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This article is a critique of the meaning and importance of some of the recently published work in the field whose development as a special area began with papers by Zuckerkandl & Pauling and Pauling & Zuckerkandl entitled Chemical Paleogenetics, Molecular Restoration Studies of Extinct Forms of Life and the paper by Margoliash on the evolution of cytochrome c. The primary emphasis is on conclusions and inferences derivable from amino acid sequences, followed bya section on nucleotide sequences. These are preceded by a brief indication of some of the literature that antedates the origin of the genetic code as it now stands. This will not treat immunoglobulin evolution, reviewed by Gally & Edelma and Smith, Hood & Fitch or related material on the evolution of enzymes and proteins reviewed by Smith and Arnheim. The functional interpretation of these changes has made significant progress [Dickerson and Takano et al]. For the evolution of metabolic pathways, see brew, Vogel, and Lejohn. [ABSTRACT FROM AUTHOR]

To show the occurrence of cytochrome c-1 in ϱ- petite mutants of Saccharomyces cerevisiae, in which its presence was questioned, low-temperature absorption spectra of various ϱ+ or ϱ- strains have been compared. Some of these strains carry nuclear mutations producing different types of cytochrome deficiencies, thus lowering the spectral interferences on cytochrome c1 and making easier its observation. The comparison was made with whole cells and with mitochondrial preparations. All the ϱ- mutants examined present a shoulder or a peak at a wavelength corresponding to cytochrome c1 in the ϱ+ strains. This absorption band at 552 nm in ϱ- mutants was further identified by isolating a cytochrome with an α absorption maximum at this wavelength. The low-temperature absorption spectrum of this cytochrome was compared to low-temperature absorption spectra of cytochrome c1 isolated from a wild-type strain, and of cytochrome c and cytochrome b2. It is similar, if not identical, to the spectrum of the wild-type cytochrome c1, and quite distinct of that of the other cytochromes. The cytochrome c1 content, in the various ϱ+ and ϱ- strains examined, is estimated with the help of a calculation procedure taking into account the spectral cross interferences of the cytochromes. In the absorption spectra of the ϱ- strains, a shoulder is recorded at about 557-558.5 nm. The nature of this shoulder is discussed. [ABSTRACT FROM AUTHOR]

Rhodopseudomonas spheroides Y cells were grown photosynthetically in a defined medium supplemented with either a limiting amount of iron or with an excess of iron. Soluble and bound cytochromes were determined in both cases. The composition of the soluble cytochromes was not altered qualitatively by iron deficiency. In both culture conditions, a number of cytochromes were isolated by chromatography and purified to various extents: cytochromes c-550 (c2 type), c', c-551, c-552, and b-559. In normal cells cytochrome c2, and to a lesser extent cytochrome c' predominated whereas the amounts of both were greatly lowered in iron deficiency. Bound cytochromes were detected by difference spectroscopy of particles after chemically or lightinduced oxidoreductions. When iron was not limiting, cells were also grown in presence of diphenylamine, which inhibited carotenoid synthesis and facilitated detection of cytochromes. Particles from those cells, as well as particles from uninhibited cells contained cytochrome c-551 (bound form of cytochrome c2), cytochrome b-560, and a minor component, possibly b-type. All reacted to illumination. In iron-deficient cells, the bound heine b was almost completely suppressed; difference spectra suggested the presence only of cytochrome c2 and c'. [ABSTRACT FROM AUTHOR]

Activity levels of the enzymes involved in ketone utilization were compared in heart, fast-twitch red, slow-twitch red, and fast-twitch white types of muscles in rats. 3-Hydroxybutyrate dehydrogenase, 3-ketoacid CoA-transferase, and acetoacetyl-CoA thiolase activities were lowest in white muscle, higher in slow red than in fast red muscle, and highest in the heart. The large differences between the four muscle types in the levels of these enzymes reflects differences in both mitochondrial content and composition. Differences in composition were evidenced by large dissimilarities between the different muscle types in the ratios of the activities of the enzymes of ketone oxidation to each other and to citrate synthase and cytochrome c. Of particular interest is the finding that, despite the fact that in the rat slow red muscle has a lower content of mitochondria than fast red muscle, 3-hydroxybutyrate dehydrogenase activity is 140% higher and 3-ketoacid CoA-transferase is 70% higher in slow red than in fast red muscle. A 14-week-long program of treadmill running induced increases in the levels of ketone utilization enzymes in all three types of skeletal muscle but not in heart; 3-hydroxybutyrate dehydrogenase became measurable, though at very low levels, in white muscle; increased 2.6-fold in slow red muscle, and 6-fold in fast red muscle. 3-Ketoacid CoA-transferase increased 2-fold in both fast red and white types of muscle, but only 26% in slow red muscle. Acetoacetyl-CoA thiolase activity increased 4045 % in all three types of skeletal muscle. In contrast, citrate synthase and cytochrome c increased approximately 2-fold in all three types of skeletal muscle. These adaptive changes tend to make skeletal muscles more like heart muscle in their enzyme patterns and may help to explain why physically trained, as compared to untrained, individuals do not develop post-exercise ketosis. [ABSTRACT FROM AUTHOR]

Complexes of cytochrome c and of an undecapeptide of the protein have been studied by means of Mössbauer spectroscopy in order to obtain information concerning the active heme site. The variation &the quadrupole splitting with the temperature pewits an evaluation of the distortions from octahedral symmetry around the iron ion in the heme group, and also an estimation of the spin-orbit constant. The effect of the different ligands such as CN-, F-, imidazol, etc., is discussed. [ABSTRACT FROM AUTHOR]

Oxidized Euglena cytochrome ƒ has an absorbance band at 695 nm which disappears upon reduction. Various treatments that cause known changes of conformation of cytochrome c appear to have similar effects on cytochrome ƒ Heating cytochrome ƒ results in the gradual disappearance of the 695-nm band; the thermodynamic parameters for this change of the tertiary structure at 26 °C and ionic strength 0.01 are: ΔF= +2.67 kcal/mole, ΔH= +33 kcal/mol and ΔS = + 102 cal × K-1 × mol-1. The pK values obtained by equilibrium titrations of the herne-linked acid and alkaline ionizations are roughly 2.1 and 9.4. Equilibrium studies of the alkaline pK reveal that more than one berne-linked proton is dissociated in this pH range. The carboxymethylated product of the cyanide complex of oxidized cytochrome ƒ has the same properties as cytochrome c carboxymethylated under the same conditions. The results suggest that a methionine sulfur is involved in iron coordination in oxidized Euglena cytochrome ƒ [ABSTRACT FROM AUTHOR]

Characterizes the properties of the assimilatory nitrate reductase from the fungus Aspergillus nidulans. Sedimentation coefficient; Molecular weight; Sucrose density gradient; Cytochrome c; Enzyme activities.

