Properties of purified carbon monoxide dehydrogenase from Clostridium thermoaceticum, a nickel, iron-sulfur protein

Carbon monoxide dehydrogenase from Clostridium thermoaceticum has been purified to homogeneity using a strict anaerobic procedure. The enzyme has a molecular weight of about 440,000 and it consists of three each of two different subunits giving the composition alpha 3 beta 3. The molecular weight of the alpha-subunit is 78,000 and that of the beta-subunit is 71,000. Pore limit gel electrophoresis gave a molecular weight of 161,000 indicating that the enzyme dissociates to form a dimer with an alpha beta structure. The dimer apparently contains per mol 2 nickel, 1 zinc, 11 iron, and 14 acid-labile sulfur. The anaerobic enzyme has an iron-sulfur type spectrum, which is changed in the presence of the substrate, CO. In the presence of oxygen, which destroys the activity or CO2, the spectrum is that of a typical iron-sulfur protein. Under acidic conditions a low molecular weight nickel factor separates from the enzyme. Viologens, methylene blue, ferredoxin, flavodoxin, and rubredoxin serve as electron acceptors. Of these rubredoxin is by far the most efficient. The enzyme has a pH optimum around 8.4. At this pH and 50 degrees C under 100% CO atmosphere, the apparent Km for methyl viologen is 3.03 mM and Vmax is 750 mumols of CO oxidized min-1 mg-1. Cyanide and methyl iodide inhibit the enzyme. CO reverses the cyanide inhibition but promotes the reaction with methyl iodide. The pure enzyme has no hydrogenase or formate dehydrogenase activity.     

Chemical and spectral properties of carbon monoxide: methylene blue oxidoreductase. The molybdenum-containing iron-sulfur flavoprotein from Pseudomonas carboxydovorans

Carbon monoxide:methylene blue oxidoreductase, the key enzyme of CO-oxidation in energy metabolism of the carboxydobacterium Pseudomonas carboxydovorans, has been isolated in good yield and purity and found to contain FAD, molybdenum, iron, and labile sulfide in the ratio of 1:1:4:4. The enzyme is, therefore, a new molybdenum-containing iron-sulfur flavoprotein, exhibiting chemical and spectral properties quite similar to those of xanthine oxidase. Analytical data on the spectral characteristics of the enzyme in the oxidized and various reduced states are presented. Carbon monoxide:methylene blue oxidoreductase turned out to be photoreducible in the presence of EDTA and urea and was subject to reoxidation by air oxygen; no flavoprotein semiquinone was formed. Unphysiological electron acceptors, e.g. methylene blue, were used as oxidizing substrates whereas NAD or NADP turned out to be ineffective. Methylene blue reduction with CO was not affected by the presence of allopurinol, and carbon monoxide:methylene blue oxidoreductase was not able to catalyze the reduction of methylene blue with xanthine, adenine, or aldehydes. CO was the only reducing substrate used by the enzyme. Carbon monoxide:methylene blue oxidoreductase formed no sulfite adduct, and the reactivity with ferricyanide or cytochrome c was significant but slow. As known for other molybdenum hydroxylases, carbon monoxide:methylene blue oxidoreductase was rapidly inactivated by methanol, but the enzyme exhibited no ability to catalyze the oxidation of NADH with methylene blue, and NAD was not able to overcome methanol inhibition.     

Isolation procedure and some properties of myeloperoxidase from human leucocytes.

1. A rapid isolation procedure with a high yield for pure myeloperoxidase (donor:H2O2 oxidoreductase, EC 1.11.1.7) from normal human leucocytes is described. The enzyme was solubilized from leucocytes with the detergent, cetyltrimethylammonium bromide, and purified to apparent homogeneity. The yield of the enzyme was 17% with an absorbance ratio A430nm/A280nm = 0.85. 2. The purified enzyme showed three isoenzyme bands after polyacrylamide gel electrophoresis; ultracentrifuge studies indicated one homogeneous band with a molecular weight of 144 000. After reduction of myeloperoxidase, sodium dodecyl sulfate gel electrophoresis resolved an intense band (63 000 daltons) and a weak band (81 000 daltons). 3. The carbohydrate content of the enzyme was at least 2.5%. Mannose, glucose and N-acetylglucosamine were present. The amino acid composition is reported. 4. The EPR spectrum exhibited a high-spin heme signal with rhombic symmetry (gx = 6.92, gy = 5.07 and gz = 1.95). Upon acidification this signal was converted into a signal with more axial symmetry (g perpendicular = 5.89). At high pH (9.5) the EPR spectrum of the enzyme only shows low-spin ferric heme resonances. The circular dichroism spectra of ferric and ferrous myeloperoxidase in the visible and ultraviolet region show maxima and minima in ellipticity.     

