D.R. Sanadi

The mechanism of oxidative phosphorylation. XIV. Purification and properties of a second energy-transfer factor.

Abstract A protein factor, designated as Factor B, was extracted from lyophilized acetone-washed bovine heart mitochondria and purified by ammonium sulfate fractionation, and ion-exchange chromatography on DEAE-cellulose and CM-cellulose. Centrifugation in a sucrose density gradient showed that the activity of the purified factor was closely associated with a symmetrical protein peak comprising approximately 70% of the protein. Its molecular weight was estimated to be 32,000, using hemoglobin and cytochrome c as markers. Factor B produces several-fold stimulation of ATP-driven NAD reduction, and of net phosphorylation coupled to NADH or succinate oxidation in ammonia particles. The stimulation of ATP-driven NAD reduction activity exceeds that given by an optimal amount of oligomycin, and in the presence of a saturation level of Factor B, oligomycin stimulation disappears. Also, Factor B stimulation is evident in urea-depleted particles which have been supplemented by Factor A. These particles show no stimulation by oligomycin. The results suggest that Factor B may participate in the energy transfer reactions between the respiratory chain and the terminal step resulting in ATP synthesis.

Studies of oxidative phosphorylation and energy-linked cytochrome b reduction with tetramethyl-p-phenylene diamine as substrate

Abstract The coupled oxidation of ascorbate-50 μ m TMPD yields P O ratios significantly greater than 1.0 with rat liver mitochondria, and P O ratios of no greater than 1.0 with heart mitochondria. The addition of either antimycin, NOQNO, rotenone or an increase in TMPD concentration to 300 μ m reduces the P O ratio with liver mitochondria to a value close to 1.0, but has no significant effect on the P O ratio obtained with heart mitochondria. It is concluded that the true value of the P O ratio supported by the ascorbate-TMPD substrate system is close to and no greater than 1.0, and that P O ratios in excess of 1.0 observed during ascorbate-50 μ m TMPD oxidation in liver mitochondria are due to the simultaneous coupled oxidation of DPN-linked endogenous substrates. The oxidation of ascorbate-TMPD by either the ETP preparation, the saline washed submitochondrial preparation (SWP) or a purified cytochrome oxidase preparation is dependent upon cytochrome c . It is concluded that the site of entry of electrons from TMPD into the respiratory chain of the ETP and SWP preparations lies between the antimycin-sensitive site and cytochrome c 1 . In suspensions of heart mitochondria and phosphorylating submitochondrial beef heart particles made anaerobic by the oxidation of ascorbate-TMPD, cytochromes c , c 1 , a and a 3 are reduced in an uncoupler-insensitive reaction. The reduction of cytochrome b in these systems is mediated by an energy-linked reversed electron transfer reaction through an antimycin-sensitive site. The energy requirement for the latter reaction may be satisfied either through an oligomycin-insensitive energy transfer pathway from high energy intermediates generated in the cytochrome c to oxygen couple of the respiratory chain, or through an oligomycin-sensitive energy transfer pathway from exogenous ATP. In heart mitochondria, cytochrome b is present almost entirely in an energy-linked form. Submitochondrial heart muscle particles contain variable proportions of two postulated forms of cytochrome b , an energy-linked form reducible by either succinate or ascorbate-TMPD, and a nonenergy-linked form reducible only by succinate.

Studies on oxidative phosphorylation: XV. Latent adenosine 5′-triphosphatase activity of factor A☆

Abstract Further purification and stabilization of the mitochondrial energy transfer factor, Factor A, is described. The eleetrophoretic purity and sedimentation properties of Factor A are shown. Purified Factor A exhibits characteristic activation of ATPase at 45 ° and cold lability of the resulting ATPase activity. Activity in energy-linked reactions is unaffected during the treatment. Heating and cooling cycles are associated with gross changes in the optical rotatory dispersion (ORD) at 232 mμ. However, the time course of ATPase activation and ORD changes are quite different. An ATPase inhibitor and ADP are recovered from Factor A following denaturation of the material at 75 dg. Activation of oligomycin-insensitive ATPase by exposure to elevated temperatures (45–60 °) has been observed in submitochondrial particles also. This ATPase remains bound to particles and declines on cooling. It is proposed that the ADP phosphorylation enzyme of mitochondria is devoid of ATPase activity unless it is exposed to environmental changes.

Studies on oxidative phosphorylation

Abstract The stimulation of energy-linked reactions by low levels of oligomycin varies greatly with the type of submitochondrial particle. Particles made by disruption of combined heavy and light mitochondria by exposure to sonic oscillation and depleted by urea treatment showed no stimulation over a wide range of oligomycin concentration. However, the mitochondrial coupling factor described previously stimulated energy-linked reactions in these particles. Similar particles from heavy mitochondria gave some stimulation with low levels of oligomycin. High stimulation was obtained with alkali-treated particles derived from heavy mitochondria. The magnitude of stimulation appeared to be related to the concentration of endogenous coupling factors. Both severely depleted and fully supplemented particles were not stimulated by oligomycin, but particles with intermediate levels of endogenous factors gave striking effects.

