Purification and Properties of Soluble Chlorophyllase from Tea Leaf Sprouts

Soluble chlorophyllase (chlorophyll-chlorophyllido-hydrolase,EC 3.1.1.14 [EC] ) was purified 650-fold from tea leaf sprouts byammonium sulfate fractionation and gel filtration through SephadexG-200 and Sepharose CL-6B. The purified enzyme showed two bandson polyacrylamide gel electrophoresis and the specific activitywas 2.6 µmol chlorophyll a hydrolyzed min^–1 mg^–1of protein. The molecular weights determined by Sepharose CL-6Bwere 910,000 and 350,000, indicating high molecular aggregates.The subunit molecular weight estimated by sodium lauryl sulfate-polyacrylamidegel electrophoresis was 38,000. The isoelectric point was 3.9.The optimum pH was 5.5 in acetate buffer and the Km value forchlorophyll a was 10 µM. This enzyme did not require athiol compound nor metal ion such as Mg²⁺. (Received January 26, 1981; Accepted April 3, 1981)     

Purification of Sulphite-Cytochrome c Reductase of Thiobacillus novellus and Reconstitution of Its Sulphite Oxidase System with the Purified Constituents

Sulphite-cytochrome c reductase (sulphite: ferricytochrome coxidoreductase, EC 1.8.2.1 [EC] ) derived from Thiobacillus novelluswas purified by chromatography on a DEAE-cellulose column andby gel filtration with a Sephadex G-100 column. Although thereductase thus purified moved as a single band both in gel filtrationand in isoelectric focusing it was always split into two bandsby polyacrylamide gel electrophoresis; the one had the enzymaticactivity and showed absorption spectrum of cytochrome, whilethe other had no activity and was colourless, in contrast withthe results reported by Charles and Suzuki [(1966) Biochim.Biophys. Acta 128: 522]. The enzymatic properties of the purifiedreductase were almost the same as those of the enzyme obtainedby Charles and Suzuki. Cytochrome c-551 free of the reductase activity was obtained.Its molecular weight was determined to be 23,000 by polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate.The cytochrome seemed to exist in the organism as a complexwith the reductase or a subunit of the enzyme. In the stateof the complex with the enzyme, the cytochrome was reduced veryquickly on addition of sulphite, while the cytochrome free ofthe reductase activity was hardly reduced by the enzyme withsulphite. A sulphite oxidase system was reconstituted with the reductase,cytochrome c-550 and cytochrome oxidase highly purified fromthe bacterium. ¹ Present address: Water Research Institute, Nagoya University,Nagoya 464, Japan ² Present address: Institute for Biological Science, SumitomoChemical Co., Ltd., Takarazuka, Hyogo 665, Japan (Received January 23, 1981; Accepted March 9, 1981)     

Soluble Dissimilatory Nitrate Reductase Containing Cytochrome c from a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

Dissimilatory nitrate reductase [nitrite: (acceptor) oxidoreductase.EC 1.7.99.4 [EC] ] from a denitrifying photosynthetic bacterium, Rhodopseudomonassphaeroides forma sp. denitrificans proved to be a soluble enzymethat could be purified 47-fold. It was labile, and containedcytochrome c, based on the results of specific staining forheme on polyacrylamide gel electrophoresis and on its absorptionspectrum. Its physiological molecular weight was determinedto be 112k, although heterogeneous molecular weights of 112k,100k, 73k and 60k were found for different preparations. Theoptimum for enzyme activity was about pH 6, and the K_m for thenitrate was 1.6 mM. As an electron donor, benzyl viologen wasvery good; but NADH, NADPH, FAD, FMN, cytochromes b₂ and c₂,dichlorophenolindophenol and phenazine methosulfate were noteffective. Bathophenanthroline and thiocyanate inhibited enzymaticactivity. The addition of 1 mM tungstate to the growing culturein place of molybdate decreased the nitrate reductase in thecells, but a further addition of 1 mM molybdate stopped it.This nitrate reductase is believed to be a molybdo-iron proteinsimilar to the enzymes from other bacteria with a nitrate respiratingability. (Received February 29, 1980; Accepted January 29, 1981)     

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; )     

Characterization of Porphobilinogen Synthase from an Aerobic Photosynthetic Bacterium, Erythrobacter sp. Strain OCh 114

