Nozomu Oshino

The role of H2O2 generation in perfused rat liver and the reaction of catalase compound I and hydrogen donors

Abstract 1. 1. Hydrogen peroxide production in hemoglobin-free perfused rat liver was measured quantitatively by analyzing the steady state level of catalase-H2O2 intermediate ( p m e ). The method is based on a relationship between the ratio of H2O2 generaztion rate ( dx n dt ) to catalase-heme concentration (e) and the concentration of methanol ( a 1 2 ) which causes a decrease in the p m e value to half of its saturation value ( p m e ). Endogenous hydrogen donor in the perfused liver was estimated to be equivalent to about 30 μ m methanol. 2. 2. The rate of H2O2 production in the perfused liver with 2 m ml -lactate and 0.3 m m pyruvate was 49 nmol/min/g wet wt liver at 30 °C. This rate corresponds to about 1.7% of the total liver respiration rate. 3. 3. Mitochondrial production of H2O2 was proved by the fact that fatty acids enhance the H2O2 production and that this enhancement was inhibited by either rotenone or antimycin A. Perfusion of antimycin A caused stimulation of H2O2 production by a factor of 1.7. In the presence of 0.3 m m octanoate, H2O2 generation increased to 170 nmol/min/g wet wt liver. The results strongly suggest that one of the major sources for H2O2 production is the mitochondrial system participating in fatty acid oxidation. 4. 4. Changes in the redox state of cytosolic pyridine nucleotides by xylitol or by lactate did not alter the H2O2 production signficantly. 5. 5. Peroxisomal H2O2 production was observed when the liver was perfused with either urate or glycolate. The maximum rate of H2O2 production in the presence of urate and glycolate were 750 and 490 nmol/min/g wet wt liver, respectively. 6. 6. The total role of H2O2 production and utilization is considered and H2O2 metabolism in the peroxisomes is compared with that of other systems. The general conclusion is that, the body regulates the intracellular H2O2 level below about 10−7 m , and with this level biological oxidations of considerable significance are possible through the catalase system.

Optical measurements of intracellular oxygen concentration of rat heart in vitro.

Abstract The optical characteristics of hemoglobin-free perfused rat heart have been examined in detail. Ethyl hydrogen peroxide is found to convert myoglobin into “ferryl compound” in the perfused heart, as is also seen in vitro. After pretreatment with ethyl hydrogen peroxide, a typical mitochondrial absorption spectrum, similar to that of isolated rat heart mitochondria, is obtained in perfused heart. The overall absorption spectrum of the heart obtained by the aerobic to anaerobic transition is a superposition of the mitochondrial spectrum on that of myoglobin. By comparing these spectra, it is found that measurement of cytochrome a + a3 at 605–620 nm is possible in spite of the absorbance change due to the oxygenation-deoxygenation of myoglobin, whereas the wavelength pairs for cytochrome c at 550-540 nm, cytochrome b at 562–575 nm and cytochrome a + a3 at 445–450 nm can not be used in the heart because of interference from the absorption change of myoglobin. The partial pressure of O2 (P50) which is required for half maximal deoxygenation (or oxygenation) of myoglobin in perfused heart is found to be 2.4 mm Hg at room temperature and the Hill constant, n, is 1.1; these values are similar to those of myoglobin purified from rat heart. The steady-state O2 titration has been performed by using absorbancy changes of myoglobin and cytochrome a + a3 as intracellular O2 indicators. In the perfused heart, the percentage change of oxygenation-deoxygenation of myoglobin parallels the oxidation-reduction of cytochrome a + a3, while the mixture of purified myoglobin and isolated mitochondria shows a deviation, reflecting the difference of O2 affinities between myoglobin and cytochrome a + a3. The results indicate that there may be an O2 gradient between cytosolic and mitochondrial compartments in the hemoglobin-free perfused heart. The absorption changes of myoglobin and of cytochrome a + a3 can be measured in a single contraction-relaxation cycle. A triple beam method was introduced to eliminate the effect of light scattering changes in these measurements. The results demonstrated that myoglobin is more oxygenated during the systolic and diastolic periods and deoxygenated in the resting period, whereas cytochrome a + a3 is more reduced in systole and diastole and oxidized in the resting state. Changing the perfusion conditions greatly alters the time course of the events which occur during the contraction-relaxation cycle of the perfused heart.