Biguanides inhibit a large scale of metabolic processes which in most cases are membrane- linked. Oxidative phosphorylation is strongly inhibited in that part of the respiratory chain located on the substrate site of cytochrome c. It is assumed that inhibition is due to physical changes within the membrane phospholipids, rather than a result of direct interaction with functional intermediates of energy conservation. The following observations have been made. 1. Binding affinity to mitochondrial, submitochondrial and other membranes is identical with that to pure phospholipids. 2. Synthetic phospholipid vesicles exactly resemble the binding properties and kinetics of mitochondrial membranes. 3. Gradual lipid extraction of mitochondrial membranes causes a proportional decrease of binding capacity but no change of affinity constants. 4. The stoichiometry shows that the capacity for inhibitor binding is indeed in the range of mitochondrial phospholipid content. 5. Binding of the inhibitors occurs to the aqueous lipid interface of the membrane. Possibilities are discussed for the mode of action of the inhibitors representing firmly fixed positive charges.

An ATP-driven rise in fluorescence yield (Q reduction) and cytochrome f oxidation in light-triggered chloroplasts are demonstrated. Both effects are shown to be dependent on the creation and maintenance of the light, triggered state. ATP-induced reduction of Q depends upon the presence of an electron donor in the dark with a redox potential up to, but not exceeding, 300 mV. The effects are abolished by uncouplers or energy transfer inhibitors. 3-(3,4Dichlorophenyl)-l,l-dimethylurea inhibits the ATP-driven reduction of Q but not that of cytochrome oxidation. A location for a coupling site and the site of action of electron-transport inhibitors is suggested on the basis of these observations. [ABSTRACT FROM AUTHOR]

1. Escherichia coli membrane vesicles isolated from strains derepressed for 2-keto-3-deoxy-D-gluconate transport system were found to be able to take up 2-keto-3-deoxy-D-gluconate and D-glucoronate when an energy source was added. The artificial pair ascorbate plus phenazine methosulfate and the physiological electron donors D-(-)-lactate and DL-glycerol 3-phosphate specifically stimulated transport activity. 2. Vesicular uptake was lacking in kdgT-mutants and was restored in membranes from a reverent strain. Km values for 2-keto-3-deoxy-D-gluconate and for D-glucuronate closely agree with those previously determined in whole cells. From the additive fact that no binding activity was detected in the periplasm, it was concluded that all 2-keto-3-deoxy-D-gluconate transport activity coded by the kdgT gene was restricted to membrane. 3. By recording difference spectra between oxidized and reduced membrane vesicles, L-glycerol 3-phosphate oxidation was shown to follow the terminal cytochrome chain common to D(-)-lactate and other substrates. D-(-)-Lactate and glycerol 3-phosphate oxidation primarily occurs via two membrane-bound, flavin-linked specific dehydrogenases and this step was found to be rate-limiting for 2-keto-3-deoxy-D-gluconate uptake in vesicles. 4. 2-Keto-3-deoxy-D-gluconate uptake is inhibited by classical electron-transfer inhibitors, by proton-conducting compounds and thiol reagents. Except for oxamate and rotenone, all inhibitors induced rapid and complete efflux of previously accumulated 2-keto-3-deoxy-D-gluconate. Considering the site of action of these inhibitors along the respiratory chain it was concluded that (a) the site coupling electron flux to substrate uptake was lying between the primary dehydrogenase and cytochrome b1; and that (b) the carrier coupling site or a coupling component, yet speculative, rather than the recognition site for 2-keto-deoxygluconate had active sulfhydryl groups. [ABSTRACT FROM AUTHOR]

Difference spectra produced by the binding of steroids to rat adrenal-mitochondrial cytochrome P-450 indicate distinct interactions with, respectively, pregnenolone, 11-deoxycorticosterone and 25-hydroxycholesterol. These so-called type I (deoxycorticosterone and hydroxycholesterol) or type II (pregnenolone) spectral changes have been compared in bovine adrenal-cortex mitochondria and in adrenal mitochondria from rats. In the latter species the animals were subjected to one of three pretreatments; rats were either given an ether stress to increase the plasma concentration of adrenocorticotrophic hormone or were injected with the protein-synthesis inhibitor cycloheximide, or kept in a quiescent state. These experiments substantiate the theory that distinct P-450 cytochromes are involved in cholesterol side-chain cleavage (P-450SCC) and steroid 11β-hydroxylation (P-45011β). A pH-sensitive cholesterol complex of cytochrome P-450SCC was quantitated by means of the pregnenolone difference spectrum. Adrenal mitochondria from ‘stressed’ rats had about twice as much of this cholesterol complex of cytochrome P-450SCCas adrenal mitochondria from rats kept quiescent or subjected to cycloheximide treatment. The pH-dpendence of the cytochrome P-450SCC-cholesterol complex was unaffected by the pretreatment of the animals. The type-I spectral change produced by 25-hydroxycholesterol was unaffected by stress or by changes in pH but was almost removed by sonication. The binding of 25-hydroxycholesterol to adrenal mitochondrial cytochrome P-450 was competitively inhibited by pregnenolone but was unaffected by deoxycorticosterone. The 25-hydroxycholesterol difference spectrum may detect a distinct form of cytochrome P-450SCC. Sonication of adrenal mitochondria from quiescent and cycloheximide-treated rats increased both the absorbance change produced by pregnenolone and the cholesterol side-chain cleavage activity to values approaching those fund in mitochondria from stressed rats. The rate of side-chain cleavage of added 25-hydroxycholesterol was greater than that observed with endogenous cholesterol and this rate was unaffected by prior stress to the rats. It is suggested that the low cholesterol side-chain cleavage activity in adrenal mitochondria from quiescent rats and rats given cycloheximide may be due to the existence within the mictochondrial membrane of specific ‘restraints’ upon the interaction between a certain fraction or pool of cholesterol and cytochrome P-450SCC. The availability (or restraint) of this cholesterol pool may be altered by the action of adrenocorticotrophic hormone, and mediated by a labile protein factor. [ABSTRACT FROM AUTHOR]

Bakers' yeast cytochrome b2 [L-(+)-lactate dehydrogenase or L-(+)-lactate cytochrome c oxidoreductase], usually purified by crystallisation according to Appleby and Morton, was shown to be a degraded form of the enzyme, arising by a proteolytic modification of the species formed in vivo. We present here an improved purification procedure and some properties of the intact enzyme. This intact cytochrome b2 is shown, by cross-linking and by gel filtration studies, to be in a tetrameric form with a molecular weight of about 230 000. The four identical subunits are composed of a single polypeptide chain associated with one haem and one flavin (FMN). The amino-acid composition is presented. Alanine and glutamic acid are shown to be respectively C- and Nterminal residues. A comparison of these results with those concerning the degraded form suggests that there is little loss of peptide material and that the gross quaternary structure of the protein is not modified by the proteolytic process. However circular dichroism studies show that the haem environment is altered. The evidence leading to propose a biglobular structure for the enzyme is discussed. [ABSTRACT FROM AUTHOR]

