K. Van Dam

The interaction between the mitochondrial ATPase (F1) and the ATPase inhibitor

1. The naturally occurring mitochondrial ATPase inhibitor inhibits the mitochondrial ATPase (F1) non-competitively. 2. The interaction between inhibitor and inhibitor-depleted F1 or submitochondrial particles is diminished when the ratio of ATP/ADP is low or when energy is generated by substrate oxidation. 3. The dissociation of the inhibitor from coupled Mg-ATP particles is promoted when substrates are being oxidized. This results in the appearance of a large uncoupler-stimulated ATPase activity. Activation of the uncoupler-stimulated ATPase activity is also achieved by incubation of the particles with ADP. 4. The ATPase activity of Mg-ATP particles is determined by the turnover capacity of F1. When endogenous inhibitor is removed, energy dissipation becomes the rate-limiting step. This energy dissipation can be activated by an uncoupler. 5. Evidence is presented for the existence of a non-inhibited intermediate F1-inhibitor complex.

Linear relation between rate and thermodynamic force in enzyme-catalyzed reactions

Starting from enzyme kinetics, it is shown that generally a linear rather than a proportional relationship exists between rate and free energy changes in biochemical processes. In the derivation the boundary condition of constant substrate plus product is used, which is appropriate for many cellular systems. An example is the ADP plus ATP concentration is mitochondrial oxidative phosphorylation, as is illustrated experimentally.

Relationship between chemiosmotic flows and thermodynamic forces in oxidative phosphorylation

A set of equations has been derived, describing quantitatively the relationships between flows and thermodynamic forces in the chemiosmotic model of oxidative phosphorylation. Experimental tests of these equations give information on the stoichiometric coupling constants between the different flows.

Synchrony and mutual stimulation of yeast cells during fast glycolytic oscillations

Cell synchrony was investigated during glycolytic oscillations in starved yeast cell suspensions at cell densities ranging from 2 × 106-5 × 107cells ml-1. Oscillations in NAD(P)H were triggered by inhibition of mitochondrial respiration when intracellular NAD(P)H had reached a steady state after glucose addition. Before macroscopic damping of the oscillations, individual yeast cells oscillated in phase with the cell population. After oscillations had damped out macroscopically, a significant fraction of the cells still exhibited oscillatory dynamics, slightly out-of-phase. At cell concentrations higher than 107cells ml-1the dependence upon cell-density of (i) the damping of glycolytic oscillations and (ii) the amplitude per cell suggested that cell-to-cell interaction occurred. Most importantly, at cell densities exceeding 107cells ml-1the damping was much weaker. A combination of modelling studies and experimental analysis of the kinetics of damping of oscillations and their amplitude, with and without added ethanol, pyruvate or acetaldehyde, suggested that the autonomous glycolytic oscillations of the yeast cells depend upon the balance between oxidative and reductive (ethanol catabolism) fluxes of NADH, which is affected by the extracellular concentration of ethanol. Based on the facts that cell (i) excrete ethanol, (ii) are able to catabolize external ethanol, and (iii) that this catabolism affects their tendency to oscillate, we suggest that the dependence of the oscillations on cell density is mediated through the concentration of ethanol in the medium.

The equilibrium between the mitochondrial ATPase (F1) and its natural inhibitor in submitochondrial particles.

Abstract 1. The reversible equilibrium between the mitochondrial ATPase (F 1 ) and its naturally occurring inhibitor in Mg-ATP submitochondrial particles has been studied under different conditions. 2. High ionic strength favours dissociation of the ATPase inhibitor as tested by ATPase and ATP-driven transhydrogenase activities. 3. Dissociation of the ATPase inhibitor results in an increased maximal velocity of the ATPase activity measured in the presence of uncoupler and an increased affinity for adenine nucleotides, in particular for ATP. 4. Association of the ATPase inhibitor with inhibitor-depleted Mg-ATP particles causes a slowing of the initial rate of succinate oxidation. 5. The antibiotic aurovertin stimulates the ATPase activity of Mg-ATP particles preinculbated in the presence of a supply of oxidative energy. Bound aurovertin impedes the association of inhibitor-deficient particles with ATPase inhibitor. 6. The fluorescence of aurovertin bound to inhibitor-containing particles is much less than that of aurovertin bound to inhibitor-depleted particles. 7. The oligomycin-sensitivity-conferring protein, added either alone or in the presence or absence of membranous components of the ATPase complex, has little or no effect on the fluorescence of the F 1 -aurovertin complex. 8. It is suggested that the ATPase inhibitor brings F 1 in a conformation denoted ∗F 1 that binds aurovertin with a low quantum yield, a decreased affinity and an increased binding capacity.

