Purification and characterization of catalase from chard (Beta vulgaris var. cicla)

Catalase is a major primary antioxidant defence component that primarily catalyses the decomposition of H(2) O(2) to H(2) O. Here we report the purification and characterization of catalase from chard (Beta vulgaris var. cicla). Following a procedure that involved chloroform treatment, ammonium sulfate precipitation and three chromatographic steps (CM-cellulose, Sephadex G-25, and Sephadex G-200), catalase was purified about 250-fold to a final specific activity of 56947 U/mg of protein. The molecular weight of the purified catalase and its subunit were determined to be 235 000 and 58 500 daltons, indicating that the chard catalase is a tetramer. The absorption spectra showed a soret peak at 406 nm, and there was slightly reduction by dithionite. The ratio of absorption at 406 and 275 nanometers was 1.5, the value being similar to that obtained for catalase from other plant sources. In the catalytic reaction, the apparent Km value for chard catalase was 50 mM. The purified protein has a broad pH optimum for catalase activity between 6.0 and 8.0. The enzyme had an optimum reaction temperature at 30 degrees C. Heme catalase inhibitors, such as azide and cyanide, inhibited the enzyme activity markedly and the enzyme was also inactivated by ?-mercaptoethanol, dithiothreitol and iodoacetamide.     

Studies on methanol — oxidizing yeast

Oxidation of methanol, formaldehyde and formic acid was studied in cells and cell-free extract of the yeast Candida boidinii No. 11Bh. Methanol oxidase, an enzyme oxidizing methanol to formaldehyde, was formed inducibly after the addition of methanol to yeast cells. The oxidation of methanol by cell-free extract was dependent on the presence of oxygen and independent of any addition of nicotine-amide nucleotides. Temperature optimum for the oxidation of methanol to formaldehyde was 35 degrees C, pH optimum was 8.5. The Km for methanol was 0.8mM. The cell-free extract exhibited a broad substrate specificity towards primary alcohols (C1--C6). The activity of methanol oxidase was not inhibited by 1mM KCN, EDTA or monoiodoacetic acid. The strongest inhibitory action was exerted by p-chloromercuribenzoate. Both the cells and the cell-free extract contained catalase which participated in the oxidation of methanol to formaldehyde; the enzyme was constitutively formed by the yeast. The pH optimum for the degradation of H2O2 was in the same range as the optimum for methanol oxidation, viz. at 8.5. Catalase was more resistant to high pH than methanol oxidase. The cell-free extract contained also GSH-dependent NAD-formaldehyde dehydrogenase with Km = 0.29mM and NAD-formate dehydrogenase with Km = 55mM.     

Microbial production of methyl ketones. Purification and properties of a secondary alcohol dehydrogenase from yeast.

Cell-free extracts derived from yeasts Candida utilis ATCC 26387, Hansenula polymorpha ATCC 26012, Pichia sp. NRRL-Y-11328 Torulopsis sp. strain A1 and Kloeckera sp. strain A2 catalyzed an NAD+-dependent oxidation of secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol) to the corresponding methyl ketones (acetone, 2-butanone, 2-pentanone, 2-hexanone). We have purified a NAD+-specific secondary alcohol dehydrogenase from methanol-grown yeast, Pichia sp. The purified enzyme is homogenous as judged by polyacrylamide gel electrophoresis. The purified enzyme catalyzed the oxidation of secondary alcohols to the corresponding methyl ketones in the presence of NAD+ as an electron acceptor. Primary alcohols were not oxidized by the purified enzyme. The optimum pH for oxidation of secondary alcohols by the purified enzyme is 8.0. The molecular weight of the purified enzyme as determined by gel filtration is 98 000 and subunit size as determined by sodium dodecyl sulfate gel electrophoresis is 48 000. The activity of the purified secondary alcohol dehydrogenase was inhibited by sulfhydryl inhibitors and metal-binding agents.     