Porphobilinogen synthase (formerly 5-aminolevulinic acid dehydratase,EC 4.2.1.24 [EC] ) was purified 7,405-fold from an aerobic photosyntheticbacterium, Erythrobacter sp. strain OCh 114. The molecular weightof the enzyme was determined to be 260,000 by Sephadex G-200gel filtration. The enzyme had a single pH optimum at 8.0 andshowed no requirement for metal ion and thiol compound for itsmaximum activity. The K_m value for 5-aminolevulinic acid was0.29 mM. 4,5-Dioxovaleric acid and levulinic acid were foundto be competitive inhibitors of the enzyme, with K_i values of0.65 and 0.80 mM, respectively. The enzyme was extremely labilein acidic pH and almost completely lost its activity within1 h at pH 6.0 and 30?C. This Erythrobacter enzyme seems to besimilar to the enzyme from the anaerobic photosynthetic bacteriumRhodobacter capsulatus in its molecular and catalytic properties. (Received February 17, 1988; Accepted May 9, 1988)     

Purification and Characterization of Sucrose Synthase from Peach (Prunus persica) Fruit

Sucrose synthase (EC 2.4.1.13 [EC] ) was purified from peach fruit(Prunus persica) to a single band of protein on SDS-PAGE byammonium sulfate fractionation, DEAE-cellulose (DE-52) chromatography,Sepharose CL-6B gel filtration, PBA-60 affinity chromatographyand Sephadex G-200 gel filtration. The molecular weight wasestimated to be 360,000 by gel filtration. The enzyme was foundto be a tetramer of identical 87-kDa subunits. The maximum activityfor the synthesis and cleavage of sucrose was observed at pH8.5 and pH 7.0, respectively. The enzymatic reaction followedtypical Michaelis-Menten kinetics in both directions, with thefollowing parameters: K_m(fructose), 4.8 mmM; K_m(UDPglucose),0.033 mM; K_m(sucrose), 62.5 mM; K_m(UDP), 0.080 mM. Other properties,such as substrate specificity and the effects of divalent cations,were also investigated. The relationship between the enzymeand the accumulation of sucrose in peach fruit is discussed. Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Chikusa, Nagoya 464, Japan. (Received May 2, 1988; Accepted September 14, 1988)     

Purification and Properties of NADH:Nitrate Reductase from the Red Alga Porphyra yezoensis

Assimilatory nitrate reductase (NADH) (EC 1.6.6.1 [EC] ) from thered alga Porphyra yezoensis was purified 5,700-fold by a combinationof polyethylene glycol (PEG) treatment, ammonium sulfate fractionation,chromatography on columns of butyl-Toyopearl 650-M, Blue SepharoseCL-6B, DEAE-cellulose (DE 52), and hydroxyapatite, gel filtrationon Sephacryl S-400. The purest preparation of the enzyme hada specific activity of 12.5 units mg^–1 protein. A singleband of protein was detected after polyacrylamide gel electrophoresisunder nondenaturing conditions. This band corresponded to aband that stained positive for reduced methyl viologen-nitratereductase activity. The molecular weight of the native enzymewas estimated to be 220,000. A single band of a protein witha molecular weight of 100,000 was detected after sodium dodecylsulfate-polyacrylamide gel electrophoresis. These results indicatethat the native nitrate reductase is composed of two identicalsubunits. The homogeneous preparation of nitrate reductase hada UV/visible spectrum typical of a b-type cytochrome. The K_mvalues for NADH and KNO₃ were 23 µM and 64 µM, respectively.The pH optimum for the reaction catalyzed by the nitrate reductasewas 8.3, while pH values that supported maximum partial activitiesranged from 7.0 to 8.5. Sulfhydryl reagents, such as p-HMB andNEM, inhibited full and NADH-utilizing partial activities, whilecyanide and azide were effective inhibitors of full and nitrate-reducingpartial activities. (Received March 3, 1993; Accepted September 6, 1993)     

Purification of NADP-Malic Enzyme and Phosphoenolpyruvate Carboxylase from Sugar Cane Leaves

