Inhibition of an aa3-Type Two-Subunit Cytochrome c Oxidase from Nitrobacter agilis by N,N'-Dicyclohexylcarbodiimide

The electron transfer activity of an aa₃-type two-subunit cytochromec oxidase of Nitrobacter agilis was inhibited by DCCD. Althoughthe activity of the purified cytochrome c oxidase dissolvedin 1% Triton X- 100 was not affected by DCCD even at a ratioof 1,000 mol DCCD per mol cytochrome aa₃, the activity of theenzyme dissolved in 0.02% Tween 20 or 0.02% Triton X-100 wasinhibited by 60% or more at a ratio of 1,000 mol DCCD per molcytochrome aa₃. The results of SDS polyacrylamide gel electrophoresisof the enzyme incubated with DCCD suggested that subunit IImight be a binding site for DCCD. (Received February 23, 1985; Accepted April 23, 1985)     

Preparation of a Photoactive Reaction Center Complex Containing Photo-reducible Fe-S Centers and Photooxidizable Cytochrome c from the Green Sulfur Bacterium Chlorobium tepidum

A photoactive reaction center (RC) complex was isolated fromthe green sulfur bacterium Chlorobium tepidum by solubilizationof membranes with Triton X-100, followed by sucrosedensity gradientcentrifugation, DEAE Bio-Gel A chromatography, and hydroxyapatitechromatography. The purified RC complex contained about 50–70bacteriochlorophyll molecules (BChl) per P840, as assayed byphotooxidafion. It showed a near-infrared BChl a absorptionpeak at 814 nm and shoulders at about 800 and 835 nm at roomtemperature. SDS-PAGE analysis revealed 6 polypeptides withapparent molecular masses of 100, 65, 41, 32, 23, and 18 kDa.The RC complex binds functional P840 and Cyt c₅₅₁, which werephotooxidized by continuous illumination at room temperature.Upon flash excitation, the bound Cyt c₅₅₁ was oxidized, andrereduced in the dark with a half-time of 16 and 400 ms in thepresence and absence of 0.1 mM 2,6-dichlorophenol indophenol,respectively, at room temperature. At 551 nm, the amount ofthe Cyt c photooxidized by continuous illumination was 60% ofthe amount determined by chemical oxidation-reduction. The functionalCyt c₅₅₁/P840 ratio was calculated to be 1.2–1.7. EPRspectroscopy at cryogenic temperatures revealed that the RCcomplex binds three photoreducible Fe-S centers designated tobe CF_A, CF_B and CF_X (C for Chlorobium). CF_A and CF_B were reducedin the dark with dithionite at pH 10. (Received May 26, 1993; Accepted October 4, 1993)     

Characterization of ATPases Associated with Various Cellular Membranes Isolated from Etiolated Hypocotyls of Vigna radiata (L.) Wilczek

Cellular membrane fractions, including endoplasmic reticum (ER),Golgi-enriched membrane, plasma membrane and tonoplasts, wereisolated from Vigna radiata seedlings. Each of these membranefractions was associated with specific ATPases which were highlydependent on Mg²⁺. ATPases of ER, Golgi-enriched membrane andplasma membrane were sensitive to vanadate but the tonoplastATPase was not. ATPases were mostly dependent on Cl¹, but aslight stimulation by K⁺ was observed in the case of ATPasesof Golgi-enriched membrane and plasma membrane. KNO₃ inhibitedtonoplast ATPase but stimulated the other ATPases. ER ATPasecan be distinguished from other ATPases by the following characteristics:specific inhibition by KNO₂ and Triton X-100, stimulation bylow concentrations of diethylstilbestrol and 4,4'-diisothiocyanostilbene-2,2'-disulfonicacid, and high sensitivity to heat. The ATPases showed typicalMichaelis-Menten kinetics and had K_m values of 0.5 to 0.6 ITIMMg²⁺-ATP for ER, Golgienriched-membrane and tonoplast ATPases,and 2.27 msi Mg²⁺-ATP for plasma membrane ATPase. ATPases ofGolgi-enriched membranes and plasma membranes had similar properties,but they were still distinguishable by the differences in theirK_m values and their responses to Triton X-100. Based on theseresults, it is postulated that each cellular membrane is associatedwith a specific ATPase in cells of V. radiata. ¹Contribution No. 3171 from the Institute of Low TemperatureScience. (Received April 22, 1988; Accepted September 28, 1988)     

