The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1 I. Spectral properties and redox potentials

1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl₁ which lacks cytochrome aa₃. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied.2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the α peaks of b-type cytochrome(s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two α peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochrome c₁. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl₁ in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa₃ was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm.3. Cytochrome aa₃ was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl₁. Cytochrome aa₃ was more susceptible to heat than cytochromes b and c,c₁.4. CO difference spectra at 77 K revealed two different Co-cytochrome complexes. The first, found only in wild type mitochondria, was a typical CO-cytochrome a₃ complex characterized by peaks at 596 and 435 nm and troughs at 613 and 450 nm. The second, found both in mitochondria of the wild type and the mutant, was a CO-cytochrome b complex with peaks at 567, 539 and 420 nm and a trough at 558-549 nm. Both complexes are photo-dissociable.5. Spectral evidence was obtained for interaction of cyanide with the a-type cytochrome (shift of the α peak at 77 K from 608 to 605 nm), but not with the b-type cytochrome.6. The mid-point potentials of the different cytochromes at neutral pH are as follows: cytochrome aa₃ 235 and 395 mV, cytochrome c,c₁ 233 mV, cytochromes b 120 mV.     

Isolation and properties of cytochrome c-553, cytochrome c-550, and cytochrome c-549, 554 from Nitrobacter agilis

Three c-type cytochromes isolated from Nitrobacter agilis were purified to apparent homogeneity: cytochrome c-553, cytochrome c-550 and cytochrome c-549, 554. Their amino acid composition and other properties were studied. Cytochrome c-553 was isolated as a partially reduced form and could not be oxidized by ferricyanide. The completely reduced form of the cytochrome had absorption maxima at 419, 524 and 553 nm. It had a molecular weight of 25 000 and dissociated into two polypeptides of equal size of 11 500 during SDS gel electrophoresis. The isoelectric point of cytochrome c-553 was pH 6.8. The ferricytochrome c-550 exhibited an absorption peak at 410 nm and the ferrocytochrome c showed peaks at 416, 521 and 550 nm. The molecular weight of the cytochrome estimated by gel filtration and by SDS gel electrophoresis was 12 500. It had an E_m(7) value of 0.27 V and isoelectric point pH 8.51. The N-terminal sequence of cytochrome c-550 showed a clear homology with the corresponding portions of the sequences of other c-type cytochromes. Cytochrome c-549, 554 possessed atypical absorption spectra with absorption peaks at 402 nm as oxidized form and at 419, 523, 549 and 554 nm when reduced with Na₂S₂O₄. Its molecular weight estimated by gel filtration and SDS polyacrylamide gel electrophoresis was 90 000 and 46 000, respectively. The cytochrome had an isoelectric point of pH 5.6. Cytochrome c-549, 554 was highly autoxidizable.     

Cytochromes and other pigments of dissimilatory sulphate-reducing bacteria

Summary A survey was made of various visible light absorption spectra of whole cells, particulate and soluble fractions and haem extracts of representative strains of all known species of sulphate-reducing bacteria. The previously accepted distinction that Desulfovibrio species contain only a c-type cytochrome whereas Desulfotomaculum species contain only a b-type cytochrome was not confirmed. The pigment contents of the genera Desulfovibrio and Desulfotomaculum were not completely distinct from each other, but both genera had characteristic spectral patterns. Reduced minus oxidized spectra of whole cells and particulate fractions showed the presence of b-type cytochromes in all Desulfotomaculum species and in Desulfovibrio africanus. However, protohaem, the prosthetic group of b-type cytochromes, occurred in haem extracts from all species, although only just detectable in the extract from Desulfovibrio vulgaris NCIB 8303. Particulate c-type cytochromes were found in Desulfotomaculum orientis, Desulfotomaculum nigrificans and all the Desulfovibrio species, but the amount in Desulfotomaculum nigrificans was very small. Only the Desulfovibrio species contained soluble c-type cytochromes. Spectral properties indicated that a d-type cytochrome might exist in species in addition to Desulfovibrio africanus, but no supporting evidence was obtained from results of haem extractions. Some spectra contained peaks which could not be identified.     