Mouse spleen cell nuclear protein kinases and the stimulating effect of dsDNA on NHP phosphorylation by cyclic AMP-independent protein kinase in vitro

cAMP-dependent (designated as enzyme I, about 68,000 daltons) and cAMP-independent protein kinase (designated as enzyme II, about 45,000 daltons) have been partially purified from the nuclei of mouse spleen cells. Both kinases phosphorylated calf thymus histones as well as non-histone proteins (NHP) and required Mg2+ (8 mM) or Mn2+ (2 mM) for maximal activity. NEM (0.5 mM), which is an inhibitor of SH-enzymes, inhibited the histone phosphorylating activity of enzyme II by more than 90%, whereas it inhibited the activity of enzyme I by less than 10%. Moreover, the activity of enzyme II was more sensitive to high temperature than that of enzyme I. Non-histone protein (CM-III protein) served as a more effective substrate for enzyme II than histones; the Km value for CM-III protein was 34.4 micrograms/ml whereas that for histone H2a (14,300 daltons) was 155 micrograms/ml (1.08 x 10(-5) M). CM-III protein phosphorylation by enzyme II in vitro was greatly stimulated by the addition of dsDNA, but not by single-stranded DNA or bacterial ribosomal RNA. However, the phosphorylation of CM-III protein by enzyme I was less than 50% of that of histones, and there was no stimulatory effect. SDS-gel electrophoresis showed that two distinct NHPs (about 13,000 and 19,000 daltons) prepared from calf thymus chromatin were preferentially phosphorylated by enzyme II in vitro in the presence of dsDNA. This finding suggests that these two NHPs may be specific phosphate acceptors of cAMP-independent protein kinase (enzyme II) in the nuclei of mouse spleen cells.     

Purification and properties of guanylate cyclase from the synaptosomal soluble fraction of rat brain.

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) was purified 2250-fold from the synaptosomal soluble fraction of rat brain. The specific activity of the purified enzyme reached 41 nmol cyclic GMP formed per min per mg protein at 37 degrees C. In the purified preparation, GTPase activity was not detected and cyclic GMP phosphodiesterase activity was less than 4% of guanylate cyclase activity. The molecular weight was approx. 480 000. Lubrol PX, hydroxylamine, or NaN3 activated the guanylate cyclase in crude preparations, but had no effect on the purified enzyme. In contrast, NaN3 plus catalase, N-methyl-N'-nitro-N-nitrosoguanidine or sodium nitroprusside activated the purified enzyme. The purified enzyme required Mn2+ for its activity; the maximum activity was observed at 3-5 mM. Cyclic GMP activated guanylate cyclase activity 1.4-fold at 2 mM, whereas inorganic pyrophosphate inhibited it by about 50% at 0.2 mM. Guanylyl-(beta,gamma-methylene)-diphosphonate and guanylyl-imidodiphosphate, analogues of GTP, served as substrates of guanylate cyclase in the purified enzyme preparation. NaN3 plus catalase or N-methyl-N'-nitro-N-nitrosoguanidine also remarkably activated guanylate cyclase activity when the analogues of GTP were used as substrates.     

Development of Microbodies in the yeast Kloeckera growing on methanol.

A number of microbodies appear regularly in methanol-grown yeast cells, but rarely in ethanol- or glucose-grown cells. When one of representative methanol-utilizing yeasts, Kloeckera sp.no. 2201 (also known as Candida bodinii), was cultured on glucose and then transferred into a methanol medium, microbodies of small size could be observed in 2-h old cells. The number of microbodies per sectioned cell reached five to six after 4 h of cultivation. Though the number of microbodies did not change during prolonged cultivation, their size became larger with the passage of cultivation time. The activities of catalase and alcohol oxidase were confirmed in the particulate fractions throughout the cultivation period, whereas the activities of formaldehyde dehydrogenase and formate dehydrogenase were not detected in the particles. The activity of isocitrate lyase was detected in the particulate fractions only at the early growth phase.     

Purification and characterization of a secondary alcohol dehydrogenase from a pseudomonad.

Growth of Pseudomonas sp. NRRL B3266 in the presence of oleic acid resulted in the induction of two enzymes: oleate hydratase, which produced 10(R)hydroxyoctadecanoate, and hydroxyoctadecanoate dehydrogenase, which catalyzed the oxidized nicotinamide adenine dinucleotide-dependent production of 10-oxooctadecanoate. This latter enzyme was purified to homogeneity and shown to consist of two polypeptide chains of about 29,000 daltons each. The enzyme had a broad substrate specificity, catalyzing the dehydrogenation of a number of 18-carbon hydroxy fatty acids. The kinetic parameters for various 10- and 12-hydroxy fatty acids were similar (Km ca. 5 micron and Vmax ca. 50 to 200 mumol/min per mg of protein). The enzyme also catalyzed the dehydrogenation of unsubstituted secondary alcohols. The effectiveness of these alcohols as substrates was highly dependent on their hydrophobicity, the Km decreasing from 9 mM for 4-heptanol to 7 micron for 6-dodecanol. Inhibition of the enzyme by primary alcohols also showed a dependence on hydrophobicity, the Ki decreasing from 350 mM for methanol to 90 micron for decanol.     