Mitochondrial functions under hypoxic conditions: The steady states of cytochrome c reduction and of energy metabolism

Abstract 1. The effect of low oxygen concentration on the oxidation-reduction states of cytochrome c and of pyridine nucleotide, on Ca 2+ uptake, on the energy-linked reduction of pyridine nucleotide by succinate, and on the rate of oxygen consumption have been examined under various metabolic conditions, using pigeon heart mitochondria. 2. The oxygen concentration required to provide half-maximal reduction of cytochrome c (p50 c ) ranges from 0.27 to 0.03 μM (0.2-0.02 Torr) depending upon the metabolic activity. There is a linear increase of the p50 c value with increasing respiratory rate. 3. The fraction of the normoxic respiration that is observed at p50 c is 70–90% under State 4 conditions, but is 30% under State 3 conditions. 4. The oxygen requirement for half-maximal reduction of pyridine nucleotide (p50 PN ) varies less than p50 c , being 0.08 μM in State 3 and 0.06 μM in the uncoupled state. 5. The ability of the mitochondria to exhibit an energy-linked reduction of pyridine nucleotide by succinate disappears at an oxygen concentration of 0.09 μM (0.06 Torr). Below this oxygen concentration, endogenous Ca 2+ begins to be released from the mitochondria. Thus, the critical oxygen concentration for bioenergetic function of mitochondria corresponds approximately to 50% reduction of pyridine nucleotide (p50 PN ).

Basic Principles of Tissue Oxygen Determination from Mitochondrial Signals

The importance of measuring intracellular oxygen concentrations in tissues has, over the years, emerged as a basic parameter in the physiology and biochemistry of living tissues. The credibility of oxyhemoglobin determinations, even as refined A/V differences, is taxed especially in cases where inhomogeneous tissues with variable oxygen demands and oxygen supply are served. The formation of lactic acid in the venous blood is often used as a criterion of anoxia, but it also lacks credibility where inhomogeneous circulatory pathways are served and in addition, is questionable from the standpoint of whether the appearance of excess lactate is an unequivocal criterion of oxygen insufficiency. To indicate my empathy with polarographic techniques as they have been developed at the Johnson Foundation, I wish to recall the pioneering works of Bronk(1) Brink (2), Davies and Remond (3) that stand as landmarks in the exploration of tissue oxygen tension by microelectrode methods. I served my apprenticeship with them.

Mitochondrial function under hypoxic conditions: The steady states of cytochrome a+a3 and their relation to mitochondrial energy states

The oxidation-reduction states of mitochondrial cytochromes were studied under low O2 concentrations. 1. 1. Relative oxidation states of cytochromes caused by increasing concentrations of O2 apparently followed the sequence of their half reduction potentials only under the uncoupled conditions. 2. 2. In the presence of antimycin A, the O2-induced reduction of cytochrome bT was seen in the difference spectrum. 3. 3. The O2 dependency of the relative reduction state of cytochrome a+a3 with respect to that of cytochrome c altered significantly depending upon the presence or absence of ATP. The most significant change in the O2 dependency was that due to cytochrome a3. 4. 4. When compared at a given low O2 concentration below 0.5 μM, the reduction states of cytochrome a+a3, as well as that of cytochrome c, were higher in the presence of ADP or uncoupler than in the presence of ATP. 5. 5. Whereas the O2 concentration required for 50% oxidation of cytochrome c (P50c) depended upon the respiratory rate, the O2 concentration required for 50% oxidation of cytochrome a3 (P50a3) required information on the energy state of mitochondria. Under conditions where the redox states of cytochrome c and a+a3 are measured continuously and as a function of the O2 concentration it may be possible to evaluate the energetic state of the mitochondria.

Heme occupancy of catalase in hemoglobin-free perfused rat liver and of isolated rat liver catalase.

Maximal heme occupancy, the maximal proportion of total catalase heme present in the form of Compound I, is found to be 0.4 both in the enzyme isolated from rat liver and in the peroxisomal enzyme as present in the intact cells of perfused rat liver. This indicates that the ratio of second order rate constants for catalatic decomposition and for formation of Compound I, k4′k1, is equal in vitro and in vivo. Catalase was isolated from rat liver, and the extinction coefficients for Compound I and for cyanide-catalase at 640 minus 660 nm were determined. The measurement of heme occupancy of catalase in hemoglobin-free perfused rat liver was made possible by wavelength scanning as well as by dual wavelength absorbance photometry. Thus, Compound I and cyanide-catalase were demonstrated in the red region and in the Soret band region. Meeting the particular needs of organ photometry, specific metabolic transitions were used to visualize specific transitions of absorbing pigments. Compound I is specifically demonstrated by its decomposition by the hydrogen donor, methanol. A measure for total catalase heme is provided by formation of cyanide-catalase. The cyanide concentrations required are well below appearance of possible interference by other cyanide-binding hemoproteins at 640–660 nm.

The b cytochromes in succinate--cytochrome c reductase from pigeon breast mitochondria.

Abstract A quick and efficient method for the preparation of succinate-cytochromc c reductase from pigeon breast mitochondria using a mixture of ionic and nonionic detergents is described. The spectral characteristics of the cytochrome components of this preparation obtained during the equilibrium potentiometric titration in a new scanning spectrophotometer show a close resemblance to those of the intact mitochondria. Two b cytochromes are present whose properties can be modified by the detergents and lyotropic anions. The most sensitive cytochrome toward any modification is cytochrome bT. Bile salts can convert cytochrome bT into a form spectrally indistinguishable from that of bK, and the lyotropic anions cause disappearance of cytoehrome bT spectrum.