Differential susceptibility of subsarcolemmal and intermyofibrillar mitochondria to apoptotic stimuli

Apoptosis can be evoked by reactive oxygen species (ROS)-induced mitochondrial release of the proapoptotic factors cytochrome c and apoptosis-inducing factor (AIF). Because skeletal muscle is composed of two mitochondrial subfractions that reside in distinct subcellular regions, we investigated the apoptotic susceptibility of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS and IMF mitochondria exhibited a dose-dependent release of protein in response to H₂O₂ (0, 25, 50, and 100 µM). However, IMF mitochondria were more sensitive to H₂O₂ and released a 2.5-fold and 10-fold greater amount of cytochrome c and AIF, respectively, compared with SS mitochondria. This finding coincided with a 44% (P < 0.05) greater rate of opening (maximum rate of absorbance decrease, V_max) of the protein release channel, the mitochondrial permeability transition pore (mtPTP), in IMF mitochondria. IMF mitochondria also exhibited a 47% (P < 0.05) and 60% (0.05 < P < 0.1) greater expression of the key mtPTP component voltage-dependent anion channel and cyclophilin D, respectively, along with a threefold greater cytochrome c content, but similar levels of AIF compared with SS mitochondria. Despite a lower susceptibility to H₂O₂-induced release, SS mitochondria possessed a 10-fold greater Bax-to-Bcl-2 ratio (P < 0.05), a 2.7-fold greater rate of ROS production, and an approximately twofold greater membrane potential compared with IMF mitochondria. The expression of the antioxidant enzyme Mn²⁺-superoxide dismutase was similar between subfractions. Thus the divergent protein composition and function of the mtPTP between SS and IMF mitochondria contributes to a differential release of cytochrome c and AIF in response to ROS. Given the relatively high proportion of IMF mitochondria within a muscle fiber, this subfraction is likely most important in inducing apoptosis when presented with apoptotic stimuli, ultimately leading to myonuclear decay and muscle fiber atrophy. reactive oxygen species; skeletal muscle; mitochondrial permeability transition pore; cytochrome c; apoptosis     

Isolation and Purification of a Ubiquinone-Cytochrome b-c1 Complex from a Photosynthetic Bacterium, Rhodopseudomonas sphaeroides

A ubiquinone-cytochrome b-c₁ complex was removed from chromatophoremembranes of a Rhodopseudomonas sphaeroides green mutant bydeoxycholate-cholate treatment of the chromatophores. The complexwas purified by ammonium sulfate fractionation and gel filtration. The molecular weight of the purified complex was 240,000 (240kD) and it was composed of seven subunits with molecular weightsof 47 kD, 42 kD, 38 kD, 32 kD, 30 kD, 24 kD and 16 kD. The complexcontained 1.54 and 3.42 nmol of cytochrome c₁ and two differentcytochrome b species per mg protein, respectively. It also contained7.07 nmol of ubiquinone, 6.37 nmol of non-heme iron and about3 nmol of carotenoids per mg protein. No flavins were detected.Heme staining indicated that the 32 kD-and 24 kD-subunits werecytochromes. The midpoint potential of cytochrome c₁ was 245 mV, and thevalues for the cytochromes b were 60 mV and –75 mV atpH 7.2. The peak of the -band of the reduced-minus-oxidizeddifference spectrum of cytochrome c₁ was located at 552.5 nm,arid peaks of the b-type cytochromes with higher and lower midpointpotentials were located at 562 nm and 563 nm. The chemical and the subunit compositions of the purified complexreported here were similar to those obtained for the inner membranesof mitochondria of various organisms. (Received April 5, 1982; Accepted June 14, 1982)     

