The Isolation and Characterization of a Cytochrome b6f Complex from the Cyanobacterium Spirulina sp.

A cytochrome b₆f complex was isolated and purified from Spirulinasp. The complex was solubilized with n-heptyl ß-D-thioglucosideand chromatographed on a DEAE-Toyopearl 650M column. The purifiedcomplex contained a small amount of chlorophyll and carotenoid.At least four polypeptides were present in the complex: cytochromef (29 kDa), cytochrome b₆(23 kDa), iron-sulfur protein (ISP,23 kDa), and a 17 kDa polypeptide. Each polypeptide was separatedfrom the complex treated with 2-mercaptoethanol or urea. Theabsorption spectra of cytochrome b₆ and cytochrome f were similarto those of Anabaena and spinach as expected. The complex wasactive in supporting ubiquinol-cytochrome c oxidoreductase activity.Fifty percent inhibition of the activity was accomplished by1 µM dibromothymoquinone (DBMIB). The K_m values for ubiquinol-2and cytochrome c (horse heart) were 5.7 µM and 7.4 µM,respectively. (Received August 15, 1988; Accepted November 14, 1988)     

Comparison between Electron Transfers through Plastocyanin in Spinach Chloroplasts and Cytocbrome C2 in Rhodopseudomonas sphaeroides

Photosynthetic electron transfers through the water-solubleperipheral membrane proteins of plastocyanin and cytochromec₂, were studied in spinach chloroplasts and the photosyntheticbacterium Rhodopseudomonas sphaeroides. In spinach chloroplasts,the rate of flash-induced oxidation of cytochrome f was highlydependent on the salt concentration in the suspending medium.The maximum rate with a half time of 200 µs was observedin the presence of 50 mas KCl or 5 mM MgCl₂. The salt effectwas similar to that on the reaction rate between P700 in thylakoidfragments and externally added plastocyanin. On the other hand,in intact cells of R. sphaeroides, in which cytochrome c₂ islocated in the periplasmic space exposed to the outer ionicenvironment, the rate of cytochrome c₁ oxidation via cytochromec₂ was almost independent of salt concentration. This independencewas a contrast to the strong dependence on salt concentrationof reactions between isolated reaction centers and cytochromec₂ These results suggest that plastocyanin reacts collisionallywith the photosystem I reaction center and cytochrome b₆f complexin a manner that is controlled by the surface electrostaticpotential. Cytochrome c₂, on the other hand, reacts with thebacterial reaction center and cytochrome bc₁ complex probablyby forming a complex prior to activation of the reaction center. ¹ Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Fukazawa 2-1-1, Setagaya, Tokyo158, Japan.     

COMPONENTS OF THE ELECTRON-TRANSFERRING SYSTEM IN ACETOBACTER SUBOXYDANS AND RECONSTRUCTION OF THE LACTATE OXIDATION SYSTEM

1. Cytochromes a₁⁵⁹⁰, b⁵⁶⁰, c₁⁵⁵⁴ and c₁⁵⁵² were isolated andpurifiedfrom a strain of Acetobacter suboxydans. The proceduresusedwere described in detail.
2. The main cytochrome band at550-560 mµ in intact cellssplitted at liquid air temperatureinto two bands, 551 mµ(strong) and 559 mµ (weak).
3. Optical and physiological properties of the four cytochromeswere investigated. Lactic dehydrogenase activity was found tobe associated with cytochrome c₁⁵⁵⁴. The two c₁-type cytochromes,especially cytochrome c₁⁵⁵⁴, persisted in their reduced formafter the purification through many steps.
4. By some combinationsof isolated components reconstruction ofthe oxygen uptake systemcould be realized.
5. The oxygen-consuming activity of purifiedoxidase preparationswas accelerated by a-tocopherol but notby Emasoll 4130 andTween 80.
6. Some discussions were made onthe nature of terminal oxidase,the role of cytochrome c₁⁵⁵²in the electron-transport system,and persistence of reducedstate of c₁-type cytochromes.
7. A possible scheme of the electron-transferringsystem of Acetobactersuboxydans was presented.