1. Light-induced absorption changes of cytochrome b559 have been studied in isolated chloroplast preparations at room temperature. 2. It was shown that cytochrome b559 could undergo both photooxidation and photoreduction, both these reactions occurring to its high-potential form. 3. In the presence of FCCP and DCMU, a reversible photoreduction could be detected which was not sensitive to the presence of oxygen and which was probably a result of a cyclic electron flow around photosystem 2. 4. It is suggested that the high-potential form of cytochrome b559, which is the native form, is mainly photooxidized by photosystem 2. The photoreduction is also mediated by photosystem 2 and can be detected only when electrons cannot flow from photosystem 2 to photosystem 1, i.e. in the presence of DCMU, or when the carriers are fully reduced in the absence of an electron acceptor. [ABSTRACT FROM AUTHOR]

The spectroscopic properties, fluorescence and circular dichroism of model system of biological membranes, cytochrome c-lipid-water systems, are reported. It is shown that strong correlations exist between the fluorescence properties of dansyl phosphatidyl ethanolamine incorporated into these systems and the type of lipid protein interactions. These interactions depend on the state of cytochrome c; they are of an electrostatic type in the case of the ferricytochrome c-lipid phase and of an hydrophobic type in the case of the ferrocytochrome c-lipid phase. These correlations are valid as well on the pure phase (in the absence of excess water) as on the suspensions obtained by homogenization of the pure phases. The following results were also shown by a circular dichroism study of the suspensions, in the Soret region. 1. The addition of lipids to ferricytochrome c leads to a structural change of the protein from the native state to an intermediate state (displacement of the sixth ligand of the heme iron-side chain of methionine-80). 2. No structural change of the protein takes place by addition of the lipids to ferrocytochrome c. 3. In the presence of the lipids the reduction of cytochrome c is accompanied by a drastic structural change from the intermediate ferricytochrome c state to the native ferrocytochrome c state. [ABSTRACT FROM AUTHOR]

The rates of cholesterol biosynthesis were studied in rat fiver microsomal preparations incubated with [14C]lanosterol in the presence of gas mixtures of either carbon monoxide + oxygen 90:10, v/v)or nitrogen + oxygen (90:10, v/v). The presence of CO in the gas mixture resulted a considerable decrease in the amount of cholesterol synthesized, and there was also a reduction in the radioactivity of the 4α-methyl sterol fraction. The maximum rate of cholesterol biosynthesis in the presence of either N2 + O2 or CO + O2 occurred when the concentration of the substrate [14C]lanosterol was greater than 25 αM. The rate of cholesterol biosynthesis was constant for at least 40 min irrespective of the nature of the gas phase. During each time period studied, however, the amount of cholesterol formed was greater in the presence of N2 O2. Removal of either ATP or NADPH from the incubation medium resulted in a significant decrease in the rate of microsomal cholesterol biosynthesis. Pre-treatment of rats with phenobarbital resulted in a 35–40% increase in the rate of microsomal cholesterol biosynthesis from lanosterol. Incubations carried out under CO + O2 in which the flasks were illuminated with high intensity fight of around 450 nm wavelength completely reversed the inhibitory effect of CO on cholesterol biosynthesis from lanosterol. [ABSTRACT FROM AUTHOR]

Candida parapsilosis grows oxidatively under aerobic conditions and fermentatively in micro-aerobic (0.2 μM oxygen) continuous culture. The amount of unsaturated fatty acids and sterol in cells and mitochondria, and the aerobic cytochromes are decreased in micro-aerobic cultures. In aerobic cells mitochondrial protein synthesis accounts for the formation of more than one-third of the proteins of the organelles; in micro-aerobic cultures, this declines to 5%. At the same time, minor but significant differences in the nature of the protein products formed by the mitochondrial system are evident in micro-aerobic and aerobic cells. [ABSTRACT FROM AUTHOR]

Cytochrome oxidase and NADH cytochrome c reductase activities were analyzed biochemically in gingival biopsy specimens obtained from 22 male patients (age 23-72) undergoing periodontal treatment. Histologically, 13 specimens exhibited mild inflammation, while 9 showed more severe inflammatory responses. Cytochrome oxidase activity was significantly greater in the mildly inflamed than in markedly inflamed tissue samples. NADH cytochrome c reductase activity on the other hand was not significantly altered by the increasing degree of inflammation. The possible implication of the effect of inflammation upon oxidative enzymes is discussed in relation to degenerative and proliferative changes occurring in both types of tissue. [ABSTRACT FROM AUTHOR]

A very rapid increase in particulate cytochrome c reductase activity during the very early stages of germination of lettuce is demonstrated. The increase in activity does not parallel water uptake or the increase in cytochrome oxidase activity. The increase is reversible on drying of imbibed seeds and is not inhibited by cyanide. It is concluded that the increase in activity is due to some kind of activation process and not to de novo synthesis of protein , Possible mechanisms of activation were investigated. It was not possible to simulate the activation process in vitro, in the isolated particulars fraction. [ABSTRACT FROM AUTHOR]

Examines the response of intestinal cytochrome with the repair of iron deficiency in infants. Results of iron deficiency in infants; Analysis on the relationship between iron deficiency repair to the rate of production of new cells; Role of cytochromes in oxidative energy production.

The article presents information on genetics and biosynthesis of cytochrome. The c-type cytochromes are defined as cytochromes containing covalent linkages between the heme side-chains and the protein. One particular group of these proteins, referred to as "eukaryotic cytocbrome c", as mammalian cytochrome c", or just as "cytochrome c", is characterized by its presence in the mitochondria of all eukaryotic organisms, by a specific redox potential, and by catalytic activity with cytoebrorne oxidase from mammalian sources. The amino acid sequences of the cytochromes c from more than 30 species are sufficiently alike and their differences are of such nature that their evolution from a common ancestral form appears firmly established.

1. The interaction of cyanide with the oxidised and reduced forms of cytochrome-c oxidase has been investigated by kinetic and equilibrium measurements at 20 °C and pH 7.4. The inhibition by cyanide of the oxidation of cytochrome c has also been studied under different conditions. 2. When the oxidised form of cytochrome oxidase is mixed with cyanide, the heine-absorption bands are changed extremely slowly in a process whose rate is independent of the concentrations of cyanide and protein. Thus, the primary binding site does not appear to be one of the cytochrome components of the oxidase. 3. With reduced cytochrome oxidase, the interaction can be described as a simple secondorder process involving the α32+ form and HCN. The stability constant for the complex is 1.8 x 10³ M-1 and the rate constant, for its formation 1.3 × 10² M-1sec-1. 4. The inhibition of the enzyme may occur in two ways. One involves the reduction of the enzyme by cytochrome c and a subsequent reaction between α32+ and the inhibitor. However, if the oxidase is preincubated with cyanide, inhibition has been reported at much lower concentrations of cyanide. This cannot be related to the much slower changes in the heine-absorption bands observed in the reaction of cyanide with the oxidised form of the enzyme, and it is suggested that it involves binding of cyanide to one of the copper ions in the oxidase. [ABSTRACT FROM AUTHOR]