Studies on the change of the respiratory system in roots in relation to the growth of plants II. Activity of iron-containing oxidases in roots of cereal crops with the aging of plants

Distribution of iron-containing oxidases in aging nodal rootsof rice and wheat was studied. Activities of cytochrome c oxidase(1.9.3.1 [EC] , cytochrome c : O₂ oxidoreductase), catalase (1.11.1.6 [EC] ,H₂O₂: H₂O₂ oxidoreductase) and peroxidase (1.11.1.7 [EC] , donor:H₂O₂ oxidoreductase) in wheat roots were comparatively higherthan were those in rice roots at corresponding stages. Cytochromec oxidase in roots remained active throughout the lives of therice and wheat crops. In rice roots, catalase seemed to playa distinct role around the panicle formation stage. Decay ofcatalase activity took place earlier than did that of peroxidaseand cytochrome c oxidase activities. In wheat roots similarenzyme activity changes were not observed. Data may suggestthat the high activity of iron containing oxidases at the panicleformation stage (I) may be chiefly due to catalase activityin rice roots. ¹Paper presented at the 14th Annual Meeting of the Society ofthe Science of Soil and Manure, Japan (1968). (Received November 21, 1968; )     

Isolation and Purification of Cytochrome b561 from a Photosynthetic Bacterium, Rhodopseudomonas sphaeroides

Cytochrome b₅₆₁ was removed from chromatophores of a photoanaerobicallygrown Rhodopseudomonas sphaeroides by deoxycholate-cholate andTriton X-100 treatments of the chromatophores. The cytochromewas purified by ammonium sulfate fractionation and gel filtration.Its molecular weight was 45,000 (45 kD) and it was composedof three subunits with molecular weights of 23 kD, 19 kD andless than 6 kD. The cytochrome preparation had absorption maximaat 414 nm in the oxidized form, and at 428, 530 and 561 nm inthe reduced form. Its pi was 4.8. The midpoint potential ofthis cytochrome was 153 mV at pH 7.0. The compound was autooxidizable,and it had cytochrome c oxidase activity. (Received May 16, 1983; Accepted September 8, 1983)     

Nitric oxide regulation of mitochondrial oxygen consumption II: Molecular mechanism and tissue physiology

Nitric oxide (NO) is an intercellular signaling molecule; among its many and varied roles are the control of blood flow and blood pressure via activation of the heme enzyme, soluble guanylate cyclase. A growing body of evidence suggests that an additional target for NO is the mitochondrial oxygen-consuming heme/copper enzyme, cytochrome c oxidase. This review describes the molecular mechanism of this interaction and the consequences for its likely physiological role. The oxygen reactive site in cytochrome oxidase contains both heme iron (a₃) and copper (Cu_B) centers. NO inhibits cytochrome oxidase in both an oxygen-competitive (at heme a₃) and oxygen-independent (at Cu_B) manner. Before inhibition of oxygen consumption, changes can be observed in enzyme and substrate (cytochrome c) redox state. Physiological consequences can be mediated either by direct "metabolic" effects on oxygen consumption or via indirect "signaling" effects via mitochondrial redox state changes and free radical production. The detailed kinetics suggest, but do not prove, that cytochrome oxidase can be a target for NO even under circumstances when guanylate cyclase, its primary high affinity target, is not fully activated. In vivo organ and whole body measures of NO synthase inhibition suggest a possible role for NO inhibition of cytochrome oxidase. However, a detailed mapping of NO and oxygen levels, combined with direct measures of cytochrome oxidase/NO binding, in physiology is still awaited. mitochondria; cytochrome oxidase     

Cytochromes in the Cultured Mycobiont of a Lichen, Cladonia vulcani Sav

Cytochromes in a cultured cells of the mycobiont of Cladoniavulcani Sav. were studied, b-and c-type cytochromes and aa₃-typecytochrome c oxidase were found in the membrane fraction, whileb- and c-type cytochromes were found in the soluble fraction.Soluble cytochrome c-549.5 was purified, and some of its molecularproperties were determined. (Received June 27, 1994; Accepted October 3, 1994)     

