Paired moving charge model of energy coupling. III. Intrinsic ionophores in energy coupling systems.

The experimental basis for the postulated role of intrinsic ionophores in mitochondrial ion transport and energy coupling is summarized. Intrinsic ionophores appear to be linked to, or contained within, specific ionophoroproteins localized in the inner membrane, and the isolation of these ionophores requires their release from the ionophoroproteins. At least ten different species of ionophores have been isolated from the mitochondrion, five of which have been wholly or in part chemically identified. Intrinsic ionophores have been implicated in the activation of inorganic phosphate in ATP synthesis and hydrolysis, and in the contol of the coupling modes. The presence of ionophores in soluble proteins such as troponin and in ATP-energized kinases has been demonstrated.     

Steady-state coupling of four membrane systems in mitochondrial oxidative phosphorylation.

According to Alexandre, Reynafarje, and Lehninger, four different membrane systems are involved, with definite stoichiometry, in the mitochondrial synthesis of ATP by electron transport, via proton transport. We adopt this model and pursue some of its thermodynamic consequences. At steady state, each of the four systems must have the same flux J through the membrane and the overall thermodynamic force X for oxidative phosphorylation is the sum of the four separate forces. From these properties, using an empirical linear flux-force relation for each system, it is easy to obtain J as a function of X. In turn, X depends on the inside [NAD+]/[NADH] and the outside [ATP]/[ADP][Pi] quotients (and on the pH inside). Thus, J is related to these quotients. The relationship we derive is similar to that described by Erecińska and Wilson, as deduced from a quite different model of oxidative phosphorylation. Proton transport is involved explicitly in three of the four systems of the present model. However, because of the steady-state stoichiometric coupling of the four systems, proton transport does not appear in the overall reaction. On the other hand, Erecińska and Wilson use, in their model, a direct connection between electron transport and ATP synthesis. The present paper demonstrates that J can be related to the quotients mentioned above without this direct connection.     

Universal energy principle of biological systems and the unity of bioenergetics.

Electronic energy (chemical bond energy) is the exclusive source of utilizable energy in biological systems. The release of this energy is mediated enzymically. The energy required to rupture a single covalent or ionic bond is prohibitively high under physiological conditions [in the range of 80-200 kcal/mol (1 kcal = 4.18 kJ)]. By the technique of the pairing of bond rupture (two juxtaposed bonds ruptured simultaneously) and the pairing of bond formation, enzymes can bypass the huge thermodynamic barrier to chemical change inherent in rupture of a single bond and operate within thermal limits. Enzymes accordingly can be conceived of as the energy machines that translate this principle. The principle of this transduction is that the energy required for forming a new covalent bond can fall within thermal limits when the original charged atom partner to the bond is displaced by a substitute charged atom under conditions in which the charge field of the bond remains constant during the substitution. In the transition from classical enzymology to energy coupling, muscular contraction, template-dependent replication, etc., new dimensions and possibilities are added to the basic enzymatic machinery. Specialized molecular devices (membranes, filaments, channels, templates, etc.) have to be introduced to make possible these extensions and permutations of enzymology. But it is demonstrable that the basis pairing principle is fully preserved during any of these modifications or extensions. Long range movement--of an ion, a filament, or a template--is the most important property introduced into classical enzymology in the transition to energy coupling systems.     

Some general principles in free energy transduction.

Chemical potentials or standard chemical potentials of bound ligands cannot be used to follow the step-by-step transfer of free energy from one ligand to another in a free energy transducing cycle. The basic difficulty is that, in most states of the cycle, separate ligand free energies are not even defined because, when ligands are bound on the enzyme, the interaction free energy of the complex cannot simply be assigned to ligands nor in general even be divided between two ligands if both are bound. This is a mutual, indivisible free energy among enzyme and ligands. Separate ligand free energies are well defined only at the complete cycle level, where the enzyme drops out of consideration (returns to its original state). Other types of free energy are also considered in order to discuss recent work of Tanford. In principle, the kinetics and mechanism can be followed in molecular or atomic detail through the steps of a transduction cycle, but the transfer of free energy from one ligand to another cannot be so followed.     