Particulate cytochromes of mung bean seedlings

Efforts have been made to solubilize cytochrome components from particulate fractions of etiolated mung bean seedlings. Low temperature spectrophotometry reveals that the cytochrome composition of mitochondria isolated from whole seedlings is the same as that reported by Bonner for mung bean hypocotyls. On the basis of the identity in position of the α-bands in low temperature difference spectra for mitochondria, for a partially purified haemoprotein from mitochondria, and for purified cytochrome b-555, it is suggested that cytochrome b-555 is an intrinsic component of mung bean mitochondria. Difference spectra show that both the mitochondrial and microsomal fractions contain at least 2 b-type cytochromes. Cytochrome b-555 is almost certainly present in the microsomes, since the low temperature difference spectrum for the cytochrome is identical with the spectrum for this particulate fraction.

By freezing and thawing mung bean mitochondria in 4% cholate and centrifuging, cytochrome oxidase activity can be concentrated in the supernatant fraction, although it is not completely solubilized. The oxidase is inhibited by high concentrations of cytochrome c. A particle-bound cytochrome c can be obtained from mitochondria by digestion with snake venom. However, the autoxidizability of the preparation indicates that the cytochrome has been solubilized in a modified form. A CO-binding pigment can be obtained from mung bean microsomes by digestion with snake venom.

     

Cytochrome aa 3 from Methylococcus capsulatus (Bath)

A cytochrome aa ₃-type oxidase was isolated with and without a c-type cytochrome (cytochrome c-557) from Methylococcus capsulatus Bath by ion-exchange and hydrophobic chromatography in the presence of Triton X-100. Although cytochrome c-557 was not a constitutive component of the terminal oxidase, the cytochrome c ascorbate-TMPD oxidase activity of the enzyme decreased dramatically when the ratio of cytochrome c-557 to heme a dropped below 1:3. On denaturing gels, the purified enzyme dissociated into three subunits with molecular weights of 46,000, 28,000 and 20,000. The enzyme contains two heme groups (a and a ₃), absorption maximum at 422 nm in the resting state, at 445 and 601 nm in the dithionite reduced form and at 434 and 598 nm in the dithionite reduced plus CO form. Denaturing gels of the cytochrome aa ₃-cytochrome c-557 complex showed the polypeptides associated with cytochrome aa ₃ plus a heme c-staining subunit with a molecular weight of 37,000. The complex contains approximately two heme a, one heme c, absorption maximum at 420 nm in the resting state and at 421, 445, 522, 557 and 601 nm in the dithionite reduced form. The specific activity of the purified enzyme was 130 mol O₂/min · mol heme a compared to 753 mol O₂/min · mol heme a when isolated with cytochrome c-557.Abbreviations MMO methan monooxygenase - sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - TMPD N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride - Na₂EDTA disodium ethylenediamine-tetraacetic acid     

Changes in cytochrome levels during growth of the yeast-like fungusAureobasidium pullulans

Cytochromes ofAureobasidium pullulans have been identified and partially characterized using low-temperature and carbon-monoxide-difference spectroscopy. The presence ofa-,b-, andc-type cytochromes is demonstrated, as are other unidentified redox components. During exponential growth in batch culture, cytochrome levels showed complex changes. Changes in respiration rates and in the levels of cytochromea+a ₃ closely paralleled cellular growth: both increased exponentially until stationary phase, when no further increase occurred. Theb- andc-type cytochromes showed biphasic increases, initially doubling every, generation time and then increasing more slowly during the stationary phase. Sensitivity of respiration to 100M potassium cyanide gradually decreased during exponential growth, falling from virtually 100% inhibition after about 20 h growth to 30% inhibition in the stationary phase. The results suggest that in stationary-phase cultures, an alternative cyanide-insensitive but salicylhydroxamic-acid-sensitive terminal oxidase also operates.     

Spectral properties of soluble and pelletable phytochrome from epicotyls of etiolated pea seedlings