Radioactive leucine was incorporated by Neurospora crassa mitochondria in vivo in the presence of cycloheximide. When the membrane protein of these mitochondria was chromatographically separated on oleyl polymethacrylic acid resin, a number of fractions were obtained which differ with respect to their contents of radioactivity and cytochromes. The highest specific radioactivity was found in the fraction containing cytochrome aa3. This fraction proved to be a pure and enzymatically active cytochrome oxidase. Its ratio of absorbance at, 280 nm (ox)/ 443 nm (red.) was 2.1. By means of sodium dodecylsuffate gel-electrophoresis, this enzyme was separated into five polypeptides with molecular weights of 30000, 20000, 13000, 10000, and 8000. Only the polypeptide with the molecular weight 20000 displayed a high specific radioactivity. [ABSTRACT FROM AUTHOR]

1. Studies were undertaken to investigate the energy coupling mechanism in autotrophically grown Pseudomonas saccharophila. The cell-free extracts from Pseudomonas saccharophila contained flavoproteins and cytochromes of b, c, a, and o type 2, Added NADH was oxidized rapidly but it was not coupled to phosphorylation. The oxidation of generated NADH, was mediated, however, by the flavoprotein and cytochrome systems and yielded P/O ratios ranging from 0.86 to 1.23 3. The oxidation of hydrogen was mediated by the cytochrome systems but NAD+ was not involved, H2 oxidation yielded P/O ratios between 0.45 and 0.73. 4. Succinate oxidation resulted in a P/O ratio of 0.15 while ascorbate oxidation was not coupled to phosphorylation. 5. The phosphorylation coupled to the oxidation of hydrogen, NADH or succinate was completely uncoupled by various uncouplers of oxidative phosphorylation and phosphate esterification associated with the oxidation of NADH was quite sensitive to the flavoprotein inhibitors such as rotenone, amytal, and atabrine These inhibitors had no effect, however, on the phosphorylation coupled to hydrogen or succinate oxidation. 6 The ATP formation associated with the oxidation of hydrogen, NADH or succinate was inhibited by antimycin A or 2-n-nonyl-4-hydroxyquinoline-N-onde as well as by cyanide or azide, [ABSTRACT FROM AUTHOR]

Cytochrome oxidase was labelled by amino acid incorporation in vivo in the presence and absence of cycloheximide and was isolated by means of Triton X-400, ammonium sulfate fractionation and DEAE -cellulose chromatography from mitochondria of Neurospora crassa. A more than 50-fold purification was achieved with a final gel-filtration step on Bio-Gel. This preparation yielded in disc electrophoresis two distinct bands. In the presence of cycloheximide the purified cytochrome oxidase was not significantly labelled. The conclusion was dawn that the protein moiety of cytochrome oxidase is produced by the cycloheximide sensitive protein synthesizing machinery on the cytoplasmic ribosomes [ABSTRACT FROM AUTHOR]

the crystalline state and in solution, cytochrome b2 (yeast L-lactate:cytochrome c oxidoreductase) is shown to form a very stable complex with cytochrome c (from horse heart or yeast). This complex is characterized by a cytochrome c/cytochrome b2 heine ratio of about 0.25 which corresponds to the binding of one cytochrome c molecule to one tetraheme enzyme unit. Its formation is pH sensitive and prevented by salts but not by antimycin A. The prosthetic flag group of cytochrome b2 is not required for the binding of cytochrome c. [ABSTRACT FROM AUTHOR]

1. A detailed procedure is described for the isolation of stable respiratory membranes of relatively high phosphorylation efficiency from nitrogen-fixing cultures of Azotobacter vinelandii (P/O, NADH = 0.90-1.10). 2. Three phosphorylation sites are detectable. These are located at the level of NADH dehydrogenase (Site I; P/2e 0.45), between the primary dehydrogenases and the cytochromes (Site II; P/2e 0.53) and on the minor terminal pathway, to the oxygen side of the c-type cytochromes (Site III; P/2e 0.22). 3. Switching of electron flow between the terminal oxidation pathways with low concentrations of KCN and 2-n-alkyl-4-hydroxyquinoline-N-oxide causes changes in oxygen uptake and phosphate esterification which are compatible with the postulated locations of the three phosphorylation sites and which demonstrate, indirectly, the non-phosphorylating nature of the b1 → a2 major terminal pathway. 4. Phosphorylation efficiences with ascorbate plus purified A. vinelandii cytochromes c4 and c5, horse heart cytochrome c and the artificial electron mediators 2,6-dichlorophenol indophenol and N,N,N',N'-tetramethyl-p-phenylene diamine indicate that the minor terminal pathway is fresher split into a slow, phosphorylating pathway (c5 → oxygen) and a faster, non-phosphorylating pathway (c4 → oxygen). [ABSTRACT FROM AUTHOR]

A method for the estimation of cytochrome concentrations in whole yeast cells was worked out with strains of Saccharomyces cerevisiae obtained in the laboratory. Differences of cytochrome c content were due to mutations affecting the nature or the quantity of synthetized cytochrome c. Respiratory deficient mutants (without cytochromes a + a3 and b) were also used. Spectra were measured directly on yeast pastes (up to 3 mm thick) with a Cary 15 recording spectrophotometer provided with a Xenon lamp. Absorption differences between the maxima of α and β peaks of cytochrome c in relation to two base lines were measured. The mean value so obtained was in accordance with Lambert's law when the thickness of the sample of the same yeast paste was changed. For a constant optical path, Beer's law has been verified with the strains of different cytochrome c contents due to genotypical differences. There is a linear relation between quantities of cytochrome c extracted and absorptions measured on whole cells pastes. A device for liquid nitrogen temperature recordings is described and the extension of the method to low temperature measurements, and to the estimation of cytochromes a + a3 concentration is discussed. [ABSTRACT FROM AUTHOR]

A simple method for detecting similarities in sequences is described. It is used: 1. To provide similarity measures for classifying 25 cytochromes by their amino acid se- quences, 2. to detect repetitions in the amino acid sequences of various proteins, 3. to detect regions of possible base-pairing in the nucleotide sequence of a nuclei acid. [ABSTRACT FROM AUTHOR]

The interaction of various substrates with the cytochrome P-450 containing monooxygenase system of rat liver microsomes has been studied. Testosterone, triamcinolone acetonide (TrA) and laurate were found to interfere with the type I spectral changes produced by the addition of hexobarbital and ethylmorphine to suspensions of microsomes suggesting that these substrates bind to a common cytochrome P-450 species. Substrates producing a type I spectral change with liver microsomes, such as ethylmorphine, hexobarbital, aminopyrine, laurate and testosterone enhanced the rate of NADPH-linked cytochrome P-450 reduction. TrA, on the other hand, did not elicit a type I spectral change and did not stimulate the rate of reduction of the cytochrome. However, TrA interfered with the stimulation of this rate by hexobarbital and aminopyrine. Furthermore, when two compounds which stimulate the rate of cytochrome P-450 reduction were present together, there were no additive effects in stimulation of this rate. In fact, there was a diminution of the stimulation observed when the more potent of the two was present alone. The results suggest that the substrates of the liver-microsomal monooxygenase system studied interact with a single cytochrome P-450 species and that the competitive inhibition they exert on each other's metabolism may be related to an interference with the binding to cytochrome P-450. [ABSTRACT FROM AUTHOR]