[54] NADH-CoQ reductase-assay and purification

Publisher Summary This chapter discusses the assay and purification of NADH-CoQ reductase. The methods for the assay of NADH-CoQ reductase activity in submitochondrial particles and soluble extracts, using the higher isoprenologes of CoQ have been devised. The assay requires strict control of several variables as the substrate is essentially in a nonaqueous phase because of its low solubility. For routine measurement of activity during the preparation of the enzyme, menadione reductase activity, also described, has been found to be useful. In the procedure, the enzyme is assayed spectrophotometrically by recording the decrease in absorbency at 340 mμ during oxidation of NADH by CoQ. A unit of reductase activity is defined as that amount of enzyme, which is necessary to produce a decrease in NADH at the initial rate of 1 micromole/minute. Specific activity is given as units per milligram of protein, the latter being determined by the biuret reaction in the case of mitochondrial particles and by the Folin method for soluble preparations, with crystalline bovine serum albumin as the standard for both. The chapter also discusses the preparation of mitochondria and submitochondrial particles. The relative activities of the NADH-CoQ reductase with different electron acceptors are tabulated.

Studies on oxidative phosphorylation: XII. Uncoupling of energy-linked reactions by 2-thenoyltrifluoroacetone

Abstract 2-Thenoyltrifluoroacetone (TFA) is shown to uncouple oxidative phosphorylation and to inhibit ATP-supported reactions. Respiration in tightly coupled rat liver mitochondria is inhibited partially by TFA. TFA releases oligomycin inhibition of phosphorylative oxidation (State 3 respiration) and inhibits dinitrophenol-stimulated ATPase; these characteristics suggest a site of action closer to the electron transport chain than the oligomycin site. ATPase activity assayed in the presence of Mg 2+ is stimulated by TFA. It is suggested that the mechanism of action of TFA on oxidative phosphorylation may relate to its aromatic and lipophilic properties.

Studies on dihydronicotinamide adenine dinucleotide ubiquinone reductase: II. Purification and properties

Abstract The ubiquinone reductase activity of a soluble extract of beef heart submitochondrial particles can be separated into two active fractions by chromatography on a hydroxylapatite column. Fraction I was further purified by ammonium sulfate fractionation and Fraction II by chromatography on DEAE-cellulose. Both fractions catalyze the oxidation of NADH by ubiquinone-10, ubiquinone-6, ubiquinone-1, menadione, cytochrome c , and ferricyanide at nearly equal rates. Both contain FMN, nonheme iron, and acid-labile sulfide in the approximate ratio of 1:4:5, and have nearly identical spectra. Sedimentation and electrophoretic analyses indicate that the fractions are better than 95% homogeneous. The molecular weights of the two preparations are very similar (near 90,000), although their electrophoretic mobilities differ. NADH causes anaerobic bleaching of the flavin (450 mμ), but the absorption peak at 550 mμ is not altered. The reductases are inhibited by Amytal and rotenone, the latter showing an unusual biphasic curve with maximal inhibition at a rotenone to enzyme flavin ratio of nearly one. Piericidin A, dicumarol, o -phenanthrolene, or Tiron do not inhibit reductase activity.

On the mechanism of oxidative phosphorylation III. Effects of arsenite and 2,3-dimercaptopropanol on coupled phosphorylation in heart mitochondria

Abstract The effects of arsenite and 2,3-dimercaptopropanol (BAL), independently and in combination, have been tested on the oxidative phosphorylation system and ATPase activities found in “heavy” beef heart mitochondria. The combined reagent (0.1 m M ) uncoupled the phosphorylation associated with the oxidation of succinate, pyruvatemalate, β-hydroxybutyrate, and glutamate. Reversal of the uncoupling could be effected by a tenfold excess of BAL. The ATPase activities were stimulated at low concentrations of the reagent and then were gradually inhibited as the concentration of the reagent was increased. Reversal by excess BAL was demonstrated.

Activation of myosin ATPase by arsenite in the presence of 2,3-dimercaptopropanol.

Abstract Arsenite (0.1–0.2 m M ) in the presence of equivalent 2,3-dimercaptopropanol (BAL) stimulated the calcium ion-activated adenosinetriphosphatase (ATPase) activity of myosin two- to threefold. Excess of the combined reagent decreased the stimulation. These compounds singly had no effect on the ATPase activity. BAL could not be replaced by 2-mercaptoethanol or glutathione for the stimulation. The stimulation was abolished if BAL or other sulfhydryl compounds were present in large excess over the arsenite. These results are analogous to the previously documented effects of arsenite-BAL on the uncoupling of oxidative phosphorylation and stimulation of the latent ATPase in mitochondria.

Studies on oxidative phosphorylation: XI. Energy-dependent reduction of diphosphophyridine nucleotide by ene-diol compounds in the presence of dyes

Energy-linked reduction of added NAD by ascorbate and dihydropyrogallol in the presence of mediator dyes in submitochondrial particles has been described. The reactions are inhibited by oligomycin and rotenone but not by malonate. Antimycin A completely inhibited NAD reduction by the ascorbate-toluidine blue couple as in the ascorbate-tetramethyl-p-phenylenediamine couple, suggesting electron entry on the oxygen side of the antimycin block. Antimycin A inhibited NAD reduction only partially when ascorbate or dihydropyrogallol was used with menadione or pyocyanine as mediators. The results suggest that electrons from these couples enter the respiratory chain on either side of the antimycin block.