Purification and characterization of type I DNA topoisomerase from Lentinus edodes

Abstract Type I DNA topoisomerase was purified from the lower eukaryote Lentinus edodes . Like the topoisomerase I from other eukaryotic cells, the L. edodes enzyme removed both positive and negative superhelical turns. The M _r of the enzyme was determined to be 71,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On gel filtration by Sephacryl S-200, the enzyme appeared to be an aggregate with a native M _r of about 235 000 daltons. No energy cofactor was required and ATP did not affect the enzyme. Activity was enhanced about 10-fold by Mg²⁺ (10 mM) and about 8-fold by KCl (100 mM).     

Plant microbody proteins, I. Purification and characterization of catalase from leaves of lens culinaris.

1) Catalase from green leaves of Lens culinaris (lentils) was investigated with respect to isoenzyme patterns. In contrast to other plants, which have been reported to contain multiple forms of catalase, only one form of this enzyme was revealed when crude extracts were subjected to starch gel electrophoresis or to polyacrylamide disc-gel electrophoresis. Furthermore, catalases from leaves, stems and cotyledons were electrophoretically identical. 2) The leaf enzyme has been purified by conventional methods to apparent homogeneity. It has a molecular weight of 225 000 (ultracentrifuge) and is composed of four identical subunits of molecular weight 54 000 (sodium dodecylsulphate gel electrophoresis). The ratio A280/A405 of the pure enzyme was found to be 1.5. The isoelectric point is at pH 5.5. The enzyme, very labile at pH-values below 7.0, is stable in Tris chloride and potassium phosphate buffers between pH 7.5 and 9.5. It is slowly inactivated by 1mM dithiothreitol and is rapidly inactivated by 1mM mercaptoethanol. 3) The catalase was shown to be the major protein component of the peroxisomal matrix. It could not be detected at the membranes of the leaf peroxisomes.     

Alcohol oxidase and catalase in peroxisomes of methanol-grown Candida boidinii.

Microbodies, designated as peroxisomes because of their enzyme complement, have been isolated from methanol-grown cells of Candida boidinii. Spheroplast lysates were separated on non-continuous Ficoll density gradients, resulting in a mitochondrial fraction and a peroxisome fraction. Estimates of purity using the mitochondrial enzyme markers suggested that the contamination of mitochondria in the peroxisome fraction was about 2-3%. As shown by electron microscopy the peroxisomes were 0.4-0.6 mum in diameter and contained crystalloid inclusions. Alcohol oxidase and catalase, which catalyse the oxidation of methanol to formaldehyde in Candida boidinii, could be localized within the peroxisomes. Gel-electrophoretic studies of the peroxisome fraction demonstrated that it contained only two predominant protein bands consistent with alcohol oxidase and catalase. No alcohol oxidase and catalase activity was found in mitochondria.     

Ultrastructure of methanol-utilizing yeast cells: appearance of microbodies in relation to high catalase activity.

Nine strains of methanol-utilizing yeasts belonging to the genera Candida, Hansenula, Kloeckera, Pichia, and Torulopsis were examined with respect to the interrelationship between their catalase content and ultrastructure. Methanol-grown cells of all the yeasts tested showed higher catalase activities than the respective ethanol- and glucose-grown cells. In connection with this, occurrence of a specific organelle surrounded by a single-unit membrane ("microbodies") was observed only in the methanol-grown cells. Several morphological differences were observed between the microbodies of methanol-utilizing yeasts and those of hydrocarbon-utilizing yeasts such as Candida tropicalis. That is, microbodies of methanol utilizers were large in size, existed in closely associated forms, and had crystalloid structures. Localization of catalase activity in these microbodies was demonstrated cytochemically by use of 3,3'-diaminobenzidene. Especially, 3,3'-diaminobenzidine reaction product accumulated heavily in crystalloids of yeast microbodies.     

Alkaline phosphatase of Thiobacillus thioparus. Partial purification and properties of the enzyme.

Soluble alkaline phosphatase from Thiobacillus thioparus cells was purified about 230-fold. The enzyme had a mol. wt. of 50 000 daltons, optimum pH at 10.5, and was heat-resistant in the presence of diethanolamine. Polyacrylamide-gel electrophoresis demonstrated contamination of the preparation with inactive proteins and the presence of two active bands. The enzyme activity was distinctly stimulated by increasing concentrations of Tris or diethanolamine. In the presence of glycine, 1 mM-Zn2+ enhanced the enzyme activity; in Tris or diethanolamine buffers the activity was stimulated by 1 mM-Mg2+ whereas Zn2+ had a strong inhibitory effect. Glycine at concentrations exceeding 25 mM also inhibited the enzyme. Specificity of the enzyme is fairly broad.     