1. Cytochrome composition and light-induced absorbance changes of cytochromes b and f in isolated chloroplasts have been measured in the α-band region utilizing dual-wavelength spectrophotometry. Three cytochromes appeared in reduced minus oxidized spectra, whereas only two of them, namely, cytochrome f and a cytochrome b appeared in light-induced difference spectra. 2. Light-induced changes of cytochrome b and cytochrome f, indicated that both are located in the part of the electron transport chain joining the two photosystems. The following sequence is supported by the evidence presented: Photosystem 2 → cytochrome b → cytochrome f photosystem 1. 3. 3-(3,4-Dichlorophenyl)-1,1-dimethyl urea was found to act in the region between photosystem 2 and cytochrome b since the photoinduced oxidation of both cytochromes but not of their reduction were observed in its presence. 4. A site for ATP formation is suggested to be localized in the same region, since in the light the presence of uncouples or photophosphorylating reagents induced a more reduced steady-state of both cytochromes. [ABSTRACT FROM AUTHOR]

Krebs cycle enzymes were repressed in Neurospora during growth on sucrose as carbon source compared with growth on acetate. In mitochondrial, cytochrome-deficient strains grown on sucrose, this glucose or catabolite repression was reduced. In mutant strains in which prom synthesis and mitochondrial respiration were inhibited, catabolite repression was enhanced. These data support the hypothesis that ATP or the cellular energy level is involved in catabolite repression. The interrelationships between energy metabolism, the energy distribution in the cell and catabolite repression are discussed. The addition of casamino acids to a culture growing on sucrose or acetate, did not cause repression of any of the Krebs cycle enzymes measured, in spite of the fact that many of the constituent amino acids can be considered as biosynthetic products of the Krebs cycle. [ABSTRACT FROM AUTHOR]

1. The rotenone-insensitive NADH-cytochrome c reductase system of rat liver mitochoncdria and isolated mitochondrial outer membrane preparations is inactivated by trypsin. The trypsin sensitivity of the system is considerably higher than that of liver microsomes when compared on equal protein basis. 2. The trypsin sensitivity of the mitochondrial system decreases with increasing ionic strength of the incubating medium, whereas that of the microsomal system increases with increasing ionic strength, except at low protein concentrations (< 0.1 mg protein/ml), in which case the effect of ionic strength is similar to that found with mitochondria. 3. Experiments with 2,6-dichlorophenolindophenol and ferricyanide as electron acceptors incate that trypsin does not inactivate the flavoprotein component of the mitchondrial system. The inactivation of the NADH-cytochrome c reductase by trypsin is accompanied by a release of cytochrome b5, similar to that found with microsomes. 4. Trypsin does not inactivate the adenylate kinase of intact mitochondria, but ready inactivates this enzyme after brief exposure of the mitochondria to a hypotonic medium containing EDTA. Respiration, respiratory control, and various translocator functions of the mitochondria which are not affected by this treatment, remain insensitive to trypsin. 5. The resets are discussed with regard to their bearing on the distinction between the mitochondrial (rotenone-insensitive) and microsomal NADH-cytochrome c reductase systems, as well as on the location of the mitochondrial NADH-cytochrome c reductase system and adenylate kinase in relation to the outer mitochondrial membrane. [ABSTRACT FROM AUTHOR]

1. The rotenone-insensitive NADH-cytochrome c reductase system of rat liver mitochondria and isolated mitochondrial outer membrane preparations is inactivated by trypsin. The trypsin sensitivity of the system is considerably higher than that of liver microsomes when compared on equal protein basis. 2. The trypsin sensitivity of the mitochondrial system decreases with increasing ionic strength of the incubating medium, whereas that of the microsomal system increases with increasing ionic strength, except at low protein concentrations (< 0.1 mg protein/ml), in which case the effect of ionic strength is similar to that found with mitochondria. 3. Experiments with 2,6-dichlorophenolindophenol and ferricynide as electron acceptors indicate that trypsin does not inactivate the flavoprotein component of the mitochondrial system. The inactivation of the NADH-cytochrome c reductase by trypsin is accompanied by a release of cytochrome b5, similar to that found with microsomes. 4. Trypsin does not inactivate the adenylate kinase of intact mitochondria, but ready inactivates this enzyme after brief exposure of the mitochondria to a hypotonic medium containing EDTA. Respiration, respiratory control, and various translocator functions of the mitochondria which are not affected by this treatment, remain insensitive to trypsin. 5. The resets are discussed with regard to their bearing on the distinction between the mitochondrial (rotenone-insensitive) and microsomal NADH-cytochrome c reductase systems, as well as on the location of the mitochondrial NADH-cytochrome c reductase system and adenylate kinase in relation to the outer mitochondrial membrane. [ABSTRACT FROM AUTHOR]

1. The labeling of cytochrome c of rat liver mitochondria and microsomes with L-[14C]lysine has been studied in vivo. The detailed analysis of "labeled microsomal cytochrome c" has shown that the labeled compound in the fraction of microsomal cytochrome c is not identical with cytochrome c. 2. The behaviour of the labeled compound in the fraction of microsomal cytochrome c during chromatography on CM-cellulose columns resembled that of apocytochrome c. 3. Using 59Fe or δ-amino-[14C]levulinic acid as labeled precursors of cytochrome c, the specific activities of cytochrome c extracted from mitochondria and microsomes were the same, 40 min after injection of the label. 4. Kinetic studies of incorporation of L-[14C]lysine into the labeled compound extracted from microsomes and cytochrome c of rat liver slices showed a precursor-product relationship between the two compounds. 5. The data are taken to indicate that only the apoprotein of cytochrome c is synthesized at the endoplasmic reticulum, whereas the attachment of the haem group occurs within the mitochondria after transfer of apocytochrome c ora derivative of it into the mitochondria. [ABSTRACT FROM AUTHOR]

