Purification of glycollate oxidase from greening cucumber cotyledons

Glycollate oxidase (glycollate: oxygen oxidoreductase, EC 1.1.3.1) was purified to apparent homogeneity from crude extracts of greening cucumber cotyledons (Cucumis sat vus). Molecular sieving and chromatofocusing resulted in 700-fold purification and specific activity of 1 kat mg^-1 protein. The enzyme exhibited a M_r of 180,000, or 700,000, respectively, and is a tetramer or 16-mer made of identical subunits of M_r 43,000. Monospecific antibodies were raised against the homogeneous protein.     

Characterization of the amine oxidase involved in the growth of Trichosporon cutaneum X4 on ethylamine as source of carbon,nitrogen and energy

The amine oxidase from Trichosporon cutaneum X₄ grown on ethylamine as carbon, nitrogen and energy source was purified to near homogeneity. The purified enzyme showed the highest resistance to heat of any amine oxidase hitherto characterized from a yeast (half-life at 62°C, 14 min). Measurement of kinetic parameters as a function of carbon chain length showed results typical of a benzylamine oxidase. Both non-denaturing- and sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed multiple bands, and dimethyl suberimidate cross-linking studies revealed that the enzyme consisted of multimers of two polypeptide chains of M_r respectively 19,000 and 26,000. The smallest structure to show activity probably contained two of each kind of subunit.Abbreviation SDS sodium dodecyl sulphate     

Purification and properties of a heat-stable and cold-labile NAD-specific glutamate dehydrogenase from Sporosarcina ureae

NAD-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.2) was purified to homogeneity from Sporosarcina ureae DSM 320; the native enzyme (M _r 250,000±25,000) is composed of subunits identical in molecular mass (M _r 42,000±3,000), suggesting a hexameric structure. In cell-free extracts and in its purified form, the enzyme was heat-stable, retaining 50% activity after 15 min incubation at temperatures up to 82°C. When exposed to low temperatures at pH values between 7.0 and 9.0. cell-free extracts and purified preparations lost enzyme activity rapidly and irreversibly. The addition of substrates, glycerol, or sodium chloride improved the stability of the enzyme with respect to cold lability and heat stability.Abbreviation NAD-GluDH nicotinamide-adenine-dinucleotide-specific glutamate dehydrogenase     

Pyranose oxidase and pyranosone dehydratase: enzymes responsible for conversion of d-glucose to cortalcerone by the basidiomycete Phanerochaete chrysosporium

Pyranose oxidase (glucose 2-oxidase) and pyranosone dehydratase were purified 27.6- and 43.9-fold respectively from mycelial extracts of the fungus Phanerochaete chrysosporium using hydrophobic interaction, anion exchange and gel filtration chromatography. The enzymes appeared substantially homogeneous on SDS-PAGE and were comprised of identical subuntis with apparent M_r values of 69 000 and 99 000 for pyranose oxidase and pyranosone dehydratase, respectively. The apparent M_r's of the native enzymes, based on equilibrium ultracentrifugation, were 308 000 and 221 000. In coupled reactions, the enzymes catalyzed conversion of d-glucose via d-glucosone (d-arabino-2-hexosulose) to the antibiotic -pyrone, cortalcerone. The latter compound was isolated as a diphenylhydrazone derivative and spectroscopically identified.Abbreviations DMAB 3-dimethylaminobenzoic acid - FPLC fast protein liquid chromatography - MBTH 3-methyl-2-benzothiazolinone hydrazone hydrochloride - PD pyranosone dehydratase - PMSF phenylmethylsulfonyl fluoride - POD pyranose oxidase     

Purification and properties of pea (Pisum sativum L.) thioredoxin f,a plant thioredoxin with unique features in the activation of chloroplast fructose-1,6-bisphosphatase

