Purification and properties of sulfhydryl oxidase from bovine pancreas

Immunofluorescent studies showed that antibodies prepared against bovine milk sulfhydryl oxidase reacted with acinar cells of porcine and bovine pancreas. A close inspection of the specific location within bovine pancreatic cells revealed that the zymogen granules, themselves, bound the fluorescent antibody. Bovine pancreatic tissue was homogenized in 0.3 M sucrose, then separated into the zymogen granule fraction by differential centrifugation. The intact zymogen granules were immunofluorescent positive when incubated with antibodies to bovine milk sulfhydryl oxidase, and glutathione-oxidizing activity was detected under standard assay conditions. Pancreatic sulfhydryl oxidase was purified from the zymogen fraction by precipitation with 50% saturated ammonium sulfate, followed by Sepharose CL-6B column chromatography. Active fractions were pooled and subjected to covalent affinity chromatography on cysteinylsuccinamidopropyl-glass using 2 mM glutathione as eluant at 37 degrees C. The specific activity of bovine pancreatic sulfhydryl oxidase thus isolated was 10-20 units/mg protein using 0.8 mM glutathione as substrate. Ouchterlony double-diffusion studies showed that antibody directed against the purified bovine milk enzyme reacted identically with pancreatic sulfhydryl oxidase. The antibody also immunoprecipitated glutathione-oxidizing activity from crude pancreatic homogenates. Western blotting analysis indicated a 90,000 Mr antigen-reactive band in both bovine milk and pancreatic fractions while sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-staining protein with an apparent Mr 300,000. Thus, we believe that sulfhydryl oxidase may exist in an aggregated molecular form. Bovine pancreatic sulfhydryl oxidase catalyzes the oxidation of low-molecular-weight thiols such as glutathione, N-acetyl-L-cysteine, and glycylglycyl-L-cysteine, as well as that of a high-molecular-weight protein substrate, reductively denatured pancreatic ribonuclease A.     

Purification and some properties of cyclohexylamine oxidase from a Pseudomonas sp.

Cyclohexylamine oxidase was purified 90-fold from cell-free extracts of Pseudomonas sp. capable of assimilating sodium cyclamate. The purified enzyme was homogeneous in disc electrophoresis, and the molecular weight was found to be approximately 80,000 by gel filtration. The enzyme catalyzed the following reaction: cyclohexylamine+O2+H2O leads to cyclohexanone+NH3+H2O2. The enzyme thus can be classified as an amine oxidase; it utilized oxygen as the ultimate electron acceptor. The pH optimum of the reaction was 6.8 and the apparent Km value for cyclohexylamine was 2.5 X 10(-4) M. The enzyme was highly specific for the deamination of alicyclic primary amines such as cyclohexylamine, but was found to be inactive toward ordinary amines used as substrates for amine oxidases. The enzyme solution was yellow in color and showed a typical flavoprotein spectrum; the addition of cyclohexylamine under anaerobic conditions caused reduction of the flavin in the native enzyme. The flavin of the prosthetic group was identified as FAD by thin layer chromatography. The participation of sulfhydryl groups in the enzymic action was also suggested by the observation that the enzyme activity was inhibited in the presence of PCMB and could be recovered by the addition of glutathione.     

Purification and some properties of a novel microbial lactate oxidase

 Geotrichum candidum was found to produce a lactate oxidase. The enzyme was purified by gel filtration and ion-exchange chromatography. The purified lactate oxidase showed a molecular mass of 50 kDa under denaturing and about 400 kDa under non-denaturing conditions. Transmission electron micro-scopy analysis confirmed an octameric structure. FMN was found to be a cofactor for this enzyme. Polarographic studies confirmed an oxygen uptake by the lactate oxidase. The enzyme showed specificity towards the L isomer of lactate and did not oxidise pyruvate, fumarate, succinate, maleate and ascorbate. It was stable at alkaline pH and also for 15 min at 45°C. The addition of glycerol and dextran 500 000 to the enzyme sample enhanced storage stability. Received: 28 September 1995/Received revision: 10 January 1996/Accepted: 15 January 1996     

Purification and some properties of sulfite oxidase from Thiobacillus neapolitanus

Summary Sulfite oxidase has been purified 100-fold from extracts of Thiobacillus neapolitanus. The enzymatic activity, measured as the rate of oxygen consumption, was stimulated some 1.5–2-fold by 3.3mm AMP throughout the course of purificationm indicating that a single enzyme is responsible for the oxidation of sulfite in the presence and absence of AMP. Reduced glutathione inhibited activity and prevented the AMP-dependent rate stimulation. The rate of sulfite oxidation in the presence and absence of AMP was first order with respect to the sulfite concentration.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday.Part of a dissertation submitted by W. P. Hempfling to Yale University in partial fulfillment of the requirements for the Ph. D. degree, 1964.     

L-alpha-Hydroxyacid oxidase isozymes. Purification and molecular properties.