Cytochromes of Mitochondria from Rice Seedlings Germinated under Water and Their Changes during Air Adaptation

Rice seeds were germinated for up to 5 days under water (submerged)and some for another day in air (air-adapted). Control seedswere germinated for 6 days throughout in air. Low-temperaturedifference spectra of shoot mitochondria were compared amongthese three types of seedlings. All cytochromes found in theaerobic seedlings were present in the submerged seedlings. However,there were some differences in the cytochromes b₅₅₃ and c ofthese two types of seedlings. The cytochrome aa₃ peak heightand cytochrome oxidase activity per mitochondrial protein increased1.6- and 2.8-fold, respectively, during air adaptation. Slightlyhigher concentrations of the b-type cytochromes than found inair-adapted mitochondria were already present in submerged mitochondria.The computed difference between the dithionite-reduced differencespectra of mitochondria from submerged seedlings before andafter air adaptation, showed that cytochromes aa₃ and c hadincreased more than cytochrome b₅₅₇ during air adaptation. (Received November 16, 1987; Accepted March 16, 1988)     

Properties of the Cytochrome c Oxidase Activity of Cytochrome b561 from Photoanaerobically Grown Rhodopseudomonas sphaeroides

Cytochrome b₅₆₁ from Rhodopseudomonas sphaeroides had cytochromec (c2) oxidase activity and a pH optimum at 6.0 for this activity.The activity was affected by the ionic strength of the reactionmixture. The apparent K_m and maximal velocity (V_max) valuesin the absence of addea salts were 14 µM and 120 nmoloxidized per min per mg protein for horse heart cytochrome c.Reduced horse heart cytochrome c was reoxidized in first-orderkinetics by this cytochrome b₅₆₁. The specific activity was0.7 s^–1 per mg protein at 20°C at the concentrationof 30 µMM cytochrome c. Activity was inhibited by KCN and NaN₃, but not by antimycin.The addition of a low concentration of KCN to the cytochromeb₅₆₁ produced a change in the absorption spectrum, evidencethat KCN interacts with the heme moiety of cytochrome b₅₆₁.Results of this and preceeding studies show that the cytochromeoxidase (cytochrome "o") described earlier (Sasaki et al. 1970)is cytochrome b₅₆₁. (Received May 16, 1983; Accepted September 8, 1983)     

Effect of Intracellular ATP Levels on the Light-Induced H+ Efflux from Intact Cells of Cyanidium caldarium

The light-induced H⁺ efflux observed at acidic pH in Cyanidiumcells was shown to be an active H⁺ transport depending on theintracellular ATP produced by cyclic photo-phosphorylation.Triton X-100 was found to act as an effective uncoupler in intactCyanidium cells without collapsing the pH gradient across theplasma membrane. Triton X-100 at 0.015% significantly reducedthe intracellular ATP levels, stimulated the p-BQ, Hill reactionand completely inhibited the light-induced H⁺ efflux. Inhibitionof the H⁺ efflux by Triton X-100 correlated well with the depressionof the apparent rale of light-induced ATP synthesis as wellas the decrease in the intracellular ATP level in light. The light-induced H⁺ efflux was completely inhibited by diethylstilbestrol,a specific inhibitor of plasma membrane ATPase, without anychanges in the intracellular ATP level, thereby suggesting theparticipation of the plasma membrane ATPase in the light-inducedH⁺ efflux. ¹The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984)     

aa3-Type Cytochrome c Oxidase Occurs in Halobacterium halobium and Its Activity is Inhibited by Higher Concentrations of Salts

Cytochrome aa₃ was found to occur in Halobacterium halobiumat an early exponential phase of growth. Some of its spectralproperties were determined with a solubilized membrane fractionof the bacterium. Cytochrome c oxidizing activity of the cytochromeaa₃ was not dependent on salt concentration, but decreased steeplywith increasing buffer concentration of the reaction mixture,just as the activity of cytochrome aa₃ from non-halophilic bacteria. (Received July 4, 1986; Accepted October 17, 1986)     