(Received May 16, 1960; )     

Chromatic Regulation of Cytochrome Composition and Electron Transfer from Cytochrome b6-f Complex to Photosystem I in Anacystis nidulans (Tx 20)

Cytochrome composition of the cyanobacterial photosyntheticsystem was studied with Anacystis nidulans (Tx 20) in relationto the chromatic regulation of photosystem composition. Comparisonof cytochrome compositions in cells with a high PS I/II ratio(3.0, grown under weak orange light) and with a low ratio (1.6,grown under weak red light) indicated that cytochrome compositionwas also changed in the chromatic regulation of photosystemcomposition. Two types of cytochrome change were observed: 1)contents of cytochromes C₅₅₃ and c₅₄₈ were changed in parallelwith the changes in PS I content, and 2) cytochrome b₅₅₃ andcytochrome b₆-f complex were held at a constant molar ratioto PS II. The molar ratio, PS II : cytochrome b₅₅₉ : cytochromeb₆-f complex : cytochrome c₅₅₃ : PS I : cytochrome C₅₄₈, inthe red-grown cells was 1 : 2.5 : 1.3 : 0.17 : 1.6 : 0.67, andthe ratio in the orange-grown cells, 1:2.4:0.9:0.32:3.0:1.2.In both types of cells, almost all cytochrome f in the cytochromeb₆-f complex was rapidly oxidized after multiple flash activation,indicating that all cytochrome b₆-f complexes in cells of bothtypes are functionally connected to PS I, even when the molarratio to PS I is largely changed. The content of cytochromeC₅₅₃ was at most 0.14 of PS I, suggesting that the cytochrometurns over several times per one turnover of PS I. ¹Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Fukazawa 2-1-1, Setagaya, Tokyo158, Japan. (Received January 20, 1986; Accepted March 17, 1986)     

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)     

Contents of Cytochromes, Quinones and Reaction Centers of Photosystems I and II in a Cyanobacterium Synechococcus sp.

The contents of photosystem I and photosystem II reaction centers,cytochrome c-553, cytochrome c-550, cytochrome f, cytochromeb-559, cytochrome b-563, plastoquinone and vitamin K₁ in thecyanobacterium Synechococcus sp. were determined. About threephotosystem I reaction centers were present for each photosystemII reaction center. The amounts of cytochromes functioning betweenthe two photosystems were approximately half those of the photosystemI reaction center. Plastocyanin was not detected, while plastoquinoneand vitamin K₁ were present in excess of other electron carriersand reaction centers. The results indicate the importance ofplastoquinone and cytochrome c-553 for cooperation of the tworeaction centers through electron transport. ¹Present address: Toray Basic Research Laboratory, 1111 Tebiro,Kamakura, Kanagawa 248, Japan. (Received June 17, 1982; Accepted January 17, 1983)     

Respiratory Activities, Cytochrome Composition and Cytochrome c Oxidase Subunit II Levels of Mitochondria from Alloplasmic Wheats Having Aegilops Cytoplasms