Thioredoxin (Td) f from pea (Pisum sativum L.) leaves was purified by a simple method, which provided a high yield of homogeneous Td f. Purified Td f had an isoelectric point of 5.4 and a relative molecular mass (M_r) of 12 kilodaltons (kDa) when determined by filtration through Superose 12, but an M_r of 15.8 kDa when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein remained fully active for several months when conserved frozen at — 20° C. The pea protein was able to activate fructose1,6-bisphosphatase (FBPase; EC 3.1.3.11), but in contrast to other higher-plant Td f proteins, was not functional in the modulation of NADP⁺-malate dehydrogenase activity. In spite of the absence of immunological cross-reactions of pea and spinach Td f proteins with the corresponding antibodies, pea Td f activated not only the homologous FBPase, but also the spinach enzyme. The saturation curves for pea FBPase, either with fructose-1,6-bisphosphate in the presence of different concentrations of homologous Td f, or with pea Td f in the presence of excess substrate, showed sigmoid kinetics; this can be explained on the basis of a random distribution of fructose-1,6-bisphosphate, and of the oxidized and reduced forms of the activator, among the four Td f- and substrate-binding sites of this tetrameric enzyme. From the saturation curves of pea and spinach Td f proteins against pea FBPase, a 4:1 stoichiometry was determined for the Td f-enzyme binding. This is in contrast to the 2:1 stoichiometry found for the spinach FBPase. The UV spectrum of pea Td f had a maximum at 277 nm, which shifted to 281 nm after reduction with dithiothreitol (s at 280 nm for 15.8-kDa M_r = 6324 M^–1 · cm^–1). The fluorescence emission spectrum after 280-nm excitation had a maximum at 334 nm, related to tyrosine residues; after denaturation with guanidine isothiocyanate an additional maximum appeared at 350 nm, which is concerned with tryptophan groups. Neither the native nor the denatured form showed a significant increase in fluorescence after reduction by dithiothreitol, which means that the tyrosine and tryptophan groups in the reduced Td f are similarly exposed. Pea Td f appears to have one cysteine residue more than the three cysteines earlier described for spinach and Scenedesmus Td f proteins.Abbreviations DDT dithiothreitol - ELISA enzyme-linked immunosorbent assay - FBPase fructose- 1,6-bisphosphatase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Td thioredoxin The authors are grateful to Mrs. Francisca Castro and Mr. Narciso Algaba for skilful technical assistance. This work was supported by grant PB87-0431 of Dirección General de Investigación Cientifica y Técnica (DGICYT, Spain).     

Diamine oxidase from millet catalyzes the oxidation of 1, 3-diaminopropane

Diamine oxidase was purified sixty-fold from millet shoots. The partially purified enzyme of 150 kDa oxidized 1, 3-diaminopropane (1, 3-DAP) to 3-aminopropionaldehyde. The K_m values were 9.1×10⁻⁵M for 1, 3-DAP and 6.3×10⁻⁴M for putrescine. Extracts of shoots of prosomillet, maize and barley also contained an activity that oxidized 1, 3-DAP.     

Purification and some properties of endopolygalacturonase from Rhizopus sp. LKN

An endopolygalacturonase of Rhizopus sp. strain LKN, one of several isolates from tempe starter (ragi), was purified 235-fold by CM-Sephadex C-50, DEAE-Sephadex A-50 ion exchange chromatographies and Sephadex G-75 gel filtration. The purified enzyme was homogeneous by SDS-PAGE with a M _r of 38.5 kDa. Its K _m value for pectic acid was 2 mg/ml. It was stable at pH 4.5 to 11 and up to 50°C, with optimum activity at pH 4.5 to 4.75 and 55 to 60°C. Some ionic compounds enhanced the enzyme activity, whereas tannic acid at 0.5 mm caused about 90% inhibition.The authors are with the Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812, Japan.     