A sensitive bacterial luminescence probe for O2 in biochemical systems

Abstract 1. 1. An oxygen assay method applicable at very low oxygen concentrations has been developed using bioluminescence as an oxygen indicator. The method can be applied with accuracy to the oxygen concentration range between 10 −6 and 10 −8 M. 2. 2. The reliability of the method was verified by determining the oxygenation state of horse heart myoglobin as a function of oxygen concentration, and comparing this curve with that obtained polarographically. The luminescence method gave a half-oxygenation concentration of 2.2·10 −6 M (1.1 mm Hg) with a Hill constant n = 1.0, while the polarographic method gave 2·3·10 −6 M (1.2 mm Hg) O 2 with n = 1.0. With a series of heme-substituted myoglobins with half-oxygenation concentrations ranging from 2.2·10 −6 (1.1 mm Hg) to 4·10 −7 M (0.2 mm Hg) O 2 , the luminescence method yielded curves with n = 1.0 in all cases. 3. 3. The oxygenation state of both yeast hemoglobin and Ascaris perienteric fluid hemoglobin were continuously measured as a function of oxygen concentration from the fully oxygenated to the fully deoxygenated states. Both hemoglobins showed a half-oxygenation value of (0.01 mm Hg) 2 · 10 −8 M which is the lowest ever to have been measured. 4. 4. The method was applied to the measurement of the redox change in mitochondrial cytochrome c . The half-reduction of cytochrome c was observed at an oxygen concentration of 7·10 −8 M. 5. 5. The oxygenation state of yeast hemoglobin was compared with the redox state of cytochrome oxidase in yeast cells as a function of oxygen concentration. Half-oxygenation of hemoglobin in yeast cells is observed at an oxygen concentration of 2·10 −8 M, which is identical to that observed in purified yeast hemoglobin and is lower than the oxygen concentration producing a 50% reduction of cytochrome a 3 (3·10 −8 M) and cytochrome a (2·10 −7 M) in yeast cells.

The properties of sulfite oxidation in perfused rat liver; interaction of sulfite oxidase with the mitochondrial respiratory chain

Abstract Sulfate oxidation in the isolated mitochondrial fraction of rat liver proceeds exclusively through the respiratory chain in a sequence of electron flow from sulfite oxidase to cytochrome c and then to cytochrome oxidase. Thus, the SO 3 2− O ratio observed is almost unity and the ATP O ratio with sulfite is half that obtained with succinate as the respiratory substrate. Direct reduction of molecular oxygen to H 2 O 2 by sulfite oxidase occurs only when the respiratory chain is inhibited by cyanide. These two reactions differ with respect to the effects of the O 2 concentration and the sulfite concentration on the activity and on the SO 3 2− O ratio. In the perfused liver, infusion of sulfite causes increased uptake of O 2 , with concomitant reduction of the mitochondrial components, such as pyridine nucleotides, flavoproteins, cytochrome c and cytochrome oxidase. The SO 3 2− O ratio observed is 1.6−1.4 and no increase in H 2 O 2 production is detected during sulfite oxidation, indicating that sulfite oxidase is located between the outer and inner membranes of mitochondrion and in contact with cytochrome c , thus providing reducing equivalents to the respiratory chain. In the presence of excess cyanide, O 2 consumption increases two-fold during sulfite oxidation but the rate of sulfite oxidation does not change significantly. Under these conditions the SO 3 2− O ratio is 0.8−0.6, indicating direct reduction of O 2 to H 2 O 2 by sulfite oxidase in the perfused liver. The production of H 2 O 2 is accompanied by a remarkable oxidation of pyridine nucleotides which is attributed to the stimulation of the glutathione-peroxidase reaction. The maximal rate of sulfite oxidation is more than 1.2 μmol/min/g wet wt in the control liver, and decreases to 0.6 μmol/min/g wet wt in the liver of the tungsten-pretreated rat which possesses 23% of the sulfite oxidase activity. As the rate of sulfite oxidation reaches its maximum, progressive inhibition of some dehydrogenases appears to occur in the perfused liver.

In vitro studies on yeast cytochrome c peroxidase and its possible function in the electron transfer and energy coupling reactions

1. Yeast cytochrome c peroxidase (ferrocytochrome c:H2O2 oxidoreductase, EC 1.11.1.5) located in the intermembrane space of the mitochondrion rapidly decomposes H2O2 generated during the operation of the respiratory chain and efficiently oxidizes ferrocytochrome c. 2. Rapid utilization of H2O2 is linked to the oxidation of ferrocytochrome c generated through the usual metabolic pathway in which phosphorylation Sites I and II are operative.