Composition of partially purified NADPH oxidase from pig neutrophils.

The superoxide (O2.-)-forming enzyme NADPH oxidase from pig neutrophils was solubilized and partially purified by gel-filtration chromatography. The purification procedure allowed the separation of NADPH oxidase activity from NADH-dependent cytochrome c reductase and 2,6-dichlorophenol-indophenol reductase activities. O2.-forming activity was co-purified with cytochrome b-245 and was associated with phospholipids. However, active fractions endowed with cytochrome b were devoid of ubiquinone and contained only little FAD. The cytochrome b/FAD ratio was 1.13:1 in the crude solubilized extract and increased to 18.95:1 in the partially purified preparations. Most of FAD was associated with fractions containing NADH-dependent oxidoreductases. These results are consistent with the postulated role of cytochrome b in O2.-formation by neutrophil NADPH oxidase, but raise doubts about the participation of flavoproteins in this enzyme activity.     

Presence of cytochrome b-245 in NADPH oxidase preparations from human neutrophils

The composition of NADPH oxidase purified by Red Sepharose chromatography of extracts from human neutrophil membranes was investigated. In contrast to that was recently reported by others, the enzyme isolated according to this procedure contained a high concentration of cytochrome b-245 and little FAD. The results reinforce the belief that cytochrome b-245 is a major component of the NADPH oxidase and plays a fundamental role in the formation of O2-by neutrophils.     

A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans

The formation of nitrite reductase and cytochrome c in Micrococcus denitrificans was repressed by O₂. The purified nitrite reductase utilized reduced forms of cytochrome c, phenazine methosulphate, benzyl viologen and methyl viologen, respectively, as electron donors. The enzyme was inhibited by KCN, NaN₃ and NH₂OH each at 1 mM, whereas CO and bathocuproin, diethyl dithiocarbamate, o-phenanthroline and ,'-dipyridyl at 1 mM concentrations were relatively ineffective. The purified enzyme contains cytochromes, probably of the c and a₂ types, in one complex. A K_m of 46 μM for NO₂⁻ and a pH optimum of 6.7 were recorded for the enzyme. The molecular weight of the enzyme was estimated to be around 130000, and its anodic mobility was 6.8·10⁻⁶ cm²·sec⁻¹·V⁻¹ at pH 4.55.

The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a K_m of 27 μM for O₂. This activity was also inhibited by KCN, NaN₃ and NH₂OH and by NO₂⁻.

A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO₂⁻. It was inhibited by smaller amounts of KCN, NaN₃ and NH₂OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a K_m for O₂ of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO₂⁻.

The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller K_m for O₂ and would therefore be expected to function more efficiently especially at low concentrations of O₂.     

刺芹侧耳芳基醇氧化酶的分离纯化及其酶学性质

分离纯化刺芹侧耳Pleurotus eryngii芳基醇氧化酶,并探究其酶学性质。通过硫酸铵盐沉、DEAE-Sepharose Fast Flow弱阴离子交换层析、Sephacryl S-200 High Resolution凝胶过滤层析和Source 15Q强阴离子交换层析,得到纯化的单一酶。经肽指纹图谱鉴定,确定其为芳基醇氧化酶,酶活回收率25.5%,纯化倍数38.2。结合SDS-PAGE和IEF-PAGE分析,确定其分子量和等电点分别为70kDa和4.2。以藜芦醇为底物,该酶最适反应pH为6.0,最适反应温度为70℃,金属离子Zn²⁺、Fe²⁺和Cu²⁺对芳基醇氧化酶的活性抑制作用明显,K_m和V_max分别为0.921mmol/L和80U/mg。     

Effects of Respiratory Burst Inhibitors on Nitric Oxide Production by Human Neutrophils