The FAD-Enzyme Monodehydroascorbate Radical Reductase Mediates Photoproduction of Superoxide Radicals in Spinach Thylakoid Membranes

The photoreduction of dioxygen in spinach thylakoid membraneswas enhanced about 10-fold by the FAD-enzyme monodehydroascorbateradical (MDA) reductase at 1 µM. The primary photoreducedproduct of dioxygen catalyzed by MDA reductase was the superoxideradical, as evidenced by the inhibition of photoreduction ofCyt c by superoxide dismutase. The apparent K_m for dioxygenof the MDA reductase-dependent photoreduction of dioxygen was100 µM, higher by one order of magnitude than that observedwith thylakoid membranes only. Glutathione reductase, ferredoxin-NADP⁺reductase, and glycolate oxidase also mediated the photoproductionof superoxide radicals in thylakoid membranes at rates similarto those with MDA reductase. Among these flavoenzymes, MDA reductaseis the most likely mediator stimulating the photoreduction ofdioxygen in chloroplasts; its function in the protection fromphotoinhibition under excess light is discussed. (Received February 24, 1998; Accepted May 19, 1998)     

Effects of growth conditions on formation of cytochrome system of a denitrifying bacterium, Pseudomonas stutzeri

Cytochrome systems in cells of a denitrifying bacterium, Pseudomonasstutzeri (VAN NIEL strain), grown under different atmosphericconditions were compared with reference to the effects of nitrateand nitrite on cytochrome synthesis. When a culture was sufficiently aerated (aerobic conditions),synthesis of all cytochrome components was repressed, regardlessof the presence or absence of nitrate and nitrite. When aeratedmoderately (semi-aerobic conditions), both soluble and paniculatecytochromes c-552 and cytochrome b-558 contents markedly increasedeven in the absence of nitrate and nitrite. Under anaerobic or semi-aerobic conditions, nitrite inducedcytochrome a₂–c synthesis. This inductive effect of nitritewas counteracted by nitrate. Nitrate also repressed particulatecytochrome c-552 synthesis to some extent but nitrite did not. ¹Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima, Japan (Received June 24, 1969; )     

Zinc deficiency enhanced NAD(P)H-dependent superoxider radical production in plasma membrane vesicles isolated from roots of bean plants

The production of superoxide radical (O₂^–) was studiedin plasma membrane vesicles isolated by aqueous polymer two-phasepartitioning from roots of zinc-sufficient and zinc-deficientbean (Phaseolus vulgaris L. cv. Prélude) plants. Thetwo populations of vesicles were highly enriched in plasma membraneand had similar composition as evidenced by the specific membranemarker enzymes. Vesicles from zinc-deficient roots showed higherrates of NAD(P)H oxidation compared to vesicles from zinc-sufficientplants. The NAD(P)H-dependent formation of O₂^– in plasmamembrane vesicles was also highly increased by zinc deficiency.For both activities, a higher response to zinc deficiency wasobserved when NADPH was used as electron source. Re-supply ofzinc to deficient plants for 24 h substantially decreased therates of NAD(P)H oxidation and 0₂^– production in isolatedvesicles. The NADPH-dependent O₂^– generation was stronglystimulated by FAD and showed a high pH optimum; it was scarcelyaffected by Triton X-100 or even inhibited in the presence ofFAD and was almost insensitive to Antimycin A. The results suggest the presence at the plasma membrane of beanroots of an O₂^– generating activity, preferentially utilizingNADPH, which is affected by the zinc nutritional status of theplant. This finding, together with previous observations oncytosolic and microsomal fractions prepared from zinc-deficientroots of different plants, is consistent with a role of zincin membrane stabilization by controlling the level of oxidizingO₂ species. Key words: NAD(P)H oxidase, superoxide radical, plasma membrane, zinc deficiency     