Isolation of Assimilatory- and Dissimilatory-Type Sulfite Reductases from Desulfovibrio vulgaris

Bisulfite reductase (desulfoviridin) and an assimilatory sulfite reductase have been purified from extracts of Desulfovibrio vulgaris. The bisulfite reductase has absorption maxima at 628, 580, 408, 390, and 279 nm, and a molecular weight of 226,000 by sedimentation equilibrium, and was judged to be free of other proteins by disk electrophoresis and ultracentrifugation. On gels, purified bisulfite reductase exhibited two green bands which coincided with activity and protein. The enzyme appears to be a tetramer but was shown to have two different types of subunits having molecular weights of 42,000 and 50,000. The chromophore did not form an alkaline ferrohemochromogen, was not reduced with dithionite or borohydride, and did not form a spectrally visible complex with CO. The assimilatory sulfite reductase has absorption maxima at 590, 545, 405 and 275 nm and a molecular weight of 26,800, and appears to consist of a single polypeptide chain as it is not dissociated into subunits by sodium dodecyl sulfate. By disk electrophoresis, purified sulfite reductase exhibited a single greenish-brown band which coincided with activity and protein. The sole product of the reduction was sulfide, and the chromophore was reduced by borohydride in the presence of sulfite. Carbon monoxide reacted with the reduced chromophore but it did not form a typical pyridine ferrohemochromogen. Thiosulfate, trithionate, and tetrathionate were not reduced by either enzyme preparation. In the presence of 8 M urea, the spectrum of bisulfite reductase resembles that of the sulfite reductase, thus suggesting a chemical relationship between the two chromophores.     

Purification and properties of 5,10-methylenetetrahydrofolate reductase, an iron-sulfur flavoprotein from Clostridium formicoaceticum

Methylenetetrahydrofolate reductase in Clostridium formicoaceticum has been purified to a specific activity of 140 mumol min-1 mg-1 when assayed at 37 degrees C, pH 7.2, in the direction of oxidation of 5-methyltetrahydrofolate with benzyl viologen as electron acceptor. The purified enzyme is judged to be homogeneous by polyacrylamide disc-gel electrophoresis and gel filtration. The enzyme which is an octamer has a molecular weight of about 237,000 and consists of four each of two different subunits having the molecular weights 26,000 and 35,000. The octameric enzyme contains per mol 15.2 +/- 0.3 iron, 2.3 +/- 0.2 zinc, 19.5 +/- 1.3 acid-labile sulfur, and 1.7 FAD. The UV-visible absorbance spectrum has a peak at 385 nm and a shoulder at 430 nm and is that of a flavoprotein containing iron-sulfur centers. The reductase, which is sensitive to oxygen, must be handled anaerobically and is stabilized by 2 mM dithionite. It catalyzes the reduction of methylene blue, menadione, benzyl viologen, rubredoxin, and FAD with 5-methyltetrahydrofolate and the oxidation of reduced ferredoxin and FADH2 with 5,10-methylenetetrahydrofolate. No activity was observed with pyridine nucleotides. It is suggested that the physiologically important reaction catalyzed by the enzyme is the reduced ferredoxin-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate.     

Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing alpha beta dimer

Azotobacter vinelandii hydrogenase has been purified to homogeneity from membranes. The enzyme was solubilized with Triton X-100 followed by ammonium sulfate-hexane extractions to remove lipids and detergent. The enzyme was then purified by carboxymethyl-Sepharose and octyl-Sepharose column chromatography. All purification steps were performed under anaerobic conditions in the presence of dithionite and dithiothreitol. The enzyme was purified 143-fold from membranes to a specific activity of 124 mumol of H2 uptake . min-1 . mg protein-1. Nondenaturing polyacrylamide gel electrophoresis of the hydrogenase revealed a single band which stained for both activity and protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands corresponding to peptides of 67,000 and 31,000 daltons. Densitometric scans of the SDS-gel indicated a molar ratio of the two bands of 1.07 +/- 0.05. The molecular weight of the native enzyme was determined by three different methods. While gel permeation gave a molecular weight of 53,000, sucrose density gradient centrifugation and native polyacrylamide gel electrophoresis gave molecular weights of 98,600 +/- 10,000 and 98,600 +/- 2,000, respectively. We conclude that the A. vinelandii hydrogenase is an alpha beta dimer (98,000 daltons) with subunits of 67,000 and 31,000 daltons. Analyses for nickel and iron indicated 0.68 +/- 0.01 mol Ni/mol hydrogenase and 6.6 +/- 0.5 mol Fe/mol hydrogenase. The isoelectric point of the enzyme was 6.1 +/- 0.01. In addition, several catalytic properties of the enzyme have been examined. The Km for H2 was 0.86 microM, and H2 evolution was observed in the presence of reduced methyl viologen. The pH profile of enzyme activity with methylene blue as the electron acceptor has been determined, along with the Km and Vmax for various electron acceptors.     

Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16.

The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.     

Thiobacillus ferrooxidans cytochrome c oxidase: purification, and molecular and enzymatic features.

Cytochrome c oxidase from Thiobacillus ferrooxidans was purified to homogeneity and some of its properties were studied. The oxidase was solubilized with n-octyl-beta-D-thioglucoside (OTG) under acidic conditions (pH 4.0) and purified by one step of ion-exchange chromatography with a CM-Toyopearl column. The absorption spectrum of the oxidase showed peaks at 420 and 595 nm in the oxidized form and at 440 and 595 nm in the reduced form. Its CO compound showed a novel absorption spectrum; a double-peaked gamma band appeared at 429 and 438 nm. The oxidase seemed to have CuA-like copper atom from its ESR and near-infrared spectra. The oxidase molecule consisted of three polypeptides with molecular weights of 53,000, 22,000, and 17,000, respectively, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme in a solution containing detergents was estimated to be 169,000 on the basis of the results obtained by gel filtration, while the molecular weight per heme alpha was estimated to be 83,700. The copper content of the oxidase was 1.01 g atom per mol of heme alpha. Therefore, the cytochrome seemed to contain one molecule of heme alpha and one atom of copper in the minimal structural unit consisting of one molecule each of the three subunits, and to occur as a dimer of the unit in the solution. The oxidase oxidized ferrocytochrome c-552 of the bacterium, and the optimal pH of the reaction was 3.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbon monoxide dehydrogenase activity in Bradyrhizobium japonicum

Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2.99.2) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. The 230-kDa enzyme was composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-kDa) subunits occurring in heterohexameric (LMS)(2) subunit composition. The 75-kDa polypeptide exhibited immunological cross-reactivity with the large subunit of the CODH of Oligotropha carboxidovorans. The B. japonicum enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labile S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yielded di(carboxamidomethyl)molybdopterin cytosine dinucleotide, identifying molybdopterin cytosine dinucleotide as the organic portion of the B. japonicum CODH molybdenum cofactor. The absorption spectrum of the purified enzyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein.     

Purified fusion enzyme between rat cytochrome P4501A1 and yeast NADPH-cytochrome P450 oxidoreductase

A genetically engineered fusion enzyme between rat P4501A1 and yeast P450 reductase in the microsomal fraction of the recombinant yeast AH22/pAFCR1 was purified. The purified enzyme showed a typical CO-difference spectrum of P4501A1 and a single band with an apparent molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This agreed with the molecular weight of 131,202 calculated from the amino acid sequence. The purified enzyme showed both 7-ethoxycoumarin o-deethylase activity and horse heart cytochrome c reductase activity in the presence of NADPH. The 7-ethoxycoumarin o-deethylase activity depended on the species of lipid used for the reconstitution of the purified fusion enzyme although the purified enzyme showed the activity without reconstitution. The purified fusion enzyme had the Km value of 26 microM for 7-ethoxycoumarin and the maximal turnover rate of 29 mol product/min/mol enzyme at 30 degrees C.     