L-alpha-Hydroxyacid oxidase isozymes from rat liver (A isozyme) and kidney (B isozyme) have been isolated in a high state of purity with specific activities of 61 and 14.7 microkatals per gram protein respectively. The subunit molecular weights determined by sodium dodecylsulphate polyacrylamide gel electrophoresis were 40000 +/- 3000; the mouse A and B isozymes were also partially purified and their subunit molecular weights shown to be 37000.     

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.     

Purification and some properties of polyphenol oxidase from root-forming carrot callus tissues

1. Polyphenol oxidase (o-diphenol : O₂ oxidoreductase; E.C.1.10.3.1 [EC] ) was isolated from the other phenolases which werepresent in root-forming carrot callus, and its properties wereexamined. 2. The enzyme was purified about 45-fold over crudeextracts (precipitates between 40–70% saturation widiammonium sulfate) by a combination of Bio-gel filtration, protein-bagfiltration, and carboxymethyl cellulose chromatography. Thepurified oxidase was homogeneous according to polyacrylamidegel electrophoresis and Sephadex gel filtration. It was confirmedby CM-cellulose chromatography that the enzyme was absent incallus tissues without accompanying redifferentiation. 3. Themolecular weight of this oxidase was estimated to be 110,000-120,000 from molecular weight-mobility profiles on polyacrylamidegels containing sodium dodecyl sulfate and molecular size-elutionvolume correlations on Sephadex G-150 columns. 4. The enzymeoxidized o-diphenols but showed no detectable activity againstmonophenols. Pyrocatechol, dopamine, caffeic acid, and chlorogenicacid were effectual substrates of the enzyme with K_m valuesranging from 10^–3 M to 10^–5M. The enzyme effectivelycatalyzed the oxidation of o-diphenols over the range of pH6.0 to 7.0 and was readily inactivated by heating. The enzymeactivity was slightly influenced by increasing ionic strength.The initial rate of the enzymic reaction was enhanced by additionof Cu²⁺, Co²⁺ and Mn²⁺ ions, and was reduced in the presenceof DTT, PCMPS, glycylglycine, and DIECA. (Received June 17, 1978; )     

Purification and some properties of the 45 kDa diphenylene iodonium-binding flavoprotein of neutrophil NADPH oxidase.

The 45 kDa diphenylene iodonium-binding flavoprotein of the human neutrophil superoxide-generating oxidase has been purified by affinity chromatography. The polypeptide was eluted from Blue Memsep or 2',5'-ADP-agarose columns with either NADP or low concentrations of the specific inhibitor diphenylene iodonium. The purified protein was shown to bind FAD at a ratio of 1.09 mol of FAD/mol of protein. The reconstituted flavoprotein had a fluorescence spectrum similar, but not identical, to that of free FAD. It had an isoelectric point of approx. 4.0. The reconstituted flavoprotein displayed no diaphorase activity towards a range of artificial electron acceptors. Polyclonal antibodies raised against the pure protein inhibited superoxide generation by solubilized oxidase in a dose-dependent manner, and inhibited superoxide generation when incubated with either cytosol or membrane fractions in a reconstituted system. These antibodies precipitated the 45 kDa polypeptide together with a haem-containing 23 kDa protein thought to be the small subunit of cytochrome b-245. Antibodies raised against cytochrome P-450 reductase also precipitated these two polypeptides. These results are consistent with the 45 kDa polypeptide being the flavoprotein of the neutrophil superoxide-generating oxidase.     

D-aspartate oxidase from beef kidney. Purification and properties

The flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been purified to homogeneity from beef kidney cortex. The protein is a monomer with a molecular weight of 39,000 containing 1 molecule of flavin. The enzyme as isolated is a mixture of a major active form containing FAD and a minor inactive form containing 6-hydroxy-flavin adenine dinucleotide (6-OH-FAD). The absorption and fluorescence spectral properties of the two forms have been studied separately after reconstitution of the apoprotein with FAD or 6-OH-FAD, respectively. FAD-reconstituted D-aspartate oxidase has flavin fluorescence, shows characteristic spectral perturbation upon binding of the competitive inhibitor tartaric acid, is promptly reduced by D-aspartic acid under anaerobiosis, reacts with sulfite to form a reversible covalent adduct, stabilizes the red anionic form of the flavin semiquinone upon photoreduction, and yields the 3,4-dihydro-FAD-form after reduction with borohydride. A Kd of 5 X 10(-8) M was calculated for the binding of FAD to the apoprotein. 6-OH-FAD-reconstituted D-aspartate oxidase has no flavin fluorescence, shows no spectral perturbation in the presence of tartaric acid, is not reduced by D-aspartic acid under anaerobiosis, does not stabilize any semiquinone upon photoreduction, and does not yield the 3,4-dihydro-form of the coenzyme when reduced with borohydride; the enzyme stabilizes the p-quinoid anionic form of 6-OH-FAD and lowers its pKa more than two pH units below the value observed for the free flavin. The general properties of the enzyme thus resemble those of the dehydrogenase/oxidase class of flavoprotein, particularly those of the amino acid oxidases.     