The red-light(R)-absorbing form of phytochrome (Pr) was detected spectrophotometrically in a 20,000 g particulate fraction prepared from a 1,000 g supernatant fraction from epicotyl tissue of pea (Pisum sativum L.) seedlings grown in the dark and only briefly exposed to dim green light. The difference spectrum of phytochrome in this fraction was essentially the same as that of soluble phytochrome from the same tissue. When the non-irradiated 20,000 g particulate fraction was incubated in the dark at 25° C, an absorbance change (decrease) of Pr after actinic red irradiation was found only in the far-red (FR) region. When the 20,000 g particulate fraction was irradiated with R and then incubated in the dark, the FR-absorbing form of phytochrome (Pfr) disappeared spectrally at a rate about half that in the soluble fraction, and the difference spectrum of the Pr which became detectable after dark incubation of the 20,000 g particulate fraction was markedly distorted. In contrast, Pfr in a 20,000 g particulate fraction prepared from tissues irradiated with R did not change optically during dark incubation at 25° C for 60 min, while Pfr in the soluble fraction from the same tissue disappeared in the dark. No dissociation of either Pr or Pfr from the 20,000 g particulate fraction was indicated during a 60-min dark incubation at 25° C, but Pfr in a 20,000 g particulate fraction prepared in vitro from R-irradiated 1,000 g supernatant fraction in the presence of CaCl₂ disappeared spectrally and the difference spectrum of Pr in the 20,000 g particulate fraction became quite distorted during the dark incubation.Abbreviations Pr red-light-absorbing form of phytochrome - Pfr far-red-light-absorbing form of phytochrome - FR far-red light - FR1 first actinic far-red light - FR2 second actinic far-red light - R red light - R1 first actinic red light - 1kS 1,000 g supernatant fraction - 20kS 20,000 g supernatant fraction - 20kP 20,000 g particulate fraction     

Inhibition of electron transfer through the cytochrome b-c 1 complex by nitric oxide in a photodenitrifier,Rhodopseudomonas sphaeroides forma sp. denitrificans

The effects of nitric oxide (NO) on electron transfer were studied with a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans. NO inhibited the oxidation of cytochrome c induced by continuous illumination in intact cells. NO inhibited the re-reduction of cytochrome c, the slow phase of the carotenoid bandshift, and the oxidation of cytochrome b after a flash illumination, suggesting that NO inhibited the photosynthetic cyclic electron transfer through the cytochrome b-c ₁ region. NO also inhibited the nitrite (NO ₂ ^- ) and NO reductions with succinate as the electron donor in intact cells, but did not inhibit the NO ₂ ^- and NO reductions in chromatophore membranes with ascorbate and phenazine methosulfate as the electron donors. NO reversibly inhibited the ubiquinol: cytochrome c oxidoreductase of the membranes, suggesting that NO inhibited the electron transfer through the cytochrome b-c ₁ region and that the cytochrome b-c ₁ complex also was involved in the electron transport in both NO ₂ ^- and NO reductions. The catalytic site of NO reduction was distinct from the inhibitory site of NO.Abbreviations UHDBT 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole - UHNQ 3-undecyl-2-hydroxy-1,4-naphthoquinone - MOPS 3-(N-morpholino)propane-sulfonic acid - PMS phenazine methosulfate - DCIP 2,6-dichlorophenol indophenol - DDC diethyl-dithiocarbamate     

Artefacts induced on <Emphasis Type="Italic">c</Emphasis>-type haem proteins by electrode surfaces

In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met–His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c ₃ from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.     

Reconstitution of photosynthetic electron transport and photophosphorylation in cytochrome-c2-deficient membrane preparation of Rhodopseudomonas capsulata.

Cytochrome c₂ was removed by washing from heavy chromatophores prepared from Rhodopseudomonas capsulata cells. The easy removal of the cytochrome could indicate that it was attached on the outside of the membrane. Therefore, the membrane was probably oriented inside out in relation to the membrane of regular chromatophores, from which cytochrome c₂ could not be removed. Washing of the heavy chromatophores caused loss of photphosphorylation activity. The activity was restored to the resolved heavy chromatophores by the supernatant obtained during the washing or by the native cytochrome c₂, which was found to be the active component in this supernatant. The activity could not be restored by other c-type cytochromes. Ascorbate, which enhanced photophosphorylation activity in the heavy chromatophores at the optimal concentration of 8 mm, restored this activity to the washed heavy chromatophores, but at an optimum concentration of 50 mm. Cytochrome c₂ and dichlorophenol indophenol reduced the optimum of the ascorbate concentration to 7 mm. This might indicate that the effect of ascorbate is mediated through cytochrome c₂. Washing the heavy chromatophores caused 70% loss of the light-induced electron transport from ascorbate and from ascorbate-reduced dichlorophenol indophenol to O₂. However, this effect was only observed with the lower concentrations of ascorbate and the dye. The activity was restored either by the supernatant obtained from the washing or by various c-type cytochromes, reduced by ascorbate. Washing the heavy chromatophores did not affect succinate oxidation in the dark. It is suggested that cytochrome c₂ is one of the cytochromes catalyzing the photosynthetic cyclic electron transport, as has been seen from its high specificity in the reconstitution experiments. Light can induce oxidation of various c-type cytochromes and other redox reagents. However, reduction was specific for cytochrome c₂ from Rps. capuslata, since it was the only one which could be both reduced and oxidized as required from a component which is part of a cyclic electron transport chain. It is also suggested that cytochrome c₂ was not part of the succinate oxidase system.     

Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b ₅₅₄ (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b ₅₅₆ (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth. Received: 21 August 1996 / Accepted: 24 October 1996     

Effect of NADP+ on light-induced cytochrome changes in membrane fragments from a blue-green alga

The effect of NADP⁺ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragments prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP⁺ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH₂) to NADP⁺. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH₂ as the electron donor, NADP⁺ had no effect on the light-induced redox changes of cytochromes: with or without NADP⁺, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an α peak at 549 nm. With 664 nm illumination and water as the electron donor, NADP⁺ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b₅₅₉. Cytochrome b₅₅₉ appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP⁺) for the electron flow from water.     

The electron transport system of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum. 1. A functional and thermodynamic analysis of the respiratory chain in aerobically and photosynthetically grown cells

Plasma membranes isolated from cells of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum grown in light or in the dark were examined. Membranes isolated from cells grown aerobically in the dark contained three b-type and two c-type membrane-bound cytochromes with E _m,7 of +180, +72 and –5 mV (561–575 nm), and +244 and +27 mV (551–540 nm), respectively. Conversely, membranes isolated from cells grown anaerobically in the light contained two b-type and five c-type haems with E _m,7 of +60 and –45 mV and +290, +250, +135, –20 and –105 mV, respectively. In addition to haems of the b- and c-type, two haems of the a-type (E _m,7 of +325 and +175 mV) were present only in cells grown in the dark. Four soluble cytochromes of the c type, but not cytochrome c ₂, along with two high-potential iron-sulfur proteins (HiPIP iso-1 and iso-2) were also identified in cells grown aerobically. Inhibitory studies showed that 85–90% of the respiratory activity was blocked by very low concentrations of cyanide, antimycin A and myxothiazol (50, 0.1 and 0.2 mM, respectively). These results taken together were interpreted to show that the oxidative electron transport chain of Rsp. salinarum is linear, leading to a membrane-bound oxidase of the aa ₃ type in cells grown in the dark, while no significant cytochrome oxidase activity is catalyzed by photosynthetic membranes. These features suggest that this halophilic species is unique among the genus Rhodospirillum and that it also differs from other facultative phototrophs (e.g., Rhodobacter species) in that it does not contain either cytochrome c ₂ or a branched respiratory chain. Received: 25 February 1997 / Accepted: 20 May 1997     

Purification, characterization and gene sequence analysis of a novel cytochrome c co-induced with reductive dechlorination activity in Desulfomonile tiedjei DCB-1

The sulfate-reducing bacterium, Desulfomonile tiedjei DCB-1, conserves energy for growth from reductive dechlorination of 3-chlorobenzoate via halorespiration. To understand this respiratory process better, we examined electron carriers from different cellular compartments of D. tiedjei. A 50-kDa cytochrome from the membrane fraction was found to be co-induced with dechlorination activity. This inducible cytochrome was extracted from the membrane fractions by Tris-HCl buffer containing ammonium sulfate at 35% saturation and was purified to electrophoretic homogeneity by phenyl superose, Mono Q, and hydroxyapatite chromatography. The purified cytochrome had a high-spin absorption spectrum. In a pH titration experiment, the absorption spectrum of the inducible cytochrome shifted to low spin at pH 13.2. The midpoint potential of the inducible cytochrome at pH 7.0 was –342 mV. The NH₂-terminal amino acid sequence of the inducible cytochrome was determined and was used to obtain inverse PCR products containing the sequence of the gene encoding the inducible cytochrome. The ORF was 1398 bp and coded for a protein of 52.6 kDa. Two c-type heme-binding domains were identified in the COOH-terminal half of the protein. A putative signal peptide of 26 residues was found at the NH₂-terminal end. The protein sequence was not found to have substantial sequence similarity to any other sequence in GenBank. We conclude that this is a c-type cytochrome substantially different from previously characterized c-type cytochromes. Received: 30 May 1997 / Accepted: 29 July 1997     