Purification and characterization of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes sp. nov.

Summary Membrane-bound alcohol dehydrogenase (ADH) was purified from the membrane fraction of an industrial-vinegar-producing strain, A. polyoxogenes sp. nov. NBI1028 by solubilization using Triton X-100 and subsequent column chromatography on DEAE-Sepharose CL-6B and hydroxyapatite. The purified enzyme was homogeneous on polyacrylamide disc gel electrophoresis (PAGE). Upon sodium dodecyl sulphate-PAGE, the enzyme showed the presence of two subunits with a molecular mass of 72 000 daltons and 44 000 daltons, respectively. The small subunit was identified as cytochrome c. In addition, absorption and fluorescence spectra showed the the presence of pyrroloquinoline quinone in the purified ADH. The ADH preferentially oxidized aliphatic alcohols with a straight carbon chain except for methanol. Formaldehyde and acetaldehyde were also oxidizable substrates. The apparent K _m for ethanol was 1.2 mM. The optimum pH and temperature were 5.0–6.0 and 40°C, respectively. p-Chloromercuribenzoic acid and heavy metals such as CuSO₄ were inhibitory to the enzyme activity. Ferricyanide was effective as an electron acceptor.Offprint requests to: M. Fukaya     

Cytochemical study of catalase activity in Candida boidinii during incubation on methanol

Catalase activity of the methanol-assimilating yeast Candida boidinii M-363 was determined cytochemically and biochemically. Electron microscopic investigations on ultrathin sections were made on cells from 16, 24, and 48h batch cultures in nutrient medium with methanol (or glucose as a control) as the sole source of carbon and energy. The electron-dense oxidation product of 3,3′-diaminobenzidine was found predominantly in the mitochondrial cristae and membranes. The mitochondria were increased in number, enlarged, sometimes aggregated, with variable form and size and they characteristically developed when the strain was grown on methanol. The significant development of these organelles and their intensive DAB staining correlated with the considerable increase in catalase activity. Biochemically, catalase in the cell-free extract was determined to be maximal along the exponential growth phase of the strain during its incubation on methanol. Enzyme analysis of the heavy mitochondrial fraction showed that it possessed catalase activity but not peroxidase activity. The results showed that not only peroxisomes but also mitochondria may be structurally and functionally responsible for the high catalase activity of some methanol-assimilating yeasts. What is more, the contribution of the mitochondria to the utilization of methanol may be significant.     

Anaerobic metabolism of the common cockle, Cardium edule. II. Partial purification and properties of lactate dehydrogenase and octopine dehydrogenase. A comparative study.

1. Octopine dehydrogenase and lactate dehydrogenase were purified 190-fold and 10-fold respectively from the adductor muscle of the marine bivalve Cardium edule by gel filtration on Sephadex G-100 and chromatography on DEAE-Sephadex A-50. 2. Lactate dehydrogenase was capable to convert D- and L-lactate, had a molecular weight of about 70 000 and 280 000 daltons, exhibits no distinct pH optimum and was not inhibited by lactate. The enzyme showed apparent Km values of 0.16 mM for pyruvate and 16 mM and 48 mM for D- and L-lactate respectively. 3. In comparison to the purified enzymes from other species, octopine dehydrogenase from Cardium edule showed similar biochemical properties : pH optima of 6.8 and 8.7 respectively, Km values of 0.9 mM (for pyruvate) and 2.0 mM (for arginine), a molecular weight of 37 000 daltons and inhibition by octopine. Electrophoretic studies on standard polyacrylamide gels showed five isoenzymes. 4. The biochemical properties of both dehydrogenases are compared to the conditions in vivo of these animals and the biological role of the octopine dehydrogenase is discussed.     

Alcohol oxidases of Kloeckera sp. and Hansenula polymorpha. Catalytic properties and subunit structures.