Diamine oxidase ofLathyrus sativus seedlings. Purification and properties

Diamine oxidase (EC 1.4.3.6) was purified from 5-day-old etiolated seedlings ofLathyrus sativus by MnCl₂ treatment, (NH₄)₂SO₄ and acetone fractionations, DEAE-Sephadex chromatography followed by gel filtration on Sephadex G-200. A single step purification of the enzyme was achieved by using an immunoaffinity column, wherein rabbit antibodies to the homogeneous diamine oxidase were coupled to CNBr-activated Sepharose. The enzyme thus obtained was homogeneous by electrophoretic, immunological and ultracentrifugal criteria. It had anM _r of 148,000 (6.46S) and was a dimer with similar sub-units (M _r 75,000). Amino acid analysis showed the absence of cysteine residues although it contained five disulphide bonds. The enzyme had copper (2.7 g atom/mol enzyme) but was not a glycoprotein. No absorption maximum in the visible region was detectable. Ethylenediamine 1,3-diaminopropane and histamine were potent competitive inhibitors for the substrate putrescine. The addition of monospecific antibodies to the enzyme increased the K_m for benzyl amine without any change in the V_max Diamine oxidase from pea seedling, partially purified, exhibited complete crossreactivity with the antibodies to theL. sativus enzyme.     

Purification and some properties of rat liver cysteine oxidase (cysteine dioxygenase).

Cysteine oxidase (cysteine dioxygenase, EC 1.13.11.20) was purified approximately 1000-fold from rat liver. The purified enzyme (protein-B) was obtained as an inactive form, which was activated by anaerobic preincubation with L-cysteine. The active form of protein-B was inactivated during aerobic incubation to produce cysteine sulfinate. This inactivation of protein-B was protected by a distinct protein in rat liver cytoplasm, namely stabilizing protein (protein-A). The Ka and Km values for L-cysteine were 0.8-10(-3) M and 1.3-10(-3) M respectively. The enzyme was strongly inhibited by Cu+ and/or Fe2+ chelating agents but not by Cu2+ chelating agent. The optimum pH of enzyme reaction was 8.5-9.5 while that of enzyme activation was 6.8-9.5, with a broad peak.     

Indoleamine 2,3-dioxygenase. Purification and some properties.

Indoleamine 2,3-dioxygenase was purified from rabbit small intestine to apparent homogeneity as judged by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The native enzyme was a monomeric protein of a molecular weight of 41,000 +/- 1,000 with an s020,w value of 3.45 S. It had a relative abundance of hydrophobic amino acids such as valine, leucine, and isoleucine, and contained approximately 5% carbohydrate by weight. The estimated content of sugar residues per mol of enzyme was: galactose, 1.2; mannose, 2.6; N-acetylglucosamine, 5.2; and sialic acid, 0.8. One mole of enzyme had 0.8 mol of protoheme IX as a prosthetic group. However, copper was not detected in a significant amount and the ratio of copper to heme was less than 0.03. EPR spectra of the nitric oxide complex of the ferrous enzyme indicated that a nitrogen atom, possibly in an imidazole group, might be coordinated as the fifth ligand of the heme coenzyme. The anisotropic g values were gx = 2.08, gy = 1.98, and gz = 2.01. A single enzyme protein catalyzed the oxygenative ring cleavage of D- and L-tryptophan, D- and L-5-hydroxytryptophan, tryptamine, and serotonin. In addition, the purified enzyme had a peroxidase activity with guaiacol and potassium iodide as hydrogen donors, but not a catalase activity.     

Purification and some properties of chlorogenic acid oxidase from apple (Malus pumila).

Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malus pumila cv. Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65,000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6-8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (-)-epicatechin and 4-methylcatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The Km value for the enzyme was found to be 122 microM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyl-dithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity.     

Purification and properties of nitroalkane oxidase from Fusarium oxysporum.

A nitroalkane-oxidizing enzyme, which was inducibly formed by addition of nitroethane to the medium was purified to homogeneity from an extract of Fusarium oxysporum (IFO 5942) with an overall yield of about 20%. The enzyme catalyzed the oxidative denitrification of 1-nitropropane as follows: CH2(NO2)CH2CH3 + O2 + H2O leads to OHCCH2CH3 + HNO2 + H2O2. In addition to 1-nitropropane, 3-nitro-2-pentanol, 2-nitropropane, and nitrocyclohexane are good substrates; the enzyme is designated "nitroalkane oxidase" (EC class 1.7.3). The enzyme has a molecular weight of approximately 185,000 and consists of four subunits identical in molecular weight (47,000). Flavin adenine dinucleotide was required for the enzyme activity and could be replaced in part by riboflavin 5'-phosphate. The maximum reactivity was found at about pH 8.0. The enzyme was inhibited significantly by HgCl2, KCN, p-chloromercuribenzoate, and N-ethylmaleimide. The Michaelis constants are as follows: 1-nitropropane, 1.54 mM; 2-nitropropane, 7.40 mM; nitroethane, 1.00 mM; 3-nitro-2-pentanol, 3.08 mM; nitrocyclohexane, 0.90 mM; and flavin adenine dinucleotide, 1.33 micrometer.