A procedure is described for the purification of phosphoenolpyruvatecarboxylase (EC 4.1.1.31 [EC] ) and NADP-dependent malic enzyme (EC1.1.1.40 [EC] ) from sugar cane leaves. Each enzyme was purified tohomogeneity as judged by sodium dodecyl sulfate-polyacrylamidegel electro-phoresis, with about 30% yield. Phosphoenolpyruvatecarboxylase was purified 54-fold. A molecular weight of 400,000and a homotetrameric structure were determined for the nativeenzyme. The purified carboxylase had a specific activity of20.0 {diaeresis}mol (mg protein)_–1 min_–1, and wasactivated by glucose-6-phosphate and inhibited by L-malate.K_m values at pH 8.0 for phosphoenolpyruvate and bicarbonatewere 0.25 and O.l0 mM, respectively. NADP-malic enzyme, 356-foldpurified, exhibited a specific activity of 71.2 {diaeresis}mol(mg protein)_–1 min_–1 and was characterized as ahomotetramer with native molecular weight of 250,000. Purifiedmalic enzyme showed an absolute specificity for NADP⁺ and requireda divalent metal ion for activity. K_m values of 0.33 and 0.008mM for L-malate and NADP⁺, respectively, were determined. Thisenzyme was inhibited by several organic acids, including ketoand amino acids; while succinate and citrate increased the enzymeactivity when assayed with 10{diaeresis}M L-malate. The effectsshown by amino acids and by citrate were dependent on pH, beinghigher at pH 8.0 than at pH 7.0. (Received October 26, 1988; Accepted February 3, 1989)     

Purification and properties of {delta}-aminolevulinic acid dehydratase from radish cotyledons

-Aminolevulinic acid dehydratase (5-aminolevulinate hydro-lyase,EC 4.2.1.24 [EC] ) was purified from greening radish cotyledons. Thefinal product was homogeneous on polyacrylamide disc gel electrophoresisand had a molecular weight, estimated by gel filtration, of282,000 daltons. The enzyme seems to require magnesium ion aswell as sulfhydryl compounds for maximum activity. EDTA anda low concentration of zinc ion markedly inhibited the activity.The optimum pH was 8.0; the Km value for -aminolevulinic acidwas 3.85?10^–4M. Levulinic acid was a competitive inhibitorof the enzyme, with a Ki of 2.14?10^–4M. These propertieswere compared with those of microorganism and animal -aminolevulinicacid dehydratases. (Received November 22, 1976; )     

Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)     

Purification and Characterization of Phosphoenolpyruvate Carboxylase of Photomixotrophically Cultured Green Tobacco Cells

Phosphoenolpyruvate (PEP) carboxylase (PEPCase, EC 4.1.1.31 [EC] )was purified to apparent electrophoretic homogeneity from photomixotrophicallycultured tobacco cells by ammonium sulfate fractionation, DEAE-Sephacel-,hydroxylapatite-, Phenyl-Sepharose CL-4B-, and Sepharose CL-6B-chromatography,and fast protein liquid chromatography on Mono Q. The purifiedenzyme had a specific activity of 32 units per mg protein, andits purity was determined by denaturing polyacrylamide gel electrophoresis.The native enzyme, with a molecular weight of about 440,000,was a tetramer of four identical subunits and showed maximumactivity at pH 8.5–9.0. Non-denaturing isoelectric focusingshowed a single band at pl 5.4. Substrate-saturation kineticsof the purified enzyme for PEP, bicarbonate, and Mg^2$ were typicalMichaelis-Menten type, with K_m-values of 60, 200, and 80µM,respectively. Most effectors which are known to influence theactivity of C₄- or bacterial PEPCase had only small effectson the activity of the purified enzyme at optimum pH, whilesome inhibitory effects by organic acids (malate, citrate andoxaloacetate) and.an activating effect by glucose-6-phosphatewere observed at a suboptimal pH of 7.5. (Received September 30, 1987; Accepted December 14, 1987)     

Purification and Properties of L-Arginine Decarboxylase of Evernia prunastri

An L-arginine decarboxylase was isolated from Evernia prunastrithallus. The enzyme was purified about 117-fold and showed apH optimum of 7.1 and a temperature optimum at 26°C. Itsmolecular weight was estimated as 300,000. The Evernia argininedecarboxylase was significantly inhibited by L-ornithine, ureaand putrescine. The K_m for L-arginine was about 12.5 mM. (Received March 9, 1981; Accepted June 26, 1981)     