Human neutrophils (PMN) activated by N-formyl-methionyl-leucyl-phenylalanine (fMLP) simultaneously release nitric oxide (.NO), superoxide anion (O₂-) and its dismutation product, hydrogen peroxide (H₂O₂). To assess whether NO production shares common steps with the activation of the NADPH oxidase, PMN were treated with inhibitors and antagonists of intracellular signaling pathways and subsequently stimulated either with fMLP or with a phorbol ester (PMA). The G-protein inhibitor, pertussis toxin (1-10 μg/ml) decreased H₂O₂ yield without significantly changing. NO production in fMLP-stimulated neutrophils; no effects were observed in PMA-activated cells. The inhibition of tyrosine kinases by genistein (1-25 μg/ml) completely abolished H₂O₂ release by fMLP-activated neutrophils; conversely, NO production increased about 1.5- and 3-fold with fMLP and PMA, respectively. Accordingly, orthovanadate, an inhibitor of phosphotyrosine phosphatase, markedly decreased -NO production and increased O_2;^- release. On the other hand, inhibition of protein kinase C with staurosporine and the use of burst antagonists like adenosine, cholera toxin or dibutyryl-cAMP diminished both H₂O₂ and NO production. The results suggest that the activation of the tyrosine kinase pathway in stimulated human neutrophils controls positively O₂^- and H₂O₂ generation and simultaneously maintains -NO production in low levels. In contrast, activation of protein kinase C is a positive modulator for O_2;^-and *NO production.     

Superoxide Radical Generation in Peroxisomal Mimbranes: Evidence for the Participation of the 18-kDa Integral Membrane Polypeptide

Peroxisomes were isolated from pea (Pisum sativum L.) leaves and the peroxisomal membranes were purified by treatment with Na₂CO₃. The production of superoxide radicals (O₂^-) induced by NADH was investigated in peroxisomal membranes from intact organelles incubated with proteases (pronase E and proteinase K). Under isoosmotic conditions, in the presence of pronase E, the production of O₂- radicals was inhibited by 80%. SDS-PAGE of peroxisomal membranes after protease treatment demonstrated a decrease in the 18-kDa PMP. This suggests that this polypeptide has a small fragment exposed to the cytosolic side of the peroxisomal membrane which is essential for O₂-production. The 18-kDa PMP was purified by preparative SDS-PAGE and in the reconstituted protein the NADH-driven production of O₂- radicals was investigated. The isolated polypeptide showed a high generation rate of superoxide (about 300 nmol O₂- × mg-¹ protein × min^-1) which was completely inhibited by 50 mM pyridine. The 18-kDa PMP was recognized by a polyclonal antibody against Cyt b₅ from human ery-throcytes. The presence of b-type cytochrome in peroxisomal membranes was demonstrated by difference spectroscopy. Results obtained show that in the NADH-dependent O₂- radical generating system of peroxisomal membranes, the 18-kDa integral membrane polypeptide, which appears to be Cyt b₅, is clearly involved in superoxide radical production.     

Purification and properties of an O2-.-generating oxidase from bovine polymorphonuclear neutrophils

A membrane-associated O2-.-generating oxidase has been purified from activated bovine polymorphonuclear neutrophils (PMN). The oxidase was extracted with Triton X-100 from a PMN membrane fraction largely devoid of lysosomal granules. The Triton extract was purified by a series of steps, including ion-exchange chromatography on DE-52 cellulose, gel filtration on Sephadex G-200, and isoelectric focusing. The O2-.-generating oxidase activity was assayed as a superoxide dismutase inhibitable cytochrome c reductase. The activity of the purified enzyme was strictly dependent on NADPH as electron donor. The purification factor with respect to the phorbol myristate acetate activated PMN was 75, and the recovery was about 6%. The reactivity of the purified oxidase was increased by 3-4-fold after incubation with asolectin. The minimum molecular weight of the oxidase, deduced from migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 65 000 +/- 3000. The optimum pH of the oxidase was 7.5, its KM,NADPH was congruent to 30 microM, and its isoelectric point was at pH 5.0. The enzyme was inhibited by low concentrations of mersalyl (half-inhibition congruent to 10 microM) and Cibacron Blue (half-inhibition less than 10 microM). It was insensitive to 1 mM cyanide. Rapid loss of activity occurred at 0-2 degrees C, concomitantly with a decrease in sensitivity to superoxide dismutase: both activity and sensitivity to superoxide dismutase could be restored by addition of asolectin. The purified oxidase contained no spectrophotometrically detectable cytochrome b, and enzymatic assay failed to detect FAD in oxidase preparations subjected to heat treatment or trypsin digestion.     