Respiratory activities, cytochrome composition and cytochromec oxidase (EC 1.9.3.1 [EC] ) subunit II (COXII) levels of isolatedmitochondria in one euplasmic and four alloplasmic lines ofwheat (Triticum aestivum), having cytoplasms of Aegilops columnaris,Ae.crassa (4x),Ae. mutica and Ae. triuncialis, which show variousgrowth abnormalities, were studied. Cytoplasm-specific variationon the respiratory activities has been revealed among the cytoplasmsof five Triticum and Aegilops species. The cyanide-resistantrespiratory pathway does not seem to contribute to the variationin activity. The low level of state 3 and cytochrome oxidaseactivities were evident in lines having Ae. columnaris and Ae.triuncialis cytoplasms, which also show growth inhibition. Thelow cytochrome oxidase activity in both cytoplasms is consistentwith the low amount of cytochrome aa₃ and the severe reductionin COXII levels. The relatively low amount of cytochrome aa₃in Ae. mutica is also consistent with the reduced level of theCOXII. On the other hand, relatively low amounts of cytochromeb (b-557) are found in the Ae. crassa cytoplasmic line. Theseresults suggest that the growth abnormalities found in the alloplasmicwheat lines are related to the variation in respiratory activitiesand cytochrome composition. In particular, growth inhibitionfound in the alloplasmic wheats having Ae. columnaris and Ae.triuncialis may be caused by the severe depression of respiratoryactivity due to the impaired cytochrome oxidase activity oftheir mitochondria. ³Present address: Department of Biochemistry, Dalhousie University,Halifax, NS, Canada B3H 4H7     

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)     

Studies on cytochromes in photosynthetic electron transport system I. photoreduction and photo-oxidation of cytochrome b559 by photosystem II in spinach chloroplasts

Light-induced redox-reactions of cytochrome b₅₅₉ in spinachchloroplasts were investigated. Illumination of chloroplastsinduced photoreduction of cytochrorne b₅₅₉ Red light (650 nm)was more effective than far-red light (725 nm), indicating thatthe photoreduction is a photosystem II-mediated reaction. Onaddition of DCMU, the photoreduction was eliminated and a photooxidationof cytochrome b₅₅₉ was observed. The rate of this photooxidationwas faster with photosystem II light than with photo-systemI light. On addition of Mn⁺⁺ the photooxidation was partly suppressed;far-red light became as effective as red light in inducing photooxidationof cytochrome b₅₉₉, in the presence of DCMU and Mn⁺⁺. Ascorbate completely suppressed photooxidation of cytochromeb⁵⁵⁹ In the presence of ascorbate, however, photooxidation wasobserved in the presence of inhibitors or after inhibitory treatmentsof chloroplasts which affected the oxidizing side of systemII. These inhibitors and inhibitory treatments, but not DCMU,decreased the redoxpotential of cytochrome b₅₅₉. Reactivationof Hill reaction in Tris-washed chloroplasts by indophenol-ascorbatetreatment was not accompanied by an abolishment of photooxidationof cytochrome b₅₅₉. A possible mechanism is proposed to account for these reactionsof cytochrome b₅₅₉ in the photosynthetic electron transportin chloroplasts. (Received April 4, 1972; )     

Cytochrome b560 in chromatophores from Chromatium vinosum

A membrane-bound cytochrome of the B-type in Chromatium chromatophores,cytochrome b₅₆₀, was reduced both by flash light activationand continuous illumination in the presence of antimycin atcontrolled ambient redox potentials. The light-minus-dark differencespectra had peaks at 560 and 430 nm, and troughs at 445 and415 nm. The reduction was observed in the ambient redox potentialfrom 400 to about 200 mV. However, below 200 mV, a re-reductionof photooxidized C-type cytochrome superimposed the reductionof cytochrome b₅₆₀ In the absence of antimycin, the reductionwas not observed, suggesting that the reoxidation of cytochromeb₅₆₀ was faster than the reduction. Dark titrations at various pH values showed that E_m7 of thecytochrome b₅₆₀ was about 40 mV and the E_m value was pH-dependent(–60 mV/pH) from pH 6 to 9. Cytochrome b₅₆₀ had a pK ataround pH 9. The content and some properties of cytochrome b₅₆₀ were similarin chromatophores from either photoautotrophically or photoheterotrophicallygrown cells. The possibility of involvement of cytochrome b₅₆₀ in the photosyntheticelectron transfer is discussed. (Received April 19, 1980; )     

Studies on the metabolism of a sulfur-oxidizing bacterium VII. Oxidation of sulfite by a cell-free extract of Thiobacillus thiooxidans