The role of cysteine proteinase and carboxypeptidase in the breakdown of storage proteins in buckwheat seeds

Two proteolytic enzymes, a cysteine proteinase and a carboxypeptidase, responsible for breakdown of the main storage protein, 13S globulin, were purified from buckwheat seedlings (Fagopyrum esculentum Moench) by (NH₄)₂SO₄ fractionation, gel-filtration on Sephadex G-150, ionexchange chromatography on DEAE-Toyopearl 650 M and chromatofocusing. The cysteine proteinase was purified 74-fold. It has a pH optimum of 5.5, a pI of 4.5 and an apparent molecular mass (M_r) of 71000. The carboxypeptidase was purified 128-fold. It has a pH optimum of 5.3, a pI of 5.8 and a M_r of 78500. Cysteine proteinase hydrolyzed the modified 13S globulin only if the reaction products were eliminated from the incubation mixture by dialysis. Storage protein degradation by the proteinase increased in the presence of carboxypeptidase. We suggest that the two enzymes complete the digestion of 13S globulin after its preliminary hydrolysis by the earlier described enzyme, metalloproteinase, present in dry buckwheat seeds.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - M_r apparent molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Purification and characterization of D-glycerate-3-kinase from maize leaves

Glycerate kinase (GK; EC 2.7.1.31) from maize (Zea mays L.) leaves was purified by a sequence of ammonium-sulfate precipitations and chromatography on diethylaminoethyl-cellulose, hydroxyapatite, Sephadex G-75SF and dye ligand (Green A) columns. The purest preparation was almost 1300-fold enriched and had a specific activity of 68 mol · min^-1 · (mg protein) ^-1. The enzyme was a monomer of a relative molecular mass (M_r) of 44 kDa (kdalton) as determined by gel filtration, electrophoresis in dissociating conditions and by immunoblots. The enzyme was only weakly recognized by polyclonal antibodies against purified spinach GK, indicating substantial differences in molecular structure of the two proteins. Highly reducing conditions stabilized GK activity and were required for activation of crude leaf enzyme. The enzyme had a broad pH optimum of 6.8–8.5, and formed 3-phosphoglycerate and ADP as reaction products. Apparent K _ms for D-glycerate and Mg-ATP were 0.11 and 0.25 mM, respectively. The enzyme was strongly affected by a number of phosphoesters, especially by 3-phosphoglycerate (K _i= 0.36 mM), fructose bisphosphates and nucleoside bisphosphates. Inhibition by 3-phosphoglycerate was competitive to Mg-ATP and noncompetitive to D-glycerate. Pyruvate was found noncompetitive to D-glycerate (K _is=4 mM). The ratio of stromal concentration of Mg-ATP to phosphoesters, particularly to 3-phosphoglycerate, may be of importance in the regulation of GK during C₄-photosynthesis.Abbreviations DEAE diethylaminoethyl - kDa kdalton - GAP-DH glyceraldehyde phosphate dehydrogenase - GK glycerate kinase - LDH lactate dehydrogenase - 2-ME 2-mercaptoethanol - M_r relative molecular mass - PEP phosphoenolpyruvate - PGA(PK) phosphoglycerate (phosphokinase) - PK pyruvate kinase - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis     

Existence of aa 3-Type Ubiquinol Oxidase as a Terminal Oxidase in Sulfite Oxidation of Acidithiobacillus thiooxidans

It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an α-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa ₃-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 °C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A₁ and myxothiazol, which are inhibitors of mitochondrial bc ₁ complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.     

Effects of Exogenous Proline and Glycinebetaine on the Salt Tolerance of Rice Cultivars

Glutathione reductase was purified from iron-grown Thiobacillus ferrooxidas AP19-3 to an electrophoretically homogeneous state. The enzyme had an apparent molecular weight of 100,000 and was composed of two identical subunits of molecular weight (M_rs, 52,000) as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A purified enzyme reduced one mole of the oxidized form of glutathione (GSSG) with one mole of NADPH to produce two moles of the reduced form of glutathione (GSH) and one mole of NADP⁺. The glutathione reductase was most active at pH 6.5 and 40°C, and had an isoelectric point at 5.1. The Michaelis constants of glutathione reductase for GSSG, NADPH, and NADH were 300, 26, and 125 μM, respectively.     