Solubilization and partial purification of aromatase from chicken ovary

Aromatase, the cytochrome P-450 that converts androgen to estrogen, has been solubilized from chicken ovarian microsomes with the nonionic detergent Emulgen 913. Following chromatography on gel filtration, anion exchange, dye affinity, and hydrophobic media, ovarian aromatase is purified up to 27-fold with 10-15% recovery. Separation of the cytochrome P-450 aromatase from NADPH cytochrome P-450 reductase is achieved during the purification. The partially purified enzyme is stable for as long as 6 months when frozen in liquid nitrogen in buffer containing dithiothreitol, glycerol, Emulgen and 150 mM KCl.     

Purification by hydrophobic chromatography of soluble cytochrome b5 of human erythrocytes

Soluble cytochrome b₅ of human erythrocytes was purified very effectively by hydrophobic chromatography using a butyl-Toyopearl 650 column. Cytochrome b₅ was adsorbed tightly on the column in the presence of 60% saturated ammonium sulfate, and was eluted at 40% saturation of ammonium sulfate in the elution buffer. The chromatography gave a good yield of cytochrome b₅ of the highest purity so far reported as estimated from the 414 nm to 280 nm absorbance ratio of the oxidized form of the cytochrome b₅. The value obtained wit the cytochrome b₅ purified in this study was 6.57, and is higher than the previously reported highest value of 6.4 (Hultquist, D.E., Dean, R.T. and Douglas, R.H. (1974) Biochem. Biophys. Res. Commun. 60, 28–34). Spectral properties including molecular absorption coefficients were determined using the cytochrome b₅ purified by this method.     

A new purification procedure and molecular properties of Pseudomonas cytochrome oxidase

A procedure has been developed for purification of the cytochrome oxidase from Pseudomonas aeruginosa (EC 1.9.3.2) using DEAE- and CM-cellulose chromatography, gel filtration and crystallization. The final preparation was found to be homogeneous according to ultracentrifugal and disc electrophoretic criteria. The crystalline preparation also exhibited nitrite reductase activity. The spectrum of the enzyme characterizes it as cytochrome cd. At 280 nm E^{1 %}_{1 cm} was 18.5 after dry weight analysis.

The molecular weight of the cytochrome oxidase was calculated to be 119000 based on a sedimentation coefficient s° _20,w = 7.36 S, diffusion coefficient D _20,w = 5.36×10⁻⁷ cm²×s⁻¹ and partial specific volume of 0.72 ml/g. The iron content of the enzyme (0.166 %) indicates that this entity contains four iron atoms per molecule. Succinylation of the enzyme produced two probably identical subunits containing both hemes c and d, having a sedimentation coefficient s° _20,w = 4.30 S and an approximate molecular weight of 65000. In dodecylsulphate-acrylamide gel electrophoresis the cytochrome oxidase also dissociates into two subunits with molecular weight of 63000.     

Purification of cytochrome b6 A tightly bound protein in chloroplast membranes

Purification of cytochrome b₆ was pursued to further develop rational technology for purification, proof of purity, and study of properties of membrane proteins. Cytochrome b₆ was purified—the first time from any source—from spinach chloroplast membranes; yield of pure cytochrome b₆ was 30% of that found in ethanol-extracted particles. The three-step procedure (pH 8) employed: (I) extraction in Triton X-100-4 M (optionally 2 M) urea, (II) chromatography in a Bio-Gel A-1.5m Column (Triton X-100-4 M urea). Without this step, subsequent electrophoresis failed. (III) Preparative disc gel electrophoresis.

