Charles S. Owen

Quantitative dependence of mitochondrial oxidative phosphorylation on oxygen concentration: A mathematical model☆

Abstract The model of Wilson and co-workers ( D. F. Wilson, C. S. Owen, and A. Holian, 1977 , Arch. Biochem. Biophys . 182 , 749–762) for the regulation of mitochondrial oxidative phosphorylation has been extended to include the dependence on oxygen tension. The derived rate expression correctly describes the observed dependence of cellular energy metabolism on oxygen tension, including the oxygen dependence at “normoxic” physiological values. Experimental evidence is presented that oxidative phosphorylation by suspensions of isolated rat liver mitochondria is also dependent on oxygen concentration up to values of at least 100 μ M .

Control of mitochondrial respiration: A quantitative evaluation of the roles of cytochrome c and oxygen

Abstract The dependence of the mitochondrial respiratory rate on the reduction of cytochrome c has been measured as a function of the exogenous [ ATP ] [ ADP ][ P i ] ratio and pH. The respiratory rate at [ ADP ] [ ADP ][ P i ] values of less than 10-1 m -1 is proportional to the reduction of cytochrome c and independent of pH from pH 6.5 to pH 8.O. The maximal turnover number (at 100% reduction) for cytochrome c is approximately 70 s−1. As the [ ATP ] [ ADP ][ P i ] ratio is increased from 10−1 m −1 to 104 m −1, the respiration at any given level of reduction of cytochrome c is progressively inhibited. Greater inhibition is observed at more oxidized levels of cytochorme c with respiratory control values for oxidation of reduced cytochrome c exceeding 10. The behavior of mitochondrial respiratory control is shown to be quantitatively consistent with a proposed mechanism in which the regulation occurs in the reaction of oxygen with cytochrome oxidase. A steady-state rate expression is derived which fits the mitochondrial respiratory rate dependence on (i) the extramitochondrial [ ATP ] [ ADP ][ P i ] ratio; (ii) the level of reduction of cytochrome c (or the intramitochondrial [ NAD + ] [NADH]) at different [ ATP ] [ ADP ][ P i ] values; (iii) the pH of the suspending medium. This rate expression appears to correctly predict the relationships of the cytoplasmic [ ATP ] [ ADP ][ P i ] ratio, the mitochondrial [ NAD + ] [ NADH ] ratio, and the mitochondrial respiratory rate in intact cells as well as suspensions of isolated mitochondria.

Magnetic labeling and cell sorting

Magnetic labeling by a magnetite-antibody conjugate has been combined with magnetic filtration (high gradient magnetic separation) to effect a rapid and efficient separation of a selected cell population from a suspension of single cells. Samples of more than 10(8) cells could be fractionated in about 5 min with complete recovery. The system has been applied to a model system using red blood cells (sheep or chicken) and commercial antibodies against species-determined cell surface antigens. Enrichments of labeled cells by factors of up to 37-fold were observed. The approach was relatively insensitive to details in the experimental protocol and to the number of unlabeled cells which were in the sample. Thus, the method was easy to use and can readily be scaled up to handle large samples containing 10(8) labeled cells in a total of 10(11) or more. It should be useful as a pre-enrichment scheme for suspensions in which cells of interest are rare and, consequently, very inefficiently sorted by fluorescence-activated instruments.

Control of respiration in isolated mitochondria: Quantitative evaluation of the dependence of respiratory rates on [ATP], [ADP], and [Pi]☆

Abstract Mitochondrial preparations with respiratory control ratios greater than 10 have been isolated from dog and pigeon heart muscle. Suspensions of these mitochondria have been tested for the qualitative and quantitative dependence of the respiratory rate on the extramitochondrial ATP, ADP, and orthophosphate (P i ) concentrations. Reciprocal dependence is observed for [ATP] and [P i ] (the [ADP] required for a given rate of oxygen consumption is proportional to [ATP] and inversely proportional to [P i ]) which indicates that these two reactants are of equal importance in regulating the mitochondrial respiratory rate. Analysis of the [ATP], [ADP], and [P i ] in suspensions of mitochondria at various respiratory rates shows that the respiratory rate is proportional to the logarithm of the [ATP]/[ADP][P i ] for values between 10 3 and 10 5 , m −1 . Similar results are obtained for suspensions of mitochondria prepared from rat brain, rat kidney, and rat liver. These data demonstrate that, in mitochondria with high respiratory control, the rate of respiration is dependent on extramitochondrial [ATP]/[ADP][P i ] and not on [ATP]/[ADP]. It is concluded that [P i ]-independent adenine nucleotide translocation across the inner mitochondrial membrane is not a rate-limiting reaction in oxidative phosphorylation.

Control of respiration by the mitochondrial phosphorylation state

The time dependence of oxygen consumption of actively respiring mitochondria has been studied in detail as adenosine diphosphate (ADP) becomes depleted, and the dependence on exogenous adenosine triphosphate (ATP) and orthophosphate (Pi) has been investigated. The results are consistent with a model having the electron transport and phosphorylation processes in chemical equilibrium with the extramitochondrial phosphorylation state, [ATP][ADP] [Pi], and the respiration rate proportional to the concentration of reduced cytochrome a3. The latter is, in turn, dependent upon the phosphorylation state. The mathematical shape of polarographic oxygen consumption curves between the region of excess ADP and the region of minimum ADP has been calculated from the model, and agreement has been found with the experimentally measured rates of oxygen consumption during ADP depletion.