1. The menaquinone of Bacillus megaterium (14581 ATCC) is localized exclusively in the cytoplasmic membrane as are the cytochromes. Menaquinone is contained in a 5 to 10-fold molar excess over cytochrome b1. 2. The constituent menaquinone is almost completely reduced to menaquinole in the anaerobic state with the substrates respired by the membrane fraction. On the addition of oxygen the menaquinole is reoxidized to menaquinone. Menaquinone and menaquinole are the only forms of MK detected in the steady states of the electron transport. 3. The redox reactions of menaquinone indicate its participation in the electron transport from the substrates α-glycerophosphate, malate and NADH, to the acceptors oxygen and fumarate. Menaquinone is in a steady state in all the flow systems established with each substrate and both acceptors, and exhibits the steady state response expected for a respiratory component. 4. The cytochrome system of this strain of B. megaterium consists of a b-type cytochrome with a redox potential of + 8mV and absorption hands dose to those of pure cytochrome b1, two cytochrome oxidases, an α-type cytochrome and cytochrome o and probably a small amount of c-type cytochrome. Cytochrome b1 is completely reducible by each of the substrates. 5. 2-n-Heptyl-4-hydroxyquinoline-N-oxide inhibits the electron flow at about the concentration of the constituent menaquinone between menaquinone and the cytochromes. The electron flow from each of the substrates to both acceptors depends completely on the reaction sensitive to the inhibitor. 6. The electron transport from α-glycerophosphate, malate and NADH to oxygen and fumarate as well as the reduction of cytochrome b1 is dependent on the presence of MK. This is demonstrated by pentane extraction and reincorporation of the MK. The reincorporation leads to the reconstitution of the original pathway, since MK and cytochrome b1 participate in the electron transport and the same sensitivity to 2-n-heptyl-4-hydroxyquinoline-N-oxide is found. 7. The position of menaquinone in the respiratory system of B. megaterium is between the dehydrogenases for α-glycerophosphate, malate and NADH and cytchrome b1, to which the fumarate reductase and the cytochrome oxidases are linked. [ABSTRACT FROM AUTHOR]

Oxygen uptake was monitored in perfused livers from normal and phenobarbital treated rats. Basal rates (200–250 μatoms × g-1 × h-1), as well as antimycin A (80–100 μatoms × g-1 x h-1) and cyanide (30–35 μatoms × g-1 × h-1) insensitive respirations were similar in both groups. The addition of aminopyrine to livers from fed, phenobarbital treated rats caused a maximal stimulation of oxygen uptake of 60 μatoms × g-1 x h-1. Half-maximal effect was observed with 0.1–0.2 mM aminopyrine. Prior addition of antimycin A or cyanide did not diminish the stimulation by aminopyrine. In livers from fasted, phenobarbital treated rats, the maximal stimulation was smaller (30–35 μatoms × g-1 × h-1), and no increase in oxygen uptake was observed in the presence of antimycin A. The data support the hypothesis that extramitochondrial NADPH generation controls mixed function oxidase activity in the intact cell. Since highest rates were observed under conditions of enhanced glycogenolysis, it is suggested that the main source of NADPH generation is via dehydrogenases of the hexosemonophosphate shunt. In the glycogen depleted state, however, it is predicted that reducing equivalents from mitochondrial oxidations are transferred into the extra- mitochondrial space. A substrate shuttle mechanism is proposed which provides these reducing equivalents in the form of malate for the extramitochondrial generation of NADPH via malic enzyme. [ABSTRACT FROM AUTHOR]

Flavoproteins of intact and fractionated mitochondria have been studied by means of spectrophotometric and fluorimetric techniques. The high and low redox potential fluorescent flavoproteins of rat liver mitochondria previously assigned to the respiratory chain are located in the soluble fraction obtained on ultrasonic disintegration. In intact mitochondria, the low potential species has a redox potential of –279 mV at pH 7.2 and 25° measured by fluorescence or absorbance, and undergoes a 2 equivalent reduction. After ultrasonic disruption of mitochondria, its redox potential is -294 mV at pH 7.2 and 25° In submitochondrial particles, low potential flavoprotein reducible by NADH in the presence of rotenone is not fluorescent, appears to have a redox potential more positive than -209 mV and is attributed mainly to cytochrome b5 reductase and cytochrome b5. Mersalyl can be used to selectively inhibit cytochrome b5 reductase, and it has been possible to estimate the concentration of the low redox potential flavoprotein free from cytochrome b5 interference. The high redox potential fluorescent flavoprotein is recovered in the soluble fraction after ultrasonic disruption of rat liver mitochondria and can be reduced by succinate in the presence of antimycin A inhibited particulate fraction. This fuorescent species is also found in rat kidney mitochondria but not in blowfly mitochondria. In submitochondrial particles, a non-fluorescent high redox potential species is found which may be flavoprotein or non-heme iron. This species has a redox potential of +30 mV at pH 7.2 and 25° and undergoes 1 equivalent reduction. Flavoprotein fluorescence measurements are subject to significant errors caused by absorption of the exciting light, and consequently do not always measure flavoproteins free from interference by other respiratory carriers, e. g. cytochromes and non-heme iron proteins. [ABSTRACT FROM AUTHOR]

Sucrose-density gradient experiments have shown it impossible to isolate microsomes from rat liver homogenate free from contaminated mitochondrial cytochrome c. The incorporation of L-[14C]lysine into cytochrome c from rat liver mitochondria and microsomes in vivo has been studied. The kinetics of the specific radioactivities of free L-lysine, that of microsomal cytochrome c and that of mitochondrial cytochrome c show precursor-product relationships. From the kinetics of radioactivity of free L-lysine and microsomal cytochrome c, the true pool-size of the latter was calculated to be 1.02 nmoles/g microsomal protein, that is about 1/270 of total rat liver cytochrome c. The turnover-time of cytochrome c in the endoplasmic reticulum was found to be 102 min. From this data a turnover-time of total rat liver cytochrome c of 19 days and a half-life of 13.2 days was calculated. [ABSTRACT FROM AUTHOR]

The standard redox potential of ubiquinone in submitochondrial particles is measured by equilibration with the succinate/fumarate system to about 65 mV at pH 7, with the redox ration, n = 2. The pH dependence is -60 mV/ΔpH. The lower value measured for bound ubiquinone is discussed in comparison with the value found for isolated ubiquinone (104 to 112 mV, pH 7) determined in ethanol - HCl. In parallel experiments the standards redox potential of cytochrome b was measured to 72.5 mV at pH 7. The pH dependence is ΔE/ΔpH = 0 at pH < 6.8 and -60 mV ΔHp at pH > 6.8 [ABSTRACT FROM AUTHOR]

Acid exonuclease activity of rat liver has been determined with the help of DNA core as substrate, The intracellular distribution of the enzyme has been investigated by differential centrifugation of homogenates and compared with those of cytochrome oxidase, acid phosphatase and glucose-6-phosphatase used as reference enzymes for mitochondria, lysosomes and endoplasmic reticulum respectively. Acid exonuclease is principally associated with mitochondrial fractions and exhibits a distribution pattern similar to that of acid phosphatase. After isopyenic centrifugation in a sucrose gradient of a total mitoehondrial fraction, acid exonuclease like acid phosphatase shows a relatively flattened distribution curve with a median equilibrium density of about 1.2. The injection to the rat of Triton WR -1339, which lowers the equilibrium density of lysosomal enzymes has a similar effect on acid exonuclease. Acid exonuclease exhibits the phenomenom of structurelinked latency. it is concluded that acid exonuclease is associated with the lysosomes in rat liver. Separation between acid DNase and acid exonuelease associated with lysosomes can be performed by chromatography on hydroxyapatite of an extract of purified granules. The complementary action of the two enzymes in lysosomal digestion of DNA is illustrated, [ABSTRACT FROM AUTHOR]