Properties of cytochrome b₆: Cytochrome b₆ migrated in undenatured form as a single band in disc electrophoresis (pH 8, 7 or 8.9). None of the limited, accepted properties of the cytochrome in particles was altered by the purification procedure: Reduced b₆ has absorption maxima (22 °C) at 434, 536, and 563 nm; at −199 °C the a absorption region shows two peaks of equal intensity at 561 and 557 nm. Cytochrome b₆ is reduced by dithionite (not by ascorbate) and is autoxidizable. The prosthetic group of b₆ is protohaemin and is fully extractable by acid-acetone. No non-haem iron is present. The millimolar extinction coefficient of reduced b₆ (563–600nm) per mole of haem is 21. The protein equivalent weight is 40000 g per mole of haem. Cytochrome b₄ is an intrinsically aggregatable molecule. The reduced cytochrome does not react with CO except when Triton X-100 is present.     

NADPH-cytochrome c reductase from rabbit peritoneal neutrophils. Purification, properties and function in the respiratory burst.

An NADPH-dependent membrane-bound flavoprotein dehydrogenase, assayed as a catalyst of electron transfer from NADPH to cytochrome c, was extracted from membranes of rabbit peritoneal neutrophils with Triton X-100 and sodium deoxycholate in the presence of diisopropylfluorophosphate as antiprotease, and purified to electrophoretic homogeneity. The purified enzyme in detergent was able to enhance the rate of formation of the superoxide anion O2- in a cell-free system, consisting of membrane and cytosolic fractions from resting neutrophils complemented with arachidonic acid, guanosine 5'-[gamma- thio]triphosphate and Mg2+. This suggested that the NADPH dehydrogenase was a component of the rabbit neutrophil oxidase complex. The purification factor of the enzyme with respect to the membrane fraction was close to 1000 and the recovery of activity was 33%. FMN and FAD were associated with the enzyme in a molar ratio close to 1. On SDS/PAGE, the enzyme migrated with a molecular mass of 77 kDa. A similar mass was determined by filtration on a molecular sieve. The isoelectric point of this enzyme was 4.7 +/- 0.1. Its activity was maximal between pH 7.5 and pH 8.5, and depended on the ionic strength of the medium, with a maximum at an ionic strength of 0.5. Reduction of cytochrome c by NADPH obeyed Michaelis-Menten kinetics with a KM value of 15 microM for cytochrome c. When NADPH was the variable substrate, a KM value of 1.9 microM for NADPH was found, but a significant deviation from Michaelis-Menten kinetics was observed at high concentrations of NADPH. Mersalyl strongly inhibited the reductase activity when added to the enzyme prior to NADPH; preincubation of the enzyme with NADPH considerably reduced the inhibitory efficiency of mersalyl. A partially proteolyzed water-soluble, active, form of enzyme with a molecular mass of 67 kDa was prepared. The proteolyzed enzyme exhibited the same specificity, and kinetic behavior with respect to NADPH, and the same dependency on the ionic strength, as the native enzyme.     

Examination of the Inhibitory Effect of Norathyriol in Formylmethionyl-Leucyl-Phenylalanine-Induced Respiratory Burst in Rat Neutrophils