Properties of the cell-free extract, prepared from a strainof Thiobacillus thiooxidans by sonic disruption followed byfractionation with centrifugatiori, were investigated with referenceto its sulfite-oxidizing activity. Without the addition of cofactors the particulate fraction(F-P)catalyzed oxidation of sulfite with oxygen or bacterial cytochromec-552 obtained from Pseudomonas stutzeri as electron acceptor.TMPD reduced by ascorbic acid was also oxidized by F-P. Thesoluble fraction(F-S) showed no activity in oxidizing sulfiteand TMPD, but stimulated TMPD oxidation by F-P. Oxygen uptake with either sulfite or TMPD as substrate was inhibitedby KCN, NaN₃, CO and c-phenanthroline. CO-Inhibition was reversedby light. Reduction of cytochrome c-552 by sulfite was insensitiveto these agents. Antimycin A markedly inhibited sulfite oxidation with eitheroxygen or cytochrome c-552 as electron acceptor, but was withouteffect on TMPD oxidation. DDC and SAO, both strong inhibitors of sulfur oxidation, didnot affect sulfite and TMPD oxidations. Cytochromes of the a, b and c types were contained in F-P. Thesecytochromes were rapidly reduced when F-P was incubated withsulfite. Cytochrome(s) of the c type was present in F-S, too. ¹VI.=References (3) ²Partly supported by a grant from the Ministry of Education ³Present address: Sanyo Women's College, Hatsukaichi, Hiroshima738, Japan ⁴Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima 734, Japan (Received May 15, 1970; )     

Isolation of the Photoactive Reaction Center Complex that Contains Three Types of Fe-S Centers and a Cytochrome c Subunit from the Green Sulfur Bacterium Chlorobium limicola f. thiosulfatophilum, Strain Larsen

The photoactive reaction center (RC) complex from the greensulfur bacterium Chlorobium limicola f. thiosulfatophilum, strainLarsen, was isolated after solubilization and ammonium sulfatefractionation followed by ion-exchange chromatography. The spectrumof the complex was almost identical with that of the similarRC complex isolated by Feiler et al. [(1992) Biochemistry 31:2608–2614] except for the presence of cytochrome c₅₅₁instead of c₅₅₃ in the latter study. A molecular ratio of BChla to P840 of the isolated RC complex was assayed to be 25–35.SDSPAGE analysis revealed that the isolated complex containedthree major polypeptides with apparent molecular masses of 68,41 and 21 kDa, respectively. The 21-kDa polypeptide was identifiedto be a heme-binding protein by staining the gel for peroxidaseactivity. The cytochrome c₅₅₁ was oxidized by flash light ina biphasic manner with half times of 90 and 390 µs, respectively,that coincided with the reduction half times of P840⁺. Threedistinct iron-sulfur centers assigned to F_A, F_B and F_x, respectively,from their g-values were detected by EPR spectroscopy at cryogenictemperature. These results suggest that the present preparationcontains a minimal functional unit of the RC of this bacterium,and that this complex appears to lie on a evolutionary linebetween RC's of purple bacteria and photosystem I. (Received August 18, 1992; Accepted October 28, 1992)     

A Cytochrome P450 Mediated Naringenin 3'-Hydroxylase from Sweet Orange Cell Cultures