Purification and some properties of carbon monoxide dehydrogenase from Acinetobacter sp. strain JC1 DSM 3803.

A brown carbon monoxide dehydrogenase from CO-autotrophically grown cells of Acinetobacter sp. strain JC1, which is unstable outside the cells, was purified 80-fold in seven steps to better than 95% homogeneity, with a yield of 44% in the presence of the stabilizing agents iodoacetamide (1 mM) and ammonium sulfate (100 mM). The final specific activity was 474 mumol of acceptor reduced per min per mg of protein as determined by an assay based on the CO-dependent reduction of thionin. Methyl viologen, NAD(P), flavin mononucleotide, flavin adenine dinucleotide, and ferricyanide were not reduced by the enzyme, but methylene blue, thionin, and dichlorophenolindophenol were reduced. The molecular weight of the native enzyme was determined to be 380,000. Sodium dodecyl sulfate-gel electrophoresis revealed at least three nonidentical subunits of molecular weights 16,000 (alpha), 34,000 (beta), and 85,000 (gamma). The purified enzyme contained particulate hydrogenase-like activity. Selenium did not stimulate carbon monoxide dehydrogenase activity. The isoelectic point of the native enzyme was found to be 5.8; the Km of CO was 150 microM. The enzyme was rapidly inactivated by methanol. One mole of native enzyme was found to contain 2 mol of each of flavin adenine dinucleotide and molybdenum and 8 mol each of nonheme iron and labile sulfide, which indicated that the enzyme was a molybdenum-containing iron-sulfur flavoprotein. The ratio of densities of each subunit after electrophoresis (alpha:beta:gamma = 1:2:6) and the number of each cofactor in the native enzyme suggest a alpha 2 beta 2 gamma 2 structure of the enzyme. The carbon monoxide dehydrogenase of Acinetobacter sp. strain JC1 was found to have no immunological relationship with enzymes of Pseudomonas carboxydohydrogena and Pseudomonas carboxydovorans.     

Crystallization and Properties of Amine Dehydrogenase from Pseudomonas sp.

An amine dehydrogenase was purified and crystallized from the cell free extract of a Pseudomonas sp., isolated from soil by means of the enrichment technique. The crystalline enzyme gave a single band on polyacrylamide gel electrophoresis and the molecular weight of the enzyme was estimated to be 100,000 by gel filtration on a Sephadex column. Upon SDS-gel electrophoresis, the enzyme was dissociated into two nonidentical subunits having molecular weights of 60,000 (dehydrogenase) and 39,000 (cytochrome c). The absorption spectrum of the enzyme showed absorption maxima at 550 nm, 524 nm, 411 nm and 280 nm, and a broad shoulder at around 350 nm, indicating that the enzyme was purified as a dehydrogenase-cytochrome c complex. The prosthetic group of the dehydrogenase was identified as covalently bound pyrroloquinoline quinone. The enzyme showed a broad substrate specificity toward various amines including aliphatic monoamines, aliphatic diamines, aromatic amines and polyamines.     

Purification and some properties of component A of the 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas species strain CBS

Pseudomonas sp. strain CBS 3 possesses a two-component enzyme system which converts 4-chlorophenylacetate to 3,4-dihydroxyphenylacetate by the incorporation of 2 atoms of molecular oxygen. Component A of this enzyme system was purified to homogeneity by a 5-step procedure. After the last purification step the enzyme was homogeneous in analytical and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the native protein was determined to be 140,000 by Sephadex G-200 and 144,000 by analytical ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that component A consists of three identical subunits with a molecular weight determined to range between 46,000 and 52,000. The isoelectric point was estimated to be 5.0. Component A shows an intensive red-brown color, and in the oxidized state it exhibits a visible absorption spectrum with a maximum at 458 nm and a shoulder at 560 nm. By reduction with sodium dithionite a new peak with a maximum at 518-520 nm is observed. The enzyme contains iron (1.6-1.8 mol/subunit) and acid-labile sulfide (1.6-1.9 mol/subunit) which suggests that component A is an iron-sulfur protein.     

