Purification and properties of soluble cytochrome b-560 from spinach leaves

A b-type cytochrome having an -band at 560 nm was isolated fromspinach leaves (Spinacia oleracea). A method is described forpreparing this cytochrome, cytochrome b-560 (spinach), in apurified state. The cytochrome has, in its reduced state, absorption bands at560 nm (), 530 nm (ß) and 427 nm (); and in the oxidizedstate at 562 nm (), 529 nm (ß) and 417 nm (). Thepyridine ferro-haemochrome prepared from cytochrome b-560 hadan -band at 556.5 nm, indicating the protohaem-nature of theprosthetic group. The cytochrome has an oxidation-reduction potential (E'⁰) of+0.13V at pH 7.0, as measured using the ferri-ferro oxalate system. The cytochrome is rapidly reduced on illumination with red orfar-red light in the presence of spinach chloroplasts and isoxidized at a slower rate in the dark. This photoreduction isinhibited by 1x10^–6 M 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU). The molecular weight of the cytochrome is 30,000 asestimated by the dextran gel filtration method. (Received December 3, 1971; )     

Purification and properties of plant cytochrome b5.

Microsomal cytochrome b5 was 352-fold purified from potato tubers with a yield of 10.4%. To our knowledge, this is the first report relating the purification of higher-plant cytochrome b5. Its Mr (16 700) and absorption spectrum are similar to those of animal and yeast cytochrome b5.     

Properties of bovine heart mitochondrial cytochrome b560

A large-scale preparation of the two-subunit protein complex (QPs) that converts succinate dehydrogenase into succinate-ubiquinone reductase from cytochrome b-c1 particles is achieved by a procedure involving Triton X-100 solubilization and calcium phosphate column chromatography at different pH values. The isolated two-subunit QPs contains 25 nmol of cytochrome b560/mg of protein and is able to reconstitute with soluble succinate dehydrogenase to form a TTFA-sensitive succinate-ubiquinone reductase. The maximum reconstitutive activity is 100 mumol of succinate oxidized per min per mg of QPs protein at 23 degrees C. Although cytochrome b560 in isolated QPs is not succinate reducible and its dithionite reduced form is reactive to carbon monoxide, cytochrome b560 is shown to be physically associated with succinate dehydrogenase by the following observations. The dithionite reduced form of cytochrome b560 in isolated QPs has a symmetrical alpha-absorption peak, which upon reconstitution with succinate dehydrogenase becomes slightly broadened and shows a shoulder at around 553 nm, identical to that of cytochrome b560 in succinate-ubiquinone reductase. Upon addition of succinate dehydrogenase to QPs, about 50% of the reduced form of cytochrome b560 in the QPs becomes insensitive to carbon monoxide treatment. The redox potential of cytochrome b560 in QPs is -144 mV which is higher than that of cytochrome b560 in succinate-ubiquinone reductase (-185 mV). Upon addition of succinate dehydrogenase, the redox potential of about 46% of the cytochrome b560 in QPs preparation becomes identical to that of cytochrome b560 in succinate-ubiquinone reductase. Cytochrome b560 in the QPs preparation shows two epr signals, g = 3.07 and g = 2.92, whereas cytochrome b560 in succinate-ubiquinone reductase exhibits only one epr signal at g = 3.46. When QPs is reconstituted with succinate dehydrogenase to form succinate-ubiquinone reductase, the g = 3.46 epr signal reappears at the expense of the g = 3.07 signal. Based on epr measurement at liquid helium temperature, about 18% of the total cytochrome b in the isolated active succinate-cytochrome c reductase is cytochrome b560, indicating that cytochrome b560 is indeed a unique cytochrome b and not a denatured product of cytochrome b562 or b565.     