A comparison between the effectiveness of uncouplers of oxidative phosphorylation in mitochondria and in different artificial membrane systems.

Abstract The concentration range in which uncouplers of oxidative phosphorylation stimulate the respiration of rat-liver mitochondria in State 4 was compared with that in which those compounds stimulate fluxes through black lipid membranes or liposomal membranes. A poor correlation exists between the effectiveness of uncouplers in mitochondria and in black lipid membranes. In contrast, a good correlation exists between uncoupling activity in mitochondria and stimulation of valinomycin-induced swelling of liposomes or stimulation of reduction by ascorbate/ferrocene of ferricyanide included in liposomes.

Reconstitution of bacteriorhodopsin in a millipore filter system.

At present it is generally accepted that bacteriorhodopsin, which is the only protein present in purple membranes of Halobacteria [ 1 ] , functions as a lightdriven proton-pump [2] . The conversion of lightenergy into chemiosmotic energy by this protein has been also demonstrated in model membrane systems, e.g., in liposome systems where bacteriorhodopsin can be incorporated either by sonication procedures [3,4] or by methods which employ detergents [5,6]. Incorporation of bacteriorhodopsin into liposomes together with a second protein, e.g., mitochondrial or bacterial adenosine triphosphatase complex [5,7] or cytochrome c oxidase [8], yielded vesicles in which the reaction, catalysed by the second protein, could be influenced by illuminating the vesicle suspension. Since dispersions of purple membranes are rather stable in decane solution, bacteriorhodopsin was easily reconstituted in black-film systems [9-121. An improved method has been put forward by the group of Skulachev, who developed the so-called planar membrane system. Bacteriorhodopsin-contalning vesicles can be associated with this thick membrane in the presence of Ca’+-ions [ 1 l-l 31 . In the present paper experiments are described using a related system, in which the planar membrane has been replaced by lipid-impregnated Millipore filters. The two big advantages of this system are its high stability and the possibility to increase drastically the membrane area.

Hexokinase regulates kinetics of glucose transport and expression of genes encoding hexose transporters in Saccharomyces cerevisiae

Glucose transport kinetics and mRNA levels of different glucose transporters were determined in Saccharomyces cerevisiae strains expressing different sugar kinases. During exponential growth on glucose, a hxk2 null strain exhibited high-affinity hexose transport associated with an elevated transcription of the genes HXT2 and HXT7, encoding high-affinity transporters, and a diminished expression of the HXT1 and HXT3 genes, encoding low-affinity transporters. Deletion of HXT7 revealed that the high-affinity component is mostly due to HXT7; however, a previously unidentified very-high-affinity component (K(m) = 0.19 mM) appeared to be due to other factors. Expression of genes encoding hexokinases from Schizosaccharomyces pombe or Yarrowia lipolytica in a hxk1 hxk2 glk1 strain prevented derepression of the high-affinity transport system at high concentrations of glucose.

Accumulation of substrates by mitochondria

Abstract 1. Substrates are accumulated in the matrix space of rat-liver mitochondria under conditions where no energy can be generated. 2. The accumulation shows saturation characteristics: a plot of the inverse of the external versus the inverse of the intramitochondrial concentration gives a straight line. 3. The maximal uptake (at infinite external substrate concentration) is finite and approximately equivalent to the intramitochondrial K + concentration. 4. Substrates inhibit competitively the uptake of other substrates. 5. The uptake of all substrates is inhibited competitively by 2,4-dinitrophenol.

Interaction between uncouplers and substrates in rat-liver mitochondria

Abstract 1. 1. The inhibition of the dinitrophenol-stimulated ATPase in rat-liver mitochondria by excess uncoupler is kinetically competitive with respect to ATP. 2. 2. Competitive inhibition of substrate oxidation by excess uncoupler is most marked when substrate and uncoupler have a net charge of the same sign. 3. 3. In the absence of an energy-generating system dinitrophenol is accumulated by mitochondria like other acidic compounds. 4. 4. Dinitrophenol accumulation is competitively inhibited by anionic compounds and not affected by cationic compounds. 5. 5. It is proposed that an acidic uncoupler enters the mitochondrion in exchange for endogenous anions and that the uncoupler can leave the mitochondrion as the uncharged acid. In the presence of an energy-generating system, the endogenous anions are possibly hydroxyl ions. These movements, together with an intramitochondrial energy-dependent splitting of water, can explain both the uncoupling and inhibitory action of uncouplers as well as the importance of lipid solubility of anionic uncouplers.