A microsomal flavonoid 3'-hydroxylase (F3'H) catalyzing themetabolism of naringenin to eriodictyol in Citrus sinensis (L.)Osbeck cv. ‘Hamlin’ cell suspension cultures wasshown to be a cytochrome P450 enzyme. This reaction requiredO₂ and NADPH and was inhibited by CO, with partial reversalof CO-inhibition by light at 450 nm. Cytochrome P450 contentranged from 10–20 pmol (mg microsomal protein)^–1.The F3'H reaction was shown to be linear in regard to proteinconcentration between 2.5 and 25 µg of microsomal protein.The optimum pH for the reaction was 7.4–7.6 and the temperatureoptimum was between 30 and 37°C. The apparent K_m and V_maxfor naringenin were 24 µM±3.2 and 81.4±7.9pmol eriodictyol min^–1 (mg protein)^–1, respectively.The microsomal F3'H was also capable of forming dihydroquercetinfrom dihydrokaempferol (40 pmol min^–1 (mg protein)^–1)and of quercetin from kaempferol (3.25 pmol min^–1 (mgprotein^–1). Cytochrome c and ketoconazole were the bestinhibitors of WH activity followed by piperonyl butoxide anda-naphthoflavone. Light was shown to be an inducer of the F3'Halmost doubling the specific activity and increasing the microsomalcytochrome P450 content by 30% over that of dark grown cells.F3'H activity was also confirmed in microsomal preparationsof young (new flush) leaves from ‘Hamlin’ treesand flavedo of ‘Hamlin’ oranges, ‘Marsh’grapefruit, and ‘Lisbon’ lemon. No activity wasobserved in older, hardened leaves and albedo of all the fruittested. Initiation of embryogenesis in the ‘Hamlin’cell suspension cultures by switching from a sucrose mediumto a glycerol-based medium resulted in the down-regulation ofF3'H. ¹Mention of a trademark, warranty, proprietary product, or vendordoes not constitute a guarantee by the U.S. Department of Agricultureand does not imply its approval to the exclusion of other productsor vendors that may also be suitable.     

Effect of Oxygen on Photosynthetic 14CO2 Fixation in Chroomonas sp. (Cryptophyta) II. Effects of Inhibitors, Uncouplers and an Artificial Electron Mediator on the Inhibition of 14CO2 Fixation by Anaerobiosis

In "air-grown" Chroomonas sp. cells, low concentrations of DCMU(less than 0.1 µM) could prevent the inhibition of ¹⁴CO₂fixation by anaerobiosis under light-saturating conditions (morethan 40 W.m^–2), with phenazine methosulfate showing asimilar effect. Antimycin A, carbonyl cyanide m-chlorophenylhydrazone(CCCP), and N,N'-dicyclohexylcarbodiimide strongly inhibitedanaerobic photosynthesis at concentrations which did not significantlyinhibit the rate under 2% O₂ at high light intensity (200 W.m^–2),although 0.2 µM CCCP stimulated the rate under 2% O₂ tosome extent. On the other hand, KCN inhibited the rate muchmore strongly under 2% O₂ than N₂, although it inhibited therate very strongly at concentrations above 5 µM both underN₂ and 2% O₂. These results suggest that the inhibition of photosynthetic¹⁴CO₂ fixation by anaerobiosis in this alga result from ATPdeficiency caused by over-reduction of electron carriers ofthe cyclic electron flow and that oxygen can prevent the over-reduction.Cyclic electron flow seems to be necessary to provide additionalATP for CO₂ reduction under anaerobic conditions, although itseems to be less necessary under aerobic conditions. (Received July 21, 1983; Accepted January 23, 1984)     

Simultaneous changes in cell quotas of microcystin, chlorophyll a, protein and carbohydrate during different growth phases of a batch culture experiment with Microcystis aeruginosa

The cell quotas of microcystin (Q_mcyst), protein (Q_prot), chlorophylla (Q_chloro) and carbohydrate (Q_carbo), as well as the net productionrates of these parameters, were determined during the exponentialand stationary phases in nine batch cultures of Microcystisaeruginosa (CYA 228) at light regimes from 33 to 53 µmolphotons m^–2 s^–1. The following results were obtained.(i) A parallel pattern was found in the changes of Q_mcyst, Q_prot,Q_chloro and Q_carbo during the entire growth cycle and significantcorrelations were recorded between Q_mcyst and Q_prot, Q_chloroand Q_carbo. (ii) The net microcystin production rate (µ_mcyst)was positively correlated with the specific cell division rate(µ_c), the chlorophyll production rate (µ_chloro)and the protein production rate. (iii) A significant inverselinear relationship was found between µ_c and Q_mcyst, i.e.cultures with a positive µ_c had a Q_mcyst between 110 and400 fg microcystin cell^–1, while declining cultures hadQ_mcyst values >400 fg microcystin cell^–1. Maximum variationin Q_mcyst within cultures was 3.5-fold. Collectively, the resultsshow that cells produced microcystin at rates approximatingthose needed to replace losses to daughter cells during divisionand that microcystin was produced in a similar way to proteinand chlorophyll, indicating a constitutive microcystin production.     