Purification and properties of a cytochrome b560-d complex, a terminal oxidase of the aerobic respiratory chain of Photobacterium phosphoreum

A cytochrome b560-d complex, a terminal oxidase in the respiratory chain of Photobacterium phosphoreum grown under aerobic conditions, was purified to near homogeneity. The purified oxidase complex is composed of equimolar amounts of two polypeptides with molecular weights of 41,000 and 54,000, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. It contains 10.2 nmol of protoheme and 22.5 nmol of iron/mg of protein. The enzyme is a "cytochrome bd-type oxidase," showing absorption peaks at 560 and 625 nm in its reduced minus oxidized difference spectrum at 77K. This oxidase combined with CO, and its CO difference spectrum at room temperature in the Soret region showed a peak at 418 nm and a trough at 434 nm. In addition, a trough at 560 nm (cytochrome b), and a trough at 620 nm and a peak at 639 nm (cytochrome d) were observed in the CO-binding spectrum. This cytochrome b560-d complex catalyzed the oxidation of ubiquinol-1 and ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride or phenazine methosulfate. The oxidase activity required phospholipids and was inhibited by the respiratory inhibitors, KCN and NaN3, and the divalent cation, ZnSO4. Formation of a membrane potential by the cytochrome b560-d complex reconstituted into liposomes was observed with the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide, on the addition of ubiquinol-1, showing that the enzyme provided a coupling site for oxidative phosphorylation.     

Phosphofructokinase from Ascaris suum. Purification and properties

A rapid and efficient procedure has been developed to purify phosphofructokinase from the muscle of the parasitic roundworm, Ascaris suum. The procedure can be accomplished in 1 day with a 420-fold purification and a 60% yield. The enzyme was shown to be homogeneous by two-dimensional electrophoresis, Sepharose 6B column chromatography, and high performance liquid chromatography utilizing a size exclusion column. The subunit molecular weight of the enzyme was found to be 95,000 by electrophoresis in the presence of sodium dodecyl sulfate. In solutions of low ionic strength, the native enzyme aggregated to species of higher molecular weight than did the rabbit muscle phosphofructokinase. In the presence of 0.2 M (NH4)2SO4, the minimum native molecular weight was determined to be 398,000 by high performance liquid chromatography and Sepharose 6B column chromatography. Therefore, the enzyme appears to be a tetramer with identical or near-identical subunits. The apparent isoelectric point of the enzyme was shown to be 7.3 to 7.4 by both column and gel isoelectric focusing. Amino acid analysis revealed a lower number of the aromatic residues Phe, Tyr, and Trp than in the rabbit muscle enzyme and this is in agreement with the lower extinction coefficient of E1%280 nm = 6.5. Analysis of the purified enzyme revealed 7.4 +/- 0.6 mol of phosphate/mol of enzyme.     

Purification and properties of a major cytochrome b peptide from baker's yeast.

A major cytochrome b peptide was purified from yeast mitochondria by a procedure involving solubilization in deoxycholic and cholic acids, ammonium sulfate fractionation, proteolytic digestion, and sucrose gradient centrifugation in the presence of Tween 80. The homogeneity of the purified protein was established by the criteria that the product was spectrally pure and yielded a single band on both sodium dodecyl sulfate polyacrylamide gel electrophoresis, and by gel isoelectric focusing. The purified cytochrome b polypeptide had absorption maxima at 562, 532, and 430 nm in the reduced form and at 525 to 570 nm and 419 nm in the oxidized form. The reduced minus oxidized difference spectra revealed absorption bands at 562, 532, and 430 nm at room temperature and 559, 529, and 429 nm at 77 K, respectively. The heme group was identified as protoheme by formation of the reduced pyridine hemochromogen. Treatment of the reduced form with carbon monoxide affected the absorption spectrum, indicating that the isolated hemoprotein was modified compared to native cytochrome b. The apparent molecular weight of the preparation was 28,000 based on sodium dodecyl sulfate polyacrylamide-gel electrophoresis and 28,800 based on sucrose gradient centrifugation. The isolated cytochrome b polypeptide showed a strong tendency to aggregate.     