Spinach nitrate reductase: purification, molecular weight, and subunit composition

Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 ± 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000.     

Purification and characterization of bisulfite reductase (desulfofuscidin) from Desulfovibrio thermophilus and its complexes with exogenous ligands

A dissimilatory bisulfite reductase has been purified from a thermophilic sulfate-reducing bacterium Desulfovibrio thermophilus (DSM 1276) and studied by EPR and optical spectroscopic techniques. The visible spectrum of the purified bisulfite reductase exhibits absorption maxima at 578.5, 392.5 and 281 nm with a weak band around 700 nm. Photoreduction of the native enzyme causes a decrease in absorption at 578.5 nm and a concomitant increase in absorption at 607 nm. When reduced, the enzyme reacts with cyanide, sulfite, sulfide and carbon monoxide to give stable complexes. The EPR spectrum of the native D. thermophilus bisulfite reductase shows the presence of a high-spin ferric signal with g values at 7.26, 4.78 and 1.92. Upon photoreduction the high-spin ferric heme signal disappeared and a typical 'g = 1.94' signal of [4Fe-4S] type cluster appeared. Chemical analyses show that the enzyme contains four sirohemes and eight [4Fe-4S] centers per mol of protein. The molecular mass determined by gel filtration was found to be 175 kDa. On SDS-gel electrophoresis the enzyme presents a main band of 44 to 48 kDa. These results suggest that the bisulfite reductase contains probably one siroheme and two [4Fe-4S] centers per monomer. The dissimilatory bisulfite reductase from D. thermophilus presents some homologous properties with desulfofuscidin, the bisulfite reductase isolated from Thermodesulfobacterium commune (Hatchikian, E.C. and Zeikus, J.G. (1983) J. Bacteriol. 153, 1211-1220).     

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

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

Purification and Properties of Sarcosine Oxidase from Cylindrocarpon didymum M–1

Sarcosine oxidase was purified to homogeneity from the cell extract of Cylindrocarpon didymum M–1, aerobically grown in medium containing choline as the carbon source. The molecular weight of the enzyme was estimated to be 45,000 by gel filtration method and 48,000 by the sodium dodecylsulfate disc gel electrophoresis method. The enzyme exhibited an absorption spectrum with maxima at 277 and 450 run and shoulders at 370 and 470 nm. The anaerobic addition of sarcosine to the enzyme resulted in the disappearance of the peak at 450 nm. The enzyme contained one mol of covalently bound FAD per mol of enzyme. Enzyme activity was inhibited by Ag⁺, Cu²⁺, Hg²⁺, p-chloromercuribenzoate and iodoacetate. The enzyme oxidized sarcosine but was inert toward choline, betaine, dimethylglycine and N-methyl amino acids. Km and V_max values for sarcosine were 1.8 ihm and 26.2 μmol/min/mg, respectively. The enzyme catalyzed the following reaction: Sarcosine+O₂+H₂O→glycine +formaldehyde+H₂O₂.     

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 investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.     

Comparative receptor study in gamete chemotaxis of the seaweeds Ectocarpus siliculosus and Cutleria multifida. An approach to interspecific communication of algal gametes

The terminal component of the electron transport chain, cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase) was purified from Bacillus subtilis W23. The enzyme was solubilized with alkyglucosides and purified to homogeneity by cytochrome c affinity chromatography. The enzyme showed absorption maxima at 414 nm and 598 nm in the oxidized form and at 443 nm and 601 nm in the reduced form. Upon reaction with carbon monoxide of the reduced purified enzyme the absorption maxima shifted to 431 nm and 598 nm. Sodium dodecylsulfate polyacrylamide gel electrophoresis indicated that the purified enzyme is composed out of three subunits with apparent molecular weights of 57 000, 37 000 and 21 000. This is the first report on a bacterial aa3-type oxidase containing three subunits. The functional properties of the enzyme are comparable with those of the other bacterial cytochrome c oxidases. The reaction catalyzed by this oxidase was strongly inhibited by cyanide, azide and monovalent salts. Furthermore a strong dependence of cytochrome c oxidase activity on negatively charged phospholipids was observed. Crossed immunoelectrophoresis experiments strongly indicated a transmembranal localization of cytochrome c oxidase.