Purification and Characterization of a Cytochrome P450 (trans-Cinnamic Acid 4-Hydroxylase) from Etiolated Mung Bean Seedlings

A Cyt P450 (P450_C4H) possessing trans-cinnamate 4-hydroxylase(C4H) activity was purified to apparent homogeneity from microsomesof etiolated mung bean seedlings. Upon SDS-polyacrylamide gelelectrophoresis, the purified preparation gave a single proteinband with a molecular mass of 58-kDa. Its specific P450 contentwas 12.6 nmol (mg protein)^–1. Using NADPH as electrondonor, purified P450_C4H aerobically converted trans-cinnamicacid to p-coumaric acid with a specific activity of 68 nmolmin^–1 nmol^–1 P450 in a reconstituted system containingNADPH-Cyt P450 reductase purified from the seedlings or rabbitliver microsomes, dilauroyl phosphatidylcholine, and cholate.This specific activity is by far the highest for reconstitutedC4H systems so far reported and provides direct evidence thatC4H activity is actually associated with a P450 protein. Inthe oxidized state P450_C4H showed a typical low-spin type absorptionspectrum with a Soret peak at 419 nm. A partial spectral shiftto the high spin state was observed when trans-cinnamic acidwas added to oxidized P450_C4H. By spectral titration, the dissociationconstant of the cinnamic acid-P450_C4H complex was determinedto be 2.8 µM. This value is similar to the K_m value (1.8µM) for trans-cinnamic acid determined in the reconstitutedsystem. (Received November 20, 1992; Accepted February 17, 1993)     

Rates of Photosynthesis in Characean Cells: I. PHOTOSYNTHETIC 14CO2 FIXATION BY NITELLA TRANSLUCENS

A study has been made of photosynthetic ¹⁴CO₂ fixation by isolated‘mature’ internodes of Nitella translucens. Experimentalconditions were similar to those used in studies of the ionicrelations of these cells. Maximum rates of photosynthesis were33–40µµmoles CO₂, fixed per cm² of surfacearea per second (equivalent to 12–15 /xmoles fixed permg chlorophyll per hour). ^l4CO₂ fixation was inhibited to thedark level by 3(3,4,dichlorophenyl)-1, 1-dimethylurea (at 0-6µM or 10µM) and by the uncoupler carbonyl cyanide-m-chlorophenylhydrazone(SµM). The presence of imidazole or ammonium sulphate(both of which uncouple ATP production in vitro) did not resultin an inhibition of 14CO2 fixation. These results are discussedin relation to published work on solute uptake by Nitella translucens.During photosynthesis there was rapid movement of ¹⁴C-labelledorganic compounds out of the chloroplasts. ¹⁴C-labelled sucrose,ammo-acids, and sugar phosphates were found in samples of vacuolarsap.     