An efficient purification with a high recovery of the inulin fructotransferase of Arthrobacter sp. A-6 from recombinant Escherichia coli

Inulin fructotransferase (IFTase, EC 2.4.1.93) of Arthrobacter sp. A-6 was purified from a cell extract of the recombinant Escherichia coli DH5 /pDFE cells carrying the IFTase gene using heat treatment followed by gel filtration. The enzyme was purified 45-fold to apparent homogeneity with a recovery of 79%. SDS-PAGE yielded a single protein band of M _r 46.5 kDa. The recombinant IFTase had a similar thermostability as the original enzyme from Arthrobacter sp. A-6.     

Enzyme antigens associated with pigeon breeder's disease. I. Isolation and characterization of basic hydrolases

A survey of the hydrolytic enzymes present in pigeon dropping extracts (PDE) has shown that this material contains a variety of proteolytic and nonproteolytic activities. These enzymes were separated into their basic and acidic components by chromatography on DEAE-cellulose. Staining of immunoprecipitates with specific chromogenic substrates demonstrated the presence of antibodies in symptomatic breeders to several of the basic enzymes in PDE. Five distinct hydrolytic activities were isolated from the basic group of enzymes. Trypsin, elastase, and two forms of collagenase were the specific proteolytic activities isolated. A phospholipase was also purified from these preparations. The purified elastase consisted of a single polypeptide chain (M _r =22,000). The purified trypsin had a molecular weight (M _r =25,000) and charge similar to those reported for elastase and, like elastase, the trypsin from PDE appeared to be composed of a single polypeptide chain. Two molecular weight forms of collagenase were found; both hydrolyzed bovine collagen. The high-molecular-weight collagenase (M _r =51,000) was shown to be a glycoprotein consisting of two polypeptides (M _r =24,000). It was readily separated from the low-molecular-weight collagenase (M _r =15,000) by gel filtration. The phospholipase (M _r =99,000) appeared to be a dimer. The relevance of these enzymes to the development of pigeon breeder's disease is discussed.     

Cloning and expression of a xylanase gene from Clostridium acetobutylicum P262 in Escherichia coli

Summary A xylanase gene from Clostridium acetobutylicum P262 was cloned on a recombinant plasmid pHZ300 which enabled Escherichia coli HB101 cells to produce intracellular xylanase activity. The xylanase gene was located on a 2 kb DNA fragment. The cloned xylanase had an apparent M _r of approximately 28 000 and an isoelectric point of approximately 10. Optimum xylanase activity was obtained at pH 6.0 at 37–43° C. Comparison with a xylanase partially purified from the culture medium of C. acetobutylicum P262 showed that the enzymes had similar characteristics and western blot analysis showed cross-reactivity between antibodies raised against the purified cloned enzyme and a polypeptide of the same M _r from C. acetobutylicum P262.     

Phosphoribulokinase from Rhodopseudomonas acidophila

Phosphoribulokinase from the nonsulfur purple bacterium Rhodopseudomonas acidophila has been purified to apparent homogeneity, using affinity chromatography on Cibacron Blue-agarose and AMP-agarose. The relative molar mass of the enzyme was determined by sucrose density gradient centrifugation to be M _r=248,000 with a sedimentation coefficient of s _20,w=10.9 S. Dodecyl sulfate polyacrylamide gel electrophoresis revealed that the enzyme consists of identical size subunits of M _r=32,000, suggesting an octameric structure of the holoenzyme. The enzyme cross-reacted with heterologous antibodies raised against phosphoribulokinase from the hydrogen bacterium Alcaligenes eutrophus. The pH optimum of the enzyme was shifted from 8.4 in the absence of the activator NADH to 7.6 in the presence of the effector. Mg²⁺ ions were the most effective divalent cations required for activity. Specificity of the enzyme for the sugar phosphate substrate ribulose 5-phosphate was high whereas a variety of nucleoside triphosphates besides ATP could serve as phosphate donors. NADH was a strong activator of the enzyme (K _a=0.05 mM) that primarily affected the maximal reaction velocity in a pH-dependent manner. The only other effector identified was phosphoenolpyruvate. It moderately inhibited the enzyme (I _0.5=0.32 mM).Abbreviation PRK phosphoribulokinase Dedicated to Prof. Dr. H. G. Schlegel on the occasion of his 60th birthday     