Starting with a wild strain of Hafnia having the phenotype: nitrate reductase A +, nitrate reductase B +, chlorate reductase C +, tetrathionate reductase +, fumarate reductase +, hydrogen lyase +, we have isolated two kinds of mutants having the following phenotypes: (1) A -, B +, C +, tetrathionate reductase +, fumarate reductase +, hydrogen lyase +; (2) A -, B -, C -, tetrathionate reductase -, fumarate reductase +, hydrogen lyase -. The wild strain and the mutants of category 1 can use nitrate, nitrite, fumarate, and tetrathionate as hydrogen acceptors under anaerobic conditions. The mutants of category 2 can also reduce nitrite and fumarate, but cannot use nitrate and tetrathionate as hydrogen acceptors. We have studied the effects of nitrate, nitrite, fumarate, and tetrathionate on the biosynthesis of cytochromes a2, b1, b-562, and c-550 using anaerobic cultures of the wild strain and several mutants of the above two categories. The following observations were made: (a) Wild strain. Neither tetrathionate nor fumarate have any effect. Nitrate, on the other hand, markedly reduces the levels of cytochromes a2, b-562, and c-550. As for nitrite, it causes a decrease in levels of cytochromes b-562 and b1, but a strong increase in that of cytochrome c-550. It is as though nitrate abohshes the effects of nitrite on the biosynthesis of cytochromes b1 and c-550. (b) Mutants of category 1. Nitrate causes the cytochrome a2 band to disappear, but does not influence the biosynthesis of cytochromes b-562 and c-550. Apart from the effect of nitrate on the formation of cytochrome a2, the different hydrogen acceptors caused no detectable spectral changes. (c) Mutants of category 2. We obtain essentially the same spectra as with the wild strain except when the cultures are nitrite-grown. In this case, nitrite has the same effect as nitrate because it represses the biosynthesis of cytochrome c-550. It is clear from tiffs work that the two mutant phenotypes studied have profound and different effects in their regulation of the biosynthesis of cytochromes. [ABSTRACT FROM AUTHOR]

NADPH-cytochrome ƒ reductase from spinach chloroplasts has been purified, and its amino acid composition is reported. The specificity of the enzyme for different electron acceptors has been studied. In addition to higher plant cytochrome ƒ Euglena gracilis cytochrome ƒ and plastocyanin are reduced. The reaction with these acceptors occurs through a two-step mechanism, whereas higher plant cytochrome ƒ forms a ternary complex with NADPH and the enzyme. The Km values for NADPH in the reactions with the different electron acceptors, the Km's for the electron acceptors and the turnover numbers are reported. The sulphydryl reagents slowly inactivate the enzyme and enhance the fluorescence of enzyme-bound FAD and of the tryptophan residues of the protein. In the presence of NADPH, the inactivation is very rapid, and FADH2 is split from the apoenzyme. Preincubation of the enzyme with NADPH inactivates reversibly all the reactions except cytochrome ƒ reductase. The results are interpreted in terms of structure to function relationships and the significance of the NADPH-cytochrome ƒ reductase in photosynthetic electron transport is also discussed. [ABSTRACT FROM AUTHOR]

The paramagnetism at 20° of cytochrome b5 at pH 7 shows that the heme iron of the enzyme is low spin both in the oxidized and the reduced state. The electron spin resonance (ESR) at low temperatures shows that there is a mixture of high and low spin heme contributing to the absorption. At high pH there is a reversible denaturation accompanied by changes of the ESR from both the high and low spin forms as well as of the light absorption. The ESR of cytochrome b5 is definitely different from that of microsomal Fex. [ABSTRACT FROM AUTHOR]

A NADPH-cytochrome c reductase (called Fraction II) has been solubilized with Triton X-100 from porcine liver microsomes and purified. 1. Fraction II can reconstitute with a cytochrome P-450 fraction (called Fraction I) solubilized with Triton X-100 from porcine kidney cortex microsomes to exhibit laurate ω-hydroxylation activity. 2. When the purified Fraction II is subjected to disc electrophoresis on polyacrylamide gel in the presence of 0.1% Triton X-100, it shows a single hand. In contrast, on electrophoresis in the absence of Triton X-100, a polydisperse state is obtained. Fraction II is estimated to have a molecular weight of about 400000 by gel-filtration with detergent, and it does not contain iron. 3. Either gel filtration on a Sephadex G-100 column or treatment with trypsin at 4 °C results in a conversion of Fraction II to a form, which migrates at a much faster rate on disc gel electrophoresis than dose Fraction II. This fast-moving form (called Fraction IIf) is indistinguishable from either steapsin or trypsin-solubilized NADPH-cytochrome c reductase, judging from several criteria including electrophoretic and sedimentation behaviors, and molecular size. 4. Fraction IIf is reconstitutively inactive in the hydroxylation, unless supplemented with spinach ferredoxin-NADP reductase. Incubation of Fraction IIf with trypsin at 30 °C results in a remarkable enhancement in the reconstitution activity in the presence of ferredoxin-NADP reductase. The results in this paper along with an earlier report [11] suggest that Fraction II may contain two different electron carrier components, possibly participating in the electron transfer from NADPH to cytochrome P-450. [ABSTRACT FROM AUTHOR]

Rat liver microsomes were peroxidized in vitro and chemical, physical and morphological changes in the membrane were related to the effects on certain membrane-bound microsomal enzymes. With increasing peroxidation, microsomal phospholipids revealed decreasing concentrations of 20:4 and 22:6 fatty acids. At a high level of peroxidation, where alterations in permeability and ultrastructural changes such as breakage and deformation of microsomal vesicles were apparent, there was also a decrease in the concentration of 18:2 fatty acids. Glucose-6-phosphatase, cytochrome P-450 and uridine-5′-diphosphate glueuronyltransferase (UDP glucuronyltransferase) (three microsomal enzymes known to be highly dependent on the integrity of the membrane for their function) responded differently to lipid peroxidation. Glucose-6-phosphatase showed an initial decrease in activity as well as in Km for glucose 6-phosphate, followed by a restoration to control values at an intermediate level of lipid peroxidation. At a high level of peroxidation, both kinetic parameters were again markedly decreased. Cytochrome P-450 concentration, on the other hand, revealed a continuous decrease with proceeding lipid peroxidation, which was only partly accounted for by a conversion to cytochrome P-420. The loss of cytochrome P-450 was paralleled by decreased rates of aminopyrine demethylation and 3,4-benzpyrene hydroxylation. Glucuronyltransferase activity, finally, was activated at low, but again normalized at high, levels of lipid peroxidation. It thus seems that although electron-microscopically observable changes in the microsomal membrane occurs only late in lipid peroxidation, chemical, physical and enzyme alterations are early phenomena during this process. The continuous loss of cytochrome P-450 and the final decrease in glucose-6-phosphatase late during peroxidation probably reflect damage to membrane lipids of vital importance for the integrity and function of these enzymes whereas the activation of UDP glucuronyltransferase early during peroxidation most likely can be ascribed to increased membrane permeability. [ABSTRACT FROM AUTHOR]