Purification and characterization of CMP-N-acetylneuraminic acid hydroxylase from pig submandibular glands

N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-giycosideby the action of CMPN-acetylneuramlnic acid (CMP-Neu5Ac) hydroxylase.This enzyme is a soluble cytochrome bs-dependent monooxygenaseand has been purified to apparent homogeneity from pig submandibularglands by precipitation with N-cetyN,N,N-trimethylam-moniumbromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose,Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12.This procedure resulted in an 8960-fold purification of thehydroxylase with a recovery of 0.8%. The molecular mass of thisprotein was shown to be 65 kDa on SDS-PAGE and 60 kDa as determinedby gel filtration on Superose S.12, which suggests that theenzyme is a monomer. The purified CMP-Neu5Ac hydroxylase isactivated by FeSO₄ and inhibited by iron-binding reagents suchas o-phenanthroline, KCN, Tiron and ferro-zine. An apparentKm of 11 µM was determined for the substrate CMP-Neu5Acusing purified hydroxylase in the presence of Triton X-100-solubilizedmicrosomes. In a reconstituted system consisting of purifiedhydroxylase, cytochrome b₅, cytochrome b₅ reductase and catalase,an apparent K_m of 3 µM was measured. The apparent K_mforcytochrome b₅ in this system was 0.24 µM. Immunizationof a rabbit with enriched and purified hydroxylase led to anantiserum that inhibited CMP-Neu5Ac hydroxylase activity andreacted with the purified 65 kDa protein on a Western blot afterSDS-PAGE. Antibodies specific for this 65 kDa protein were isolatedand showed a strong reaction with the purified CMP-Neu5Ac hydroxylasefrom mouse liver after immunoblotting. Initial experiments withthis monospecific antibody suggest that the activity of thehydroxylase in a particular tissue correlates with the amountof immuno-reactive protein. cytochrome b₅ N-glcoloylneuraminic acid hydroxylase pig submandibular gland mucin sialic acid     

Specific Inhibition of Electron Transfer for Nitrate Reduction by a Low Concentration of Cyanide in Rhodobacter sphaeroides f. s. denitrificans

The effects of cyanide on the electron flow in NO₃^– andNO₂^– reductions and photosynthetic electron transfer wereinvestigated with intact cells of a photodenitrifier, Rhodobactersphaeroides f. s. denitrificans. In the presence of 30 µMKCN, electron transfer for NO₃^– reduction was inhibitedby about 70% and the concomitant H translocation was completelyinhibited. However, neither NO₂^– reduction nor photosyntheticcyclic electron transfer was affected at 30 µM. Theseresults suggested that the electron transfer pathway to NO₃^–has, in addition to a b-type cytochrome and the nitratereductase,a component sensitive to a low concenration of cyanide whichis not involvedin the cytochrome bc₁ complex. (Received April 13, 1987; Accepted July 23, 1987)     

Purification and properties of a dissimilatory nitrite reductase of a denitrifying phototrophic bacterium

Nitrite reductase [nitric-oxide : (acceptor) oxidoreductase,EC 1.7.2.1 [EC] ] from a denitrifying phototrophic bacterium, Rhodopseudomonassphaeroides forma sp. denitrificans, was purified. The molecularweight of the enzyme, estimated by gel-filtration, was 80,000.Sodium dodecyl sulfate polyacrylamide gel electrophoresis ofthe purified enzyme showed a single 39,000 molecular weightband, indicating that the enzyme was composed of two subunitsof identical molecular weight. The oxidized form of the enzymeexhibited maximum absorption at 280 nm, 450 nm and 590 nm, andthe reduced form only at 280 nm. The ESR spectrum of a frozensolution of the oxidized enzyme showed a typical spectrum patternof a copper protein, suggesting that two types of Cu²⁺ existedwithin the enzyme. Estimates with an atomic absorption spectrophotometer,revealed two copper atoms per molecule. The optimum pH of theenzyme was 7.0. Km for nitrite was estimated to be 51 µM,and the optimum temperature, 30?C. The enzyme was inhibitedby CO, potassium cyanide and diethyldithiocarbamate and activatedby monoiodoacetate. Phenazine methosulfate, 2,6-dichlorophenolindophenol,horse heart cytochrome c, and cytochrome c₂ from this bacteriumwere suitable electron donors. The enzyme also showed cytochromec oxidase activity. (Received May 4, 1978; )