The invisible copper of cytochrome c oxidase: pH and ATP dependence of its midpoint potential and its role in the oxygen reaction☆

Abstract Formation of the CO compound has been studied in intact mitochondria, submitochondrial particles and isolated cytochrome oxidase. The reaction requires the prior reduction of both cytochrome a 3 and one other single-electron acceptor. It is inferred that the second acceptor is the “invisible” copper which is undetectable by both optical and spin resonance spectroscopy. The overall process can be viewed as two single electron steps plus a ligand binding reaction. At high concentrations of CO, when titrations are performed at oxidation-reduction potentials significantly above the midpoints of either cytochrome a 3 or “invisible” copper, appearance of the CO compound follows a strict n = 2 (2-electron) relationship. Its midpoint potential is also dependent on the prevailing concentration of CO and is increased by approx. 30 mV for each tenfold increase in the level of CO. At redox potentials approaching the midpoints of cytochrome a 3 or “invisible” copper, significant deviations from n = 2 behavior are apparent which are readily detectable experimentally using low CO concentrations. A mathematical analysis of this model is presented and the oxidation-reduction properties of the CO compound are utilized to determine the midpoint potential of the “invisible” copper. This value is estimated to be 340 ± 10 mV at pH 7.8, independent of pH and the prevailing sol [ATP] [ADP] × [ P 1 ] ratio. By analogy with the observations on CO binding, the primary intermediate in the oxidase reaction with oxygen is concluded to be a bridged a 3 2+ - O 2 - Cu 1+ complex. The initial reduction of molecular oxygen can then proceed via a thermodynamically favorable two-electron step to form a bridged peroxide intermediate. Subsequent reduction to water may later occur by way of two single-electron steps or one two-electron step.

Immunospecific NMR contrast agents

Most NMR contrast agents suggested to date have been paramagnetic. These agents, which include the transition and lanthanide metal ions as well as stable organic free radicals, do not provide effective contrast at concentrations much below 1 mM. However, the use of macromolecular ferromagnetic and superparamagnetic particles provides, for the first time, an NMR relaxation agent that is effective at subnanomolar concentrations. Two different sized superparamagnetic particles have been coupled to monoclonal antibodies with high affinity for a neuroblastoma-specific cell surface antigen. The specific binding of these particles, both in vivo and in vitro is demonstrated and the consequences for immunospecific NMR contrast are discussed.

Heme—Heme interaction in cytochrome c oxidase: The cooperativity of the hemes of cytochrome c oxidase as evidenced in the reaction with CO

Abstract Isolated and purified cytochrome c oxidase from beef heart muscle mitochondria (Kuboyama et al. (1972) J. Biol. Chem. 247 , 6375–6383) is shown to be very similar to the hemoprotein in situ with respect to its EPR absorption properties and the half-reduction potentials of the hemes and copper. The half-reduction potentials of cytochromes a and a 3 in the purified cytochrome c oxidase are 205 mV and 360 mV, respectively, and these values are the same in the presence and absence of cytochrome c . Low-temperature EPR spectra show that the binding of CO to reduced cytochrome a 3 changes the oxidized cytochrome a from high spin ( g 6) to low spin ( g 3). In samples at 5–8 °K the photodissociation of the reduced cytochrome a 3  CO compound shifts the spectrum of the oxidized low-spin cytochrome a to a lower g value and converts approximately 5% of the low-spin form to a high-spin form. The heme-heme interaction demonstrated in this reaction is very fast as evidenced by the fact that even at 5 °K the measured change in oxidized cytochrome is complete within 5 msec.

Two dimensional diffusion theory: Cylindrical diffusion model applied to fluorescence quenching

A value for the diffusion constant of fluorescent quenchers can be inferred by fitting measured and theoretical fluorescence decay curves. In this paper, a formalism is presented describing diffusion‐dependent fluorescence quenching in systems which are effectively two dimensional in nature. This model predicts somewhat different values for the diffusion constant from those one obtains by fitting the same experimental curve to the usual Smoluchowski model for diffusion in an isotropic (’’spherical’’) system.

Comparison of spectrum-shifting intracellular pH probes 5′(and 6′)-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7, 1′(3′H)-isobenzofuran]-3′-one and 2′,7′-biscarboxyethyl-5(and 6)-carboxyfluorescein

The dyes carboxy-SNARF-1 and BCECF are fluorescent probes of intracellular pH that exhibit changes in spectral shape upon proton binding which allow one to use measurements of fluorescence at two or more wavelengths in order to measure pH without artifacts associated with variability in dye loading, etc. In evaluating these dyes for this study, whole spectra, rather than measurements at two wavelengths, were analyzed. For BCECF, the effects of the intracellular milieu were minimal: both the pH-sensitive excitation spectrum and the pKa agreed closely with values found in extracellular solution. In contrast, both the spectra and the pKa for the emission spectrum-shifting carboxy-SNARF-1 showed significant differences between intracellular and extracellular dye. As a result, extremely misleading values for intracellular pH will be obtained if one attempts to use extracellular dye to calibrate intracellular carboxy-SNARF-1 measurements. Multiple origins were found for the discrepancy: (i) the intracellular dye was found to be significantly quenched, with the deprotonated form being more strongly quenched than the protonated form; and (ii) the pKa for the equilibrium with intracellular hydrogen ions was shifted by +0.2 pH units. These effects were readily reversed by disruption of the cell, but were not due to sequestering of dye in an acidic cell compartment.