意蜂工蜂酸性磷酸酶的纯化及其酶学特性

从意蜂Apis mellifera工蜂体内分离提纯酸性磷酸酶（ACPase, EC3.1.3.2）,并对其性质进行了研究。将工蜂酸性磷酸酶的初提物经分段盐析、DEAE-Sepharose FF离子交换层析及Sephadex G-200 凝胶过滤等纯化步骤,得到经聚丙烯酰胺凝胶电泳为单一蛋白区带的酶液。提纯倍数为77.24,酶液比活力为16.22 U/mg（对硝基苯磷酸二钠作底物）。利用凝胶过滤法测定酶的相对分子质量为135 kD,SDS-PAGE测定酶的亚基相对分子质量为63 .1 kD。酶的等电点为4.46和4.79。非还原/还原（NR/R）单向、双向SDS-PAGE显示酶分子含有链内二硫键。对二级结构圆二色谱分析显示,酶分子中α-螺旋占13.84%,β-折叠占25.68%,无规则卷曲占56.34%。氨基酸组成分析结果表明, 酸性磷酸酶约含有507个氨基酸残基,富含门冬氨酸残基。     

Purification and Properties of Manganese Superoxide Dismutase from Norway Spruce (Picea abies L. Karst)

A manganese-containing superoxide dismutase (SOD; EC 1.15.1.1 [EC] )was purified to electrophoretic homogeneity from seeds of Norwayspruce (Picea abies L.). The apparent molecular mass of thepurified enzyme was 86 kDa, as determined by gel filtration.The subunit molecular mass, estimated by SDS-polyacrylamidegel electrophoresis, was 22 kDa both in the presence and inthe absence of 2-mercaptoethanol. Thus, the native enzyme isa homotetramer with subunits that were not linked by disulfidebonds. The isoelectric point of this Mn-SOD was 5.5. The specificactivity of the Mn-SOD was strongly pH-dependent and was 400units per nmol SOD at pH 7.8 and 30 units per nmol SOD at pH10.4. The first 25 amino acid residues in the amino terminalregion of spruce Mn-SOD exhibited a high degree of sequencehomology to those of Mn-SODs from other organisms. In Mn-deficientneedles the activity of Mn-SOD was only half of that in non-deficientneedles, whereas the activity of CuZn-SOD was doubled. (Received May 20, 1994; Accepted October 31, 1994)     

Purification and characterization of {delta}-aminolevulinic acid dehydratase from Chlorella regularis

-Aminolevulinic acid dehydratase (-aminolevulinic acid hydrolyaseEC 4.2.1.24 [EC] ) which catalyzes the formation ofporphobilinogenfrom two molecules of -aminolevulinic acid (ALA) was purifiedfrom Chlorella regularis 737-fold by acetone and ammonium sulfatefractionations, DEAE-cellulose column chromatography, and SephadexG-200 gel filtration. The enzyme had an optimum pH of 8.5 inTris-HCl buffer and required either Mg²⁺ or Mn²⁺ for its maximumactivity. The Km values for Mg²⁺, Mn²⁺ and ALA were 15 µM,10µM, and 0.5 mM, respectively. The enzyme was not activatedby thiol compounds, but was inhibited by p-chloromercuribenzoate.The molecular weight estimated by gel filtration was 316,000and the isoelectric point was 5.25. (Received October 18, 1978; )     

Purification and Properties of Arginase from Soybean, Glycine max, Axes

Arginase (EC 3.5.3.1) was purified to homogeneity from cytosol of soybean, Glycine max, axes by chromatographic separations on Sephadex G-200, DEAE-sephacel, hydroxyapatite, and arginine-affinity columns. The molecular weight of the enzyme estimated by pore gradient gel electrophoresis was 240,000, while sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a single band at the molecular weight of 60,000. The optimal pH for activity was 9.5 and the K_m value was 83 millimolar. The enzyme was stimulated by polyamines such as putrescine.     