Norathyriol, aglycone of a xanthone C-glycoside mangiferin isolated from Tripterospermum lanceolatum, concentration dependently inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O₂^˙−) generation and O₂ consumption in rat neutrophils. In cell-free oxygen radical generating system, norathyriol inhibited the O₂^˙− generation during dihydroxyfumaric acid (DHF) autoxidation and in hypoxanthine-xanthine oxidase system. fMLP-induced transient elevation of [Ca²⁺]_i and the formation of inositol trisphosphate (IP₃) were significantly inhibited by norathyriol (30 μM) (about 30 and 46% inhibition, respectively). Norathyriol concentration dependently suppressed the neutrophil cytosolic phospholipase C (PLC). In contrast with the marked attenuation of fMLP-induced protein tyrosine phosphorylation (about 70% inhibition at 10 μM norathyriol), norathyriol only slightly modulated the phospholipase D (PLD) activity as determined by the formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt). Norathyriol did not modulate the intracellular cyclic AMP level. In the presence of NADPH, the phorbol 12-myristate 13-acetate (PMA)-activated particulate NADPH oxidase activity was suppressed by norathyriol in a concentration-dependent manner and the inhibition was noncompetitive with respect to NADPH. Norathyriol inhibited the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free NADPH oxidase system at the same concentration range as those used in the suppression of PMA-activated particulate NADPH oxidase activity. Taken together, these results suggest that the scavenging ability of norathyriol contributes to the reduction of generated O₂^˙−, however, the inhibition of O₂^˙− generation from neutrophils by norathyriol is attributed to the blockade of PLC pathway, the attenuation of protein tyrosine phosphorylation, and to the suppression of NADPH oxidase through the interruption of electrons transport.     

Catabolism of indole-3-acetic acid in citrus leaves: identification and characterization of indole-3-aldehyde oxidase

A new enzyme, named indole-3-aldehyde oxidase (IAldO), was identified in citrus ( Citrus sinensis L. Osbeck cv. Shamouti) leaves. The enzyme was partially purified by (NH₄)₂SO₄ fractionation. Sephadex G-200 gel filtration and DEAE-cellulose ion exchange chromatography. IAldO catalyzes the oxidation of indole-3-aldehyde (IAld) to indole-3-carboxylic acid (ICA) with the production of H₂O₂. The enzyme is highly specific for IAld. The apparent K_M of the enzyme for IAld is 19 μ M . The optimum oxidation of IAld occurs at pH 7. 5. The molecular mass of the enzyme, as determined by Sepharose-6B gel filtration, is about 200 kDa. Based on inhibitor studies, it is concluded that IAldO is not a flavin-linked oxidase and there is no requirement for free sulfhydryl groups or divalent cations for maximum activity. The enzyme is strongly inhibited by benzaldehyde. Ethylene pretreatment, wounding and aging of leaf tissues did not affect enzyme activity, suggesting that the enzyme is constitutive in citrus tissues.     

Purification of cytochrome b-245 from human neutrophils.

The low potential cytochrome b (b-245) of the microbicidal oxidase of phagocytic cells has been purified from neutrophils from patients with chronic myeloid leukaemia. Cells were homogenized in the presence of proteinase inhibitors and centrifuged to remove the cytoplasm. The pellets containing membranes, granules and other organelles (15 mg/ml) were then washed with buffered sodium cholate (5 mg/ml). Residual pellets were subsequently solubilized with the non-ionic detergent Triton N 101 (10 mg/ml) which extracted about 60% of the cytochrome b. About 10% of the cytochrome b was of mitochondrial origin which was removed on a column of n-amino-octyl-Sepharose that did not adsorb cytochrome b-245. Cytochrome b-245 was chromatographed on a column of heparin-agarose and eluted with NaCl to give a peak specific content of 11-16 nmol of cytochrome b-245/mg of protein, representing a 140-200-fold purification with a recovery of 15%. This technique results in the purification of approx. 100-150 nmol of highly purified cytochrome b-245 from (3-5) X 10(11) cells within 4 days. The most purified material gave a broad band with an apparent Mr of between 68 000 and 78 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, but gel filtration indicated an aggregated form of the protein in Triton N101 . Purified protein (14 nmol of haem/mg of protein) did not contain FAD or FMN and had no NADPH-dependent O2--generating activity.     

