytes by ultrastructural analysis for acid phosphatase activity. Reduction of the intracellular ATP content by addition of atractyloside or fructose decreased the flux through the autophagic proteolytic pathway to a similar extent (40-50%). Unexpectedly, in the presence of atractyloside the volume density of autophagosomes was reduced by 65%, whereas in the presence of fructose this reduction was only 20%. The volume density of lysosomes was not significantly affected by either of the two compounds. It is concluded that partial ATP depletion by fructose not only inhibits sequestration of cytoplasmic material in autophagosomes, but also affects the fusion between autophagosomes and lysosomes. Since fructose, in contrast to atractyloside, does not affect the cytosolic phosphate potential, it is proposed that autophagic sequestration is more sensitive to changes in the cytosolic phosphate potential whereas the fusion between autophagosomes and lysosomes is more responsive to changes in the ATP concentration.

Regulation of Mitochondrial Respiration in Liver

In studies on the control of mitochondrial respiration carried out in the past 10 years, particular attention has been focussed on cytochrome c oxidase and the adenine nucleotide translocator as rate-controlling steps. On the basis of the observation that the first two phosphorylation sites of the respiratory chain are in near equilibrium with the extramitochondrial phosphate potential, Wilson and coworkers1–5 have concluded that regulation of respiration occurs at the cytochrome c oxidase step, the effective rate of this reaction being dependent on the extramitochondrial phosphate potential. According to this model, the adenine nucleotide translocator does not exert significant control on respiration. In contrast, Davis and coworkers6–8, Kunz and coworkers9–11, Lemasters and Sowers12, and our own group13, 14 have concluded that the adenine nucleotide trans-locator is a rate-controlling step for mitochondrial oxidative phosphorylation. The uncertainty about the precise role of the adenine nucleotide translocator and of the cytochrome c oxidase step in controlling respiration is due to the difficulty of quantifying the contribution of various steps to control of a metabolic pathway such as mitochondrial oxidative phosphorylation. Kacser and Burns15–17 and Heinrich and Rapoport18–20 have developed a theoretical framework for quantifying the amount of control that a particular step in a metabolic pathway exerts on flux through that pathway.