Purification and properties of a diheme cytochrome b561 of the Escherichia coli respiratory chain

A new b-type cytochrome, cytochrome b561 (Murakami, H., Kita, K., Oya, H., and Anraku, Y. (1984) Mol. Gen. Genet. 196, 1-5) was purified to near homogeneity from the cytochrome b561-amplified Escherichia coli K12 strain HM204/pAM5029. The purified cytochrome b561 was a single polypeptide with a molecular weight of 18,000, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point was determined to be 9.6. The difference spectrum of the cytochrome at 77 K shows a major alpha-absorption peak at 561 nm and a minor peak at 555 nm. The absolute spectrum at room temperature of the oxidized form of the cytochrome had an absorption peak at 414 nm, and that of the reduced form had peaks at 562, 530, and 428 nm. The oxidation-reduction potential of the cytochrome was estimated to be +20 mV. The cytochrome contained 91.2 nmol of heme/mg of protein, showing that it was a cytoplasmic membrane-bound, b-type diheme cytochrome.     

Purification and some properties of the small subunit of cytochrome b558 from human neutrophils

We have attempted to purify the heme moiety of cytochrome b558 from human neutrophils. Cytochrome b558 was solubilized from the crude membrane fraction which was pretreated with both 1 M potassium phosphate buffer and 1% octyl glucoside at low ionic strength. The solubilization of cytochrome b558 was carried out efficiently with 1.6% octyl glucoside in the presence of 100 mM phosphate buffer. Solubilized cytochrome b558 was purified by hydroxylapatite, DEAE-Sephacel, and Mono Q fast protein liquid chromatography. The specific content of purified cytochrome b558 was 37 nmol/mg of protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified cytochrome b558 revealed a single band of 20,000 Da. The large subunit of cytochrome b558, which has been reported by others, could not be found in purified cytochrome b558 even with silver staining. The amino acid composition of the heme-containing moiety of cytochrome b558 was abundant in hydrophobic amino acids. The circular dichroism spectra of both oxidized and reduced b558-type cytochromes exhibited bilobed bands with wavelengths of crossover points closely corresponding to those of the maxima in the optical absorbance spectra at the Soret region. Furthermore, there were some differences in the shoulders and peak widths of CD spectra between oxidized and reduced b558-type cytochromes. These results indicate that this method provides the purification of the small subunit of human cytochrome b558 which is the heme-carrying subunit of cytochrome b558, and suggest that cytochrome b558 has heme-heme interaction and some conformational changes in the alternation of the redox state.     

Purification and properties of fructose diphosphate aldolase from Ascaris suum muscle

A fructose diphosphate aldolase has been isolated from ascarid muscle and crystallized by simple column chromatography and an ammonium sulfate fractionation procedure. It was found to be homogeneous on electrophoresis and Sephadex G-200 gel filtration. This enzyme has a fructose diphosphate/fructose 1-phosphate activity ratio close to 40 and specific activity for fructose diphosphate cleavage close to 11. K_m values of ascarid aldolase are 1 × 10⁻⁶m and 2 × 10⁻³m for fructose diphosphate and fructose 1-phosphate, respectively. The enzyme reveals a number of catalytic and molecular properties similar to those found for class I fructose diphosphate aldolases. It has C-terminal functional tyrosine residues, a molecular weight of 155,000, and is inactivated by NaBH₄ in presence of substrate. Data show the presence of two types of subunits in ascarid aldolase; the subunits have different electrophoretic mobilities but similar molecular weights of 40,000. Immunological studies indicate that the antibody-binding sites of the molecules of the rabbit muscle aldolase A or rabbit liver aldolase B are structurally different from those of ascarid aldolase. Hybridization studies show the formation of one middle hybrid form from a binary mixture of the subunits of ascarid and rabbit muscle aldolases. Hybridization between rabbit liver aldolase and ascarid aldolase was not observed. The results indicate that ascarid aldolase is structurally more related to the mammalian aldolase A than to the aldolase B.     

Purification of cytochrome b558 from bovine polymorphonuclear neutrophils

A 110 fold purification of cytochrome b558 from resting bovine neutrophils has been achieved. The plasma membrane bound cytochrome b was extracted with aminoxide WS35, a non ionic detergent. The purification procedure included liquid column chromatography on CM-C50 Sephadex, chromatofocusing on the anion exchanger PBE94, and gel filtration on P30 Biogel. The purified preparation was characterized by an heme to protein (nmol/mg) ratio of 7.7. The isoelectric point of cytochrome b was at pH 6.5. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate three bands corresponding to apparent Mr 64,000, 56,000 and 20,000 were revealed by staining with Coomassie Blue.