Partial purification and properties of extracellular chitinase produced by Acremonium obclavatum,an antagonist to the groundnut rust,Puccinia arachidis

An extracellular chitinase of Acremonium obclavatum was partially purified. It had an M _r of 45 kDa on SDS-PAGE, and was optimally active at pH 3 to 4 and 50°C. Hg and Mn (10 mm) inhibited activity. The chitinase hydrolysed colloidal chitin more rapidly than crude chitin or isolated A. obclavatum cell walls. The partially-purified enzyme inhibited uredospore germination and germ-tube growth of Puccinia arachidis.The authors are with the Centre for Advanced Study in Botany, University of Madras, Guindy campus, Madras 600 025, India     

Purification and characterization of halohydrin hydrogen-halide lyase from a recombinant Escherichia coli containing the gene from a Corynebacterium sp.

Summary An enzyme catalyzing the interconversion of 1,3-dichloro-2-propanol (DCP) to epichlorohydrin (ECH) was purified from Escherichia coli JM109/ pST001, which carried the gene from Corynebacterium sp. N-1074. The enzyme was crystallized by the addition of ammonium sulphate. The enzyme had a relative molecular mass (M_r) of about 105 000 and consisted of four subunits identical in M_r (approx. 28 000). The enzyme catalysed both the transformation of various halohydrins into the corresponding epoxides with liberation of halide and its reverse reaction. These facts indicated that the enzyme was halohydrin hydrogen-halidelyase. Offprint requests to: T. Nagasawa     

Influence of avocado (Persea americana) Cx-cellulase on the structural features of avocado cellulose

Avocado (Persea americana Mill.) fruit produce copious quantities of the enzyme Cx-cellulase (EC 3.2.1.4) during ripening. The possibility that Cx-cellulase is able to disrupt cellulose microfibril oranization was investigated using molecular weight (M_r), x-ray diffraction, and ultrastructural analyses of cell walls from unripe avocado fruit incubated with the purified enzyme. Results indicate that Cx-cellulase causes a downshift in the M_r of unbranched cell-wall polymers in the M_r range of 10⁶–10⁷ Da. There is an increase in the proportion of crystalline cellulose, and cellulose fibrils appear to lose cohesiveness in response to enzyme activity. We propose that Cx-cellulase attacks avocado cellulose at accessible sites in the peripheral and integral noncrystalline regions of the microfibril, resulting in a loss of cohesiveness within the fibril structure and an alteration in the binding of associated cell-wall matrix polysaccharides. The initial loss of avocado mesocarp firmness during fruit ripening may be linked to the onset of Cx-cellulase activity.Abbreviations CMC carboxymethylcellulose - DMAC dimethylacetamide - DS developmental stage - M molecular weight - XG xyloglucan     

Determination of diamine oxidase in lentil seedlings by enzymic activity and immunoreactivity

A competitive radioimmunoassay for the quantitation of diamine oxidase (EC 1.4.3.6) from Lens culinaris is reported. Specific antibodies raised in rabbits immunized with a homogeneous preparation of the enzyme were incubated with purified ¹²⁵I-enzyme and with either unlabeled diamine oxidase or plant material. Antigen-antibody complexes were isolated from the mixture by incubation with Staphylococcus protein A. The sensitivity of the test was about 5 nanograms in terms of enzyme protein. This assay was applied to the determination of the enzyme in extracts from lentil shoots grown either in the dark or in the light. Diamine oxidase activity and enzyme protein (as determined by radioimmunoassay) were measured during 7 days after germination. Both enzymic activity and enzyme protein declined slowly in the dark and rapidly in the light. These results indicate that fluctuation of the enzymic activity in this organ, both in the light and in the dark, are mediated via changes in the amount of the enzyme protein and not via the action of an inhibitor.