The effect of plastocyanin on the light-induced steady-state changes of cytochrome f in chloroplast particles that were depleted of plastocyanin has been studied. It was observed that the extent of cytochrome f photooxidation in sonic particles in the steady-state during a short exposure to long-wave red light was approximately 30% of that in chloroplasts. The addition of a catalytic amount of plastocyanin to the sonic particles stimulated cytochrome f photooxidation to 80% of its steady-state value in untreated chloroplasts. The reduction of cytochrome f by short-wave red light was not inhibited by sonic treatment of chloroplasts. The rate of cytochrome f photooxidation in the presence of a catalytic amour of detergenttreated chloroplasts was proportional to the amount of plastocyanin added. The enhancement of cytochrome f photooxidation either in sonic panicles or in digitonin, treated particles after an addition of plastocyanin was decreased in the presence of the copperchelating agent sodium diethyldithiocarbamate. Plastocyanin preincubated with sodium diethyldithiocarbamate showed less ability to stimulate cytochrome c photooxidation by detergent-treated chloroplasts. [ABSTRACT FROM AUTHOR]

A detailed study of the polypeptide fragments formed, and of their evolution during the first stages of the trypsinolysis of two homologous active cytochrome b2 molecules, was carried out, These cytochromes are complex flavoheme-L-lactate dehydrogenases extracted from different yeasts and having different molar activities. Our purpose was to gather information concerning the bonds, which, in the native proteins, are the most sensitive to trypsin (termed here protease-hypersensitive bonds). The products resulting from the fist hydrolytic events were thus analysed using disc-electrophoresis in sodium dodecylsulfate according to the method of Shapiro et al. (1967). Electrophoretic patterns were related to the molecular weight and to the amount of each intermediate species as a function of the extent of reaction. Schemes of the series of events, (including several rate constants) were derived from this set of data. A tentative map of the homologous loci of protease-hypersensitive bonds along the chains of the two cytochrome b2 molecules is proposed. Considering that such bonds which are very reactive towards trypsin are certainly located on flexible and thus loosely structured regions at the surface of the molecule, this map gives a first insight into the overall secondary and tertiary structure of the chain which includes globules of high internal stability. important finding was that the relative reactivities of the various protease-hypersensitive bonds identified, depend on conditions such as ionic strength and the binding of a ligand which probably changes the conformation. The protein transitions are therefore possibly described in terms of modulation of the loci of these bonds and of their reactivity. [ABSTRACT FROM AUTHOR]

As has been known for some time, proteolysis of cytochrome b2 yields a small protein fragment (cytochrome b2 core) which still carries the heme. This paper describes the separation of the various bands which can be seen upon electrophoresis of the small hemoprotein. The isoelectric points of the two main bands are 6.40 and 5.55. Amino acid analyses show that they have about 80 and 100 residues, respectively. End group studies using in particular carboxypeptidase C demonstrated that both bands have lysine as N- and C-terminal residues. Altogether, the data indicated that the various bands come from splits of the main chain at different positions. The significance of these results is discussed in relation with what is currently known about the structure of cytochrome b2. [ABSTRACT FROM AUTHOR]

Mitoehondria from maternally inherited mutants of Neurospora crassa mi-1 (poky) and mi-3 were examined with respect to the components of the respiratory chain, their properties and arrangement. Furthermore the sites of oxidative phosphorylation were localized. 1. An evaluation procedure which permits the quantitative determination of the various b-type and c-type eytochromes is given. 2. All mutant mitochondria contain the same cytochromes as wild type mitochondria, such as cytochrome a,a3, cytochrome c and c1 and cytochrome b562 and b556 (the indices refer to the maxima at low temperature). Yet the molar ratios of the cytochromes; a :c : c1: b562 : b556 differ widely: in the wild type the ratios are 1.0:2.9:1.0:0.9:0.9; in mi-1 the ratios are <0.05:3.6:1.0:0.05:0.3; and in mi-3 the ratios are 0.3:3.2:1.0:0.9.0.8. The content (μmol/g protein) of the reference cytochrome c1 varies slightly from 0.34 in the wild type to 0.39 in mi-1 and 0.32 in mi-3, The main feature of the cytochrome pattern in mi-1 is a very low content of cytochrome a,a3 and b562; besides that the content of cytochrome c is increased by one third of the normal value. In mi-3 only the content of cytochrome a,a3 is diminished. 3. The comparatively low content of ubiquinone in the wild type is increased 8-fold in mi-1 and 5-fold in mi-3. The content of mitochondrial NAD remains largely unchanged. 4. Both mitochondria from mi-1 and mi-3 show antimycin- and cyanide-insensitive respiration. The share of uninhibited respiration depends on the activity of the different dehydrogenases and on the ratio of the capacities of both oxidase pathways. The insensitive respiration decreases with the age of the hyphae culture. 5. The ADP/O ratio in mi-1 mitochondria is lowered, due to the cyanide-insensitive oxidase pathway. This pathway is found to include one phosphorylation step with substrates linked to endogenous NADH. Exogenous NADH and NADPH are oxidized similarly to succinate, involving two phosphorylation sites on the cyanide-sensitive path but no phosphorylation site on the insensitive path. 6. Inhibitors distinguish the two oxidase pathways: the normal pathway is sensitive to cyanide and antimycin, the second pathway to salicyihydroxamate. NAP-linked and ubiquinone-linked substrates are oxidized by both pathways whereas tetramethyl-p-phenylenediamine plus ascorbate is oxidized only by the normal cyanide-sensitive pathway. 7. The redox state of ubiquinone in relation to antimycin and salicylhydroxamate indicates participation of ubiquinone in both paths of the mi-1 mutant. 8. In wild type, cytochrome b562 is largely oxidized in anaerobic uncoupled state but can be reduced to about 60-70% by ATP, whereas cytochrome b556 is largely reduced under both conditions Cytochrome b562 is completely reduced in the antimycin-inhibited state and becomes oxidized on reduction of cytochrome c1. In analogy to mammalian mitochondria cytochrome is defined as cytochrome bT and cytochrome b556 as cytochrome bK. In contrast to the wild type, cytochrome b558 in mi-1 remains largely oxidized even with antimycin present. 9. Titration with antimycin gives nonlinear curves with a titer of complete inhibition at about equimolar ratio of antimycin to cytochrome bT. In the mi-1 mutant the antimycin titer strongly decreases parallel to the loss of cytochrome bT. [ABSTRACT FROM AUTHOR]

The specific binding of anions to oxidized horse-heart cytochrome c has been studied by two methods: measuring the effect of ion concentration on the redox potential, and directly by gel filtration. The anions investigated were: chloride, phosphate, ADP and sulfanilate. The binding constants estimated are in the range of 1-10 mM-1, and the number of sites per protein molecule are 1 to 2. The specificity of anion binding to the oxidized protein is demonstrated. [ABSTRACT FROM AUTHOR]