1. Alcohol oxidase (alcohol: oxygen oxidoreductase) of a thermophilic methanol-utilizing yeast, Hansenula polymorpha DL-1, was isolated in crystalline form. 2. This alcohol oxidase of H. polymorpha was more stable to heat than was the enzyme of Kloeckera sp. This difference in heat stability is compatible with the difference in growth temperatures for both yeasts. 3. The crystalline alcohol oxidases of both yeast oxidized the lower primary alcohols (C-2 to C-4) as well as methanol. The apparent Km values for the methanol of Kloeckera and H. polymorpha enzymes were 0.44 and 0.23 mM, respectively. The enzymes could also oxidize formaldehyde to formate, and were inactivated by relatively low concentrations of hydrogen peroxide. 4. The molecular weight for both enzymes was calculated to be about 670000. Each enzyme is composed of eight identical subunits (molecular weight 83000) and contains eight moles of FAD as the prosthetic group. The NH2-terminal and COOH-terminal amino acids of H. polymorpha enzyme were identified as alanine and phenylalanine, respectively. The octameric subunits model of each enzyme was confirmed by electron micrographs, which showed an octad aggregate, composed of two tetragons face to face.     

Physiological Studies of Methane- and Methanol-Oxidizing Bacteria: Comparison of a Primary Alcohol Dehydrogenase from Methylococcus capsulatus (Texas Strain) and Pseudomonas Species M27

A primary alcohol dehydrogenase has been purified from Methylococcus capsulatus (Texas strain). The purified enzyme catalyzes the oxidation of methanol and formaldehyde to formate; other primary alcohols are oxidized to their corresponding aldehydes. Ammonium ions are required for enzyme activity. The enzyme has a molecular weight of 120,000 daltons and consists of two 62,000 molecular-weight subunits which dissociate at acidic pH. The enzyme is similar to an alcohol dehydrogenase enzyme isolated from Pseudomonas sp. M27.     

Purification and characterization of catalase from a facultative alkalophilic Bacillus

Catalase was purified to an electrophoretically homogeneous state from the facultative alkalophilic bacterium, Bacillus YN-2000, and some of its properties were studied. Its molecular weight was 282,000 and its molecule was composed of four identical subunits. The enzyme contained two protoheme molecules per tetramer. The enzyme showed an absorption spectrum of typical high-spin ferric heme with a peak at 406 nm in the oxidized form and peaks at 440, 559, and 592 nm in the reduced form. In contrast to the typical catalases, the enzyme was reduced with sodium dithionite, like peroxidases. The enzyme showed an appreciable peroxidase activity in addition to high catalase activity. The amino acid composition of Bacillus YN-2000 catalase was very similar to those of catalase from Neurospora crassa and peroxidase from Halobacterium halobium. The catalase content in the soluble fraction from the bacterium was higher with the cells grown at pH 10 than with the cells grown at lower pHs (pH 7-9).     

Purification and properties of alcohol oxidase from Poria contigua.

1. Alcohol oxidase (alcohol:oxygen oxidoreductase) was purified 22-fold from the brown rot fungus Poria contigua. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis, and by sedimentation in an ultracentrifuge. The molecular weight was calculated to be 610000 +/- 5000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels and electron microscopic analysis indicate that the enzyme is an octamer composed of eight probably identical subunits, each having a molecular weight of 79 000. The enzyme contains eight mol FAD/mol as the prosthetic group. 2. This alcohol oxidase oxidizes not only methanol but also lower primary alcohols (C2-C4), 2-propin-1-ol and formaldehyde. The apparent Km value for methanol is 0.2 mM, and that for formaldehyde 6.1 mM. Sodium azide was found to be a competitive inhibitor with respect to methanol. 3. The enzyme from the fungus Poria contigua is immunologically different from the alcohol oxidase isolated from the methanol-utilizing yeast Candida boidinii. Furthermore antiserum raised against this enzyme did not cross-react with the alcohol oxidase from the white rot fungus Polyporus obtusus.     

Characterization of a manganese-containing catalase from the obligate thermophile Thermoleophilum album.

A manganese-containing catalase has been characterized from Thermoleophilum album NM, a gram-negative aerobic bacterium obligate for thermophily and n-alkane substrates. The level of catalase in cells was increased about ninefold by growth in the presence of paraquat (2.5 microM), a superoxide-generating toxicant. Superoxide dismutase levels were unaffected by this compound. The enzyme was purified from cultures grown in the presence of paraquat to greater than 95% homogeneity and had an Mr of 141,000. The enzyme was composed of four subunits, and each had an Mr of 34,000. There were 1.4 +/- 0.4 atoms of manganese present per subunit. The catalase had a Km for hydrogen peroxide of 15 mM and a Vmax of 11 mM/mg. Peroxidase activity, as measured with p-phenylenediamine, copurified with the catalase. Inhibitors of heme-catalase were weak inhibitors of the T. album enzyme. The optimum pH for catalase activity was 8 to 9. The enzyme was stable from pH 6.5 to 11 and retained activity at assay temperatures from 25 to 80 degrees C. The catalase was stable for 24 h of incubation at 60 degrees C.