Arginine metabolism in Saccharomyces cerevisiae. Some general properties of yeast arginase

Cells of Saccharomyces cerevisiae ATCC 9763 were cultured aerobicallyat 30?C in defined media containing arginine as a sole sourceof nitrogen. After 5 hr of growth arginase (L-arginine ureohydrolaseEC 3.5.3.1 [EC] ) was extracted by sonication and partially purifiedby gel filtration. The specific enzyme activity was increasedthirty times as a result of Sephadex G-200 treatment. The geltreated enzyme was stable for at least 3 days when stored at2?C. Enzyme activity was maximal at pH 9.2 and was increasedby preincubation with divalent metallic ions. The apparent Michaelisconstant for arginine was 12.5 mM in glycinate buffer and 250mil in phosphate buffer. The enzyme was strongly inhibited byL-ornithine. The kinetics of this inhibition revealed that ornithinecompeted with arginine for the active site. The molecular weightof yeast arginase was estimated to be 120000?10000 by determinationof elution volumes from Sephadex G-200. The general propertiesof the yeast enzyme are discussed in relation to (a) arginasesfrom other sources and (b) regulation of the Krebs-Henseleitcycle in Saccharomyces. (Received January 27, 1973; )     

Purification and Properties of an Extracellular Endo-1,4-r{beta}-Glucanase from Suspension-Cultured Poplar Cells

An endo-1,4-rß-glucanase (EC 3.2.1.4 [EC] ) was purifiedto apparent homogeneity from the culture medium of poplar (Populusalba L.) cells by sequential anion-exchange, hydrophobic, andgel-filtration chromatography. The preparation of extracellularrß-glucanase was homogeneous on SDS-polyacrylamidegel electrophoresis (PAGE) and native PAGE. The molecular weight,as determined by SDS-PAGE was 50,000, whereas that determinedby gel filtration was 40,000. The isoelectric point (pI) was5.5. The purified enzyme catalyzed the endohydrolysis of carboxy-methylcellulosewith a pH optimum of 6.0 and a k_m of 1.0 mg ml^–1. Theenzyme specifically cleaved the 1,4-rß-glucosyl linkagesof carboxymethylcellulose, swollen cellulose, lichenan and xyloglucan,although the last was hydrolyzed more slowly than the othertested substrates. The activity of the endo-1,4-rß-glucanaseincreased up to the early stage of the mid-logarithmic phaseof growth and then decreased rapidly, suggesting that the rß-glucanaseis induced before cell development. (Received April 28, 1993; Accepted July 19, 1993)     

Alcohol Dehydrogenase from Cultured Rice Cells

Rice cells in suspension culture had high alcohol dehydrogenaseactivity during the logarithmic growth phase (3rd to 5th day).Ethanol was accumulated both in the cells and in the medium.The highest amount of ethanol was accumulated on the 4th dayin cells (10 µmoles/g fresh weight) and during the stationarygrowth phase (8th day) (180 mM, ca. 1%) in the medium. The enzymewas isolated from the cell extract and purified 36-fold witha 14% yield by ammonium sulfate fractional precipitation, andchromatography on DEAE-Sephadex, Sephadex G-150 and Blue Dextran-Sepharose.The purified enzyme was homogeneous, as judged by its sedimentationvelocity, and poly acrylamide gel, starch gel and SDS-polyacrylamidegel electrophoreses. Its molecular weight was 76,000 distributedin two, identical 37,000 subunits. The isoelectric point wasat pH 5.5. The enzyme contained 2.1 g atoms of zinc, 12 freeSH groups and 3 to 4 SS bonds per molecule. The pH optimum forethanol oxidatioa was pH 9.5 and for acetaldehyde reductionpH 6.0. The K_m values for ethanol, NAD^$, acetaldehyde and NADHwere 64.5 mM, 47.1 µM, 1.3 mM and 9.5 µM. The aminoacid composition, substrate specificity, and the effects ofchelators, SH reagents and sugar metabolic intermediates alsoare reported. (Received August 25, 1981; Accepted December 7, 1981)     

Purification and Some Properties of Alcohol Acetyltransferase from Banana Fruit

Isoamyl acetate, one of the major characteristic aroma compoundsof banana fruit (Musa sapientum L.), was synthesized by thecondensation of acetyl-CoA and isoamyl alcohol under the actionof alcohol acetyltransferase, which was found to be localizedin the soluble fraction of pulp cells. The activity of thisenzyme increased with the ripening of banana fruit. The enzyme was purified about 62-fold. The purified enzyme wasvery labile at pHs lower than pH 7.0 but relatively stable atpH 7.5{small tilde}9. The enzyme was most active at 30C andpH 8.5. The molecular weight was estimated to be about 40,000by gel filtration. Its K_m values for acetyl-CoA and isoamylalcohol were 50 µM and 0.4 mM, respectively. (Received January 28, 1985; Accepted May 30, 1985)