Membranes of Rhodopseudomonas sphaeroides VII. Photochemical properties of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes

Previous pulse-chase studies have shown that bacteriochlorophyll a-protein complexes destined eventually for the photosynthetic (chromatophore) membrane of Rhodopseudomonas sphaeroides appear first in a distinct pigmented fraction. This rapidly labeled material forms an upper band when extracts of phototrophically grown cells are subjected directly to rate-zone sedimentation. In the present investigation, flash-induced absorbance changes at 605 nm have demonstrated that the upper fraction is enriched two-fold in photochemical reaction center activity when compared to chromatophores; a similar enrichment in the reaction center-associated B-875 antenna bacteriochlorophyll complex was also observed. Although b- and c-type cytochromes were present in the upper pigmented band, no photoreduction of the b-type components could be demonstrated. The endogenous c-type cytochrome (E_m = +345 mV) was photooxidized slowly upon flash illumination. The extent of the reaction was increased markedly with excess exogenous ferrocytochrome c but only slightly in chromatophores. Only a small light-induced carotenoid band shift was observed. These results indicate that the rapidly labeled fraction contains photochemically competent reaction centers associated loosely with c-type and unconnected to b-type cytochrome. It is suggested that this fraction arises from new sites of cytoplasmic membrane invagination which fragment to form leaky vesicles upon cell disruption.     

Intracellular localisation of hexokinase in Cuscuta reflexa

In Cuscuta reflexa 16% of the hexokinase activity was associated with the particulate fraction and the rest in the 105 000 g, 1 hr supernatant. In a sucrose gradient, hexokinase activity banded with an organelle at a mean density of 1.20 g cm^?3, coinciding with the mitochondrial marker, cytochrome c oxidase. Fractionation of isolated mitochondria by digitonin showed the presence of the enzyme in the outer membrane along with its marker rotenone-insensitive NADH cytochrome c reductase. No latent form of hexokinase was detected.     

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.Abbreviations IEF isoelectric focusing - M _r molecular weight - pI isoelectric point     

ENZYME-STRUCTURE RELATIONSHIPS IN THE ENDOPLASMIC RETICULUM OF RAT LIVER : A Morphological and Biochemical Study

Subfractionation of preparations of rat liver microsomes with a suitable concentration of sodium deoxycholate has resulted in the isolation of a membrane fraction consisting of smooth surfaced vesicles virtually free of ribonucleoprotein particles. The membrane fraction is rich in phospholipids, and contains the microsomal NADH-cytochrome c reductase, NADH diaphorase, glucose-6-phosphatase, and ATPase in a concentrated form. The NADPH-cytochrome c reductase, a NADPH (or pyridine nucleotide unspecific) diaphorase, and cytochrome b₅ are recovered in the clear supernatant fraction. The ribonucleoprotein particles are devoid of, or relatively poor in, the enzyme activities mentioned. Those enzymes which are bound to the membranes vary in activity according to the structural state of the microsomes, whereas those which appear in the soluble fraction are stable. From these findings the conclusion is reached that certain enzymes of the endoplasmic reticulum are tightly bound to the membranes, whereas others either are loosely bound or are present in a soluble form within the lumina of the system. Some implications of these results as to the enzymic organization of the endoplasmic reticulum are discussed.     

A Novel Type of D-Mannitol Dehydrogenase from Acetobacter xylinum: Occurrence,Purification, and Basic Properties

We purified a novel type of D-mannitol dehydrogenase, which contains a c-type cytochrome and an unknown chromophore in the soluble fraction of an acetic acid bacterium, Acetobacter xylinum KU-1, to homogeneity. The enzyme showed the maximum activity at pH 5 and 40°C. It was stable up to 60°C at pH 6, and was inhibited by Hg²⁺ and p-quinone (K_i = 0.18 mm). The molecular weight of the enzyme was about 140,000, and those of the subunits were 69,000, 51,000, and 20,000; the enzyme is hetero-trimeric and contained 8 g-atoms of Fe per mole. The α-helix content was estimated to be about 52.9%. The enzyme catalyzed phenazine methosulfate dependent oxidation of d-mannitol with an apparent K_m of 98 μm (for d-mannitol) and V_max of 213 μmol/min/mg. The reduced form of the enzyme showed the absorption maxima at 386, 416, 480, 518, 550, and 586 nm, which are attributable to a c-type cytochrome in the enzyme.