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U₂). Analysis with marker enzymes indicated that the U₂ fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.Abbreviations L₁ first lower phase - NR nitrate reductase - NRA nitrate-reductase activity - PM plasma membrane - T:p Triton X-100 (octylphenoxy polyethoxyethanol) to protein ratio - U₂ second upper phase     

NADH-Linked Ferricyanide and Cytochrome c Reduction Activities in Corn Root Plasma Membrane

Corn root plasma membrane catalyzed NADH reduction of ferricyanideand cytochrome c over a wide pH range. At pH 7.5, apparent K_msof NADH-cytochrome c pair were significantly lower than thoseof NADH-ferricyanide pair. FMN and polylysine respectively enhancedthe reduction of ferricyanide and cytochrome c. Yet, polyaspartatedecreased the ferricyanide reduction. NADH oxidation observedin the presence of both ferricyanide and cytochrome c was significantlyslower than the sum of rates obtained with individual acceptors.The results suggest that the membrane may contain differentbut not totally independent reduction sites for cytochrome cand ferricyanide. (Received April 13, 1993; Accepted August 23, 1993)     

Effect of the hydrophile-lipophile balance of non-ionic detergents (Triton X-series) on the solubilization of biological membranes and their integral b-type cytochromes

The solubilization of four integral membrane proteins (i.e. cytochrome b-561 of the chromaffin granule membrane, cytochrome b₅ of the endoplasmic reticulum and the mitochondrial b-type cytochrome(s) as well as cytochrome c oxidase) has been studied at 0 °C using the non-ionic detergents of the Triton X-series having the common hydrophobic 4(1,1,3,3-tetramethylbutyl)phenoxy (t-octyl-phenoxy) group and a variable average number ( ) of polar ethylene oxide units added. Following a pre-extraction of peripheral membrane and matrix proteins with low and high salt concentration and a weak non-ionic detergent (Tween 20, average hydrophile-lipophile balance ( ), the amount of heme proteins solubilized by subsequent Triton X-solutions was measured. With the detergents tested the degree of solubilization decreased in the sequence cytochrome b-561 >cytochrome b₅ >mitochondrial cytochrome(s) b and parallelled the effect of the detergents on light scattering and the phospholipid to protein ratio of the three membranes. For all the b-cytochromes, the solubilizing power of the detergent increased with decreasing average length of the polar ethylene oxide chain and the hydrophile-lipophile balance as long as clouding did not occur (e.g. Triton X-114, and ). Thus, the greatest difference in the degree of solubilization of the three cytochromes was observed with Triton X-405 ( and ). All the cytochromes were most efficiently solubilized (i.e. approx. 90%) by Triton X-100 ( and ).     

Uridine phosphorylase activity of isolated plasma membranes of rat liver.

Plasma membranes were isolated from rat liver homogenates either by differential centrifugation or by fractionation in discontinuous sucrose density gradients. Both membrane preparations contained about 17% of the total uridine phosphorylase (EC 2.4.2.3) activity and 44% of the total 5'-nucleotidase (EC 3.1.3.5). The enrichment factor for uridine phosphorylase in the fractions prepared by differential centrifugation was about 2.8 and by the gradient method, as much as 11.0; the respective enrichment factors for 5'-nucleotidase were 1.8 and 9.5. Uridine phosphorylase activity of isolated plasma membrane fractions was stimulated 2.5-fold by 0.1% Triton X-100. Unlike the cytosol enzyme, uridine phosphorylase of plasma membranes showed little or no deoxyuridine-cleaving activity. Contamination of the membrane fractions by thymidine phosphorylase (EC 2.4.2.4) of the cytosol was negligible. The other subcellular organelles obtained by either procedure and characterized by marker enzyme activities were found not to contain significant uridine phosphorylase activity; the cytosol fractions contained just over 70% of the total uridine phosphorylase activity with an enrichment of only about 2.8-fold. The activity of the cytosol enzyme was not stimulated by Triton X-100.