Assessment of mitochondrial energy coupling in vivo by 13C/31P NMR

The recently cloned uncoupling protein homolog UCP3 is expressed primarily in muscle and therefore may play a significant role in the regulation of energy expenditure and body weight. However, investigation into the regulation of uncoupling protein has been hampered by the inability to assess its activity in vivo. In this report, we demonstrate the use of a noninvasive NMR technique to assess mitochondrial energy uncoupling in skeletal muscle of awake rats by combining (13)C NMR to measure rates of mitochondrial substrate oxidation with (31)P NMR to assess unidirectional ATP synthesis flux. These combined (31)P/(13)C NMR measurements were performed in control, 10-day triiodo-l-thyronine (T(3))-treated (model of increased UCP3 expression), and acute 2,4-dinitrophenol (DNP)-treated (protonophore and mitochondrial uncoupler) rats. UCP3 mRNA and protein levels increased 8.1-fold (+/- 1.1) and 2.8-fold (+/- 0.8), respectively, in the T(3)-treated vs. control rat gastrocnemius muscle. (13)C NMR measurements of tricarboxylic acid cycle flux as an index of mitochondrial substrate oxidation were 61 +/- 21, 148 +/- 25, and 310 +/- 48 nmol/g per min in the control, T(3), and DNP groups, respectively. (31)P NMR saturation transfer measurements of unidirectional ATP synthesis flux were 83 +/- 14, 84 +/- 14, and 73 +/- 7 nmol/g per s in the control, T(3), and DNP groups, respectively. Together, these flux measurements, when normalized to the control group, suggest that acute administration of DNP (mitochondrial uncoupler) and chronic administration of T(3) decrease energy coupling by approximately 80% and approximately 60%, respectively, and that the latter treatment correlates with an increase in UCP3 mRNA and protein expression. This NMR approach could prove useful for exploring the regulation of uncoupling protein activity in vivo and elucidating its role in energy metabolism and obesity.     

Sticky ions in biological systems.

Aqueous gel sieving chromatography on Sephadex G-10 of the Group IA cations (Li+, Na+, K+, Rb+, Cs+) plus NH4+ as the Cl- salts, in combination with previous results for the halide anions (F-, Cl-, Br-, I-) as the Na+ salts [Washabaugh, M.W. & Collins, K.D. (1986) J. Biol. Chem. 261, 12477-12485], leads to the following conclusions. (i) The small monovalent ions (Li+, Na+, F-) flow through the gel with water molecules attached, whereas the large monovalent ions (K+, Rb+, Cs+, Cl-, Br-, I-) adsorb to the nonpolar surface of the gel, a process requiring partial dehydration of the ion and implying that these ions bind the immediately adjacent water molecules weakly. (ii) The transition from strong to weak hydration occurs at a radius of about 1.78 A for the monovalent anions, compared with a radius of about 1.06 A for the monovalent cations (using ionic radii), indicating that the anions are more strongly hydrated than the cations for a given charge density. (iii) The anions show larger deviations from ideal behavior (an elution position corresponding to the anhydrous molecular weight) than do the cations and dominate the chromatographic behavior of the neutral salts. These results are interpreted to mean that weakly hydrated ions (chaotropes) are "pushed" onto weakly hydrated surfaces by strong water-water interactions and that the transition from strong ionic hydration to weak ionic hydration occurs where the strength of ion-water interactions approximately equals the strength of water-water interactions in bulk solution.     

A critique of the chemosmotic model of energy coupling.

The chemosmotic model provides a framework for visualizing energy-coupled reactions (vectorial reaction sequences, membrane-dependent gradient formation, and charge separation of reacting species) and a mechanism for energy coupling (indirect coupling between the driving and driven reaction sequences mediated by a membrane potential or a protonmotive force). The mechanistic parameters of this model have been examined from four standpoints: compatibility with the experimental realities, supporting evidence that is unambiguous, compatibility with the enzymic nature of energy coupling, and the capability for generating verifiable predictions. Recent developments that have clarified the mechanism of ion transport, the nature of the protonic changes that accompany energy coupling, and the enzymic nature of energy coupling systems have made such an examination both timely and necessary. After weighing the available evidence, it has been concluded that the chemosmotic principle of indirect coupling has no basis in fact and that it is physically unsound in respect to the mechanism of energy coupling and enzymic catalysis.     

Ion-transport chain of cytochrome oxidase: the two chain-direct coupling principle of energy coupling.

Cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) couples the aerobic oxidation of ferrocytochrome c to the cyclical transport of monovalent cations or to the active transport of monovalent and divalent cations. This transport capability is mediated by an intracomplex ion-transport chain of two protein-bound molecules of cardiolipin per molecule of cytochrome oxidase. Cardiolipin in a two-phase system shows the identical ionophoric pattern as does the cytochrome oxidase coupled system. A molecular model of the cardiolipin chain suggests the possibility of a cage-like structure through which cations can be transferred from phosphate group to phosphate group. The ion-transport chain and the electron-transport chain are anchored to the same set of subunits (I+IV); the close proximity of the two chains argues for the direct coupling of electron and cation flow. The ion-transport chain of cytochrome oxidase provides an introduction to the molecular mechanisms by which ions are moved across membranes in energy-coupling systems.     