Purification and properties of a neutral invertase from the roots of Cichorium intybus

Multiple activity peaks of neutral invertase (EC 3.2.1.26) were found in chicory roots ( Cichorium intybus L. var. foliosum cv. Flash). The main activity peak was purified by a combination of anion-exchange chromatography, hydrophobic interaction chromatography, chromatofocusing and gel filtration. This protocol produced a 77-fold purification and a specific activity of 1.6 μmol (mg protein)⁻¹ min⁻¹. The mass of the enzyme was 260 kDa as estimated by gel filtration and 65 kDa on SDS-PAGE. Optimal activity was found between pH 7 and 7.5. The purified enzyme exhibited hyperbolic saturation kinetics with a K_m between 10 and 20 mM for sucrose. No other products than glucose and fructose could be detected. Raffinose was hydrolyzed at a rate of 2.4% relative to sucrose whereas the enzyme did not hydrolyze maltose, cellobiose, trehalose, 1-kestose, 1.1-nystose or inulin. Neutral invertase activity was completely inhibited by HgCl₂ and AgNO₃ and partially inhibited by CoCl₂, and ZnSO₄ (1 mM). Pyridoxal phosphate (K_i≅ 500 μ M ), Tris (K_i≅ 1.2 m M ), glucose and fructose (K_i≅ 16 m M ) were strong inhibitors of the enzyme. Fructose and Tris behaved as competitive inhibitors. A possible role for the enzyme's activity in vivo is discussed.     

Purification of a cytochrome aa3 terminal oxidase from protoplast membrane vesicles of Micrococcus luteus

Abstract A cytochrome aa₃ terminal oxidase was isolated from protoplast membrane vesicles of Micrococcus luteus grown under aerobic conditions. The purified complex showed similarities to cytochrome c oxidase (EC 1.9.3.1) of the electron transport chain of mitochondria and many prokaryotes. The enzyme was solubilized by subsequent treatment with the detergents CHAPS and n-dodecyl-β-d-maltoside and purified by ion-exchange chromatography using poly-L-lysine agarose and TMAE-fractogel-650 (S) columns, followed by hydroxyapatite chromatography. The purified complex is composed of two major subunits with apparent molecular masses of 54 and 32 kDa. After purification the isolated enzyme contains 12.1 nmol of heme A (mg protein)⁻¹ and exhibits absorption maxima at 424 nm and 598 nm in the oxidized state and at 442 nm and 599 nm in the reduced state. The CO-difference spectrum shows peaks at 428 and 590 nm which is indicative of heme a ₃, furthermore oxygen consumption was found to be sensitive to cyanide.     

Partial purification of the cyanide-resistant alternative oxidase of skunk cabbage (Symplocarpus foetidus) mitochondria.

A partial purification of the cyanide-resistant, alternative oxidase from skunk cabbage (Symplocarpus foetidus L.) spadix mitochondria is described. Skunk cabbage mitochondria were solubilized in N,N-bis-(3-D-glucon-amido-propyl)deoxycholamide and the alternative oxidase was purified using a batch DEAE-cellulose treatment, followed by precipitation with Extracti-Gel and chromatography on Sephadex G-200. Following pooling and concentrating of the most active fractions from the gel filtration column, a 20- to 30-fold purification of the alternative oxidase was obtained, with no evidence of contamination by cytochrome c oxidase (complex IV) or cytochrome c reductase (complex III). Polyacrylamide gel electrophoresis of the partially purified oxidase showed major polypeptides at 36 and 29 kD, both of which react with monoclonal antibodies raised against the Sauromatum guttatum alternative oxidase. The purified oxidase fraction showed no absorbance in the visible spectral region, and addition of sodium borohydride induced no absorbance changes in the ultraviolet region. The purified alternative oxidase catalyzed the four-electron reduction of oxygen to water in the absence of citrate, but catalyzed an apparent two-electron reduction of oxygen to hydrogen peroxide in the presence of 0.7 M citrate.