Evidence for the involvement of coupling factor B in the H+ channel of the mitochondrial H+-ATPase.

Membrane energization by ATP has been measured in vesicles containing purified bovine heart mitochondrial H+-ATPase (ATP synthase) with the voltage-sensitive dye oxonol VI. The dithiol chelator, Cd2+, and the thiol oxidant, copper o-phenanthroline, produced discharge of the membrane potential when added at the steady state and inhibited its establishment when added prior to energization by ATP. These effects, which were reversed by dithiothreitol, were not accompanied by an increase in the nonspecific H+ permeability of the membrane. Passive H+ conduction in proteoliposomes containing F0 (hydrophobic segment of ATP synthase) was assayed by the quenching of 9-aminoacridine fluorescence after establishing a K+ diffusion potential. This conductance was blocked by Cd2+, an inhibitor of coupling factor B (FB). Labeling of F0 with 115Cd2+ at the concentrations that inhibited the F0 conductance followed by gel electrophoresis yielded a single radioactive band with a molecular weight corresponding to FB, the presence of which in the F0 preparation was confirmed by immunoblot staining. The data offer strong evidence that FB is an essential component of the H+ channel of F0, because H+ conduction through the channel is inhibited by chemical modification of FB.     

Plasma mitochondrial coupling factor 6 in patients with acute myocardial infarction.

Previous studies have shown that mitochondrial coupling factor 6 (CF6) is an endogenous peptide that inhibits prostacyclin (PGI2) synthesis in vascular endothelial cells. In this study, we measured the plasma CF6 level of patients with acute myocardial infarction (AMI) to observe dynamic changes of CF6. All patients showed elevated plasma CF6 levels upon admission for treatment of AMI. Their CF6 levels peaked approximately 72 h after the onset of AMI and remained high for 7 days. At 7 days, their CF6 levels decreased to the level seen upon admission, but not to within a normal range. Hyperlipidemic patients had significantly greater CF6 levels at 24 h after onset of AMI than patients with a normal lipid profile. On admission, the plasma CF6 level in patients with a cardiac function of Killip class > or =II was higher than that in patients with a Killip class I cardiac function. At 3 days after the onset of AMI, the plasma CF6 levels of patients with a creatinine kinase (CK) peak value > or =1,500 units/l were significantly higher than those of patients with a CK peak value <1,500 units/l (p =0.05). At 7 days after the onset of AMI, the plasma CF6 levels of patients who received no reperfusion were significantly higher than those of patients who received a successful reperfusion. The plasma CF6 levels of AMI patients at admission, at 24 h, and at 3 days after onset of symptoms correlated positively with the cardiac function by Killip classification, respectively. At 24 h after onset of AMI, the plasma CF6 levels correlated positively with plasma total cholesterol levels and low-density lipoprotein levels. At 3 days, the plasma level of CF6 correlated positively with the plasma CK peak value and correlated negatively with left ventricular ejection fraction. These results suggest that the plasma CF6 level was elevated in patients with AMI.     

Steady-state properties of coupled systems in mitochondrial oxidative phosphorylation.

At steady state there is effective coupling among various otherwise independent membrane and internal mitochondrial systems that share the same substrates or ligands (e.g., H+, Ca2+, Pi, ADP, ATP). The number of different systems, coupled through shared substrates or ligands, is no doubt very large. But, just as an infinite series can be approximated by a finite number of terms, here the number of systems included in the analysis can be limited, as an approximation. In two previous papers, the basic but oversimplified set of four tightly coupled systems was studied. These are: respiratory chain; reverse ATPase; proton-phosphate cotransport; and ADP-ATP exchange. Essentially as illustrations of the methodology required for a more realistic analysis, two much more complicated examples are formulated here: eight tightly coupled systems, and the original four systems but with the tight coupling relaxed in two of these four.     

Kinematics of biological aging. II]

Using privation instead of physical time, a function of the theoretical vitality is found which depends on the parameter x. It is given by the quotient of growth-rate to age-rate. Consequences which could result from a possible continuity of such a relation are discussed.     

Population biological principles of drug-resistance evolution in infectious diseases

The emergence of resistant pathogens in response to selection pressure by drugs and their possible disappearance when drug use is discontinued are evolutionary processes common to many pathogens. Population biological models have been used to study the dynamics of resistance in viruses, bacteria, and eukaryotic microparasites both at the level of the individual treated host and of the treated host population. Despite the existence of generic features that underlie such evolutionary dynamics, different conclusions have been reached about the key factors affecting the rate of resistance evolution and how to best use drugs to minimise the risk of generating high levels of resistance. Improved understanding of generic versus specific population biological aspects will help to translate results between different studies, and allow development of a more rational basis for sustainable drug use than exists at present.