Purification and Properties of Nitrite Reductase from Spinach Leaves

Nitrite reductase (EC 1.6.6.4) has been purified 730-fold from spinach leaves. The enzyme catalyzes the reduction of nitrite to ammonia, with the use of reduced form of methyl viologen and ferredoxin. A stoichiometry of one molecule of nitrite reduced per molecule of ammonia formed has been found. KCN at 2.5×10^-4 m inhibited nitrite reductase activity almost completely. Purified enzyme was almost homogeneous by disk electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 61,000 from gel filtration. Nitrite reductase, in the oxidized form, has absorption maxima at 276, 388 and 573 mμ. Both methyl viologen and ferredoxin linked nitrite reductase activities of the enzyme were inactivated on exposure to low ionic strength.     

Purification and Characterization of an Aminopeptidase, the Enzyme-Hydrolyzing Alanine-p-nitroanilide (APAase), from Euonymus Leaves

An aminopeptidase, the enzyme-hydrolyzing alanine-p-nitroanilide(APAase), from leaves of Euonymus alatus f. ciliato-dentatuswas purified about 1,400-fold by a combination of ion-exchangeand gel filtration column chromatographies. Polyacrylamide gelelectrophoresis showed the purified APAase to be homogenous.The molecular weight of this APAase was estimated to be about100,000, and the optimum pH for its hydrolytic activity againstalanine-p-nitroanilide (APA) was 8.6–9.0. APAase hydrolyzedalanine-ß-naphthylamide (alanine-NA), glycine-NA,lysine-NA and arginine-NA. It was inhibited slightly by p-chloromercuribenzoate(PCMB) and iodoacetic acid and was not activated by thiol reagents.Therefore, a sulfhydryl group could not be present at the activesite of APAase. APAase was inhibited strongly by 1,10-phenanthroline,but was unaffected by EDTA. Of the metal ions tested, Hg²⁺,Zn²⁺ and Mn²⁺ strongly inhibited its activity, and Ca²⁺ stimulatedit to some extent. (Received November 15, 1984; Accepted March 12, 1985)     

Purification and Characterization of an Aminopeptidase, LPAase 2, from Euonymus Leaves

An aminopeptidase, LPAase 2, from the leaves of Euonymus alatusf. ciliato-dentatus was purified about 240-fold by a combinationof DEAE-cellulose and Sephadex G-100 column chromatographies.The molecular weight of LPAase 2 was estimated to be about 62,000,and the optimum pH for the hydrolytic activity against leucinep-nitroanilide(LPA) was 7.6. LPAase 2 hydrolyzed LPA, leucine-rß-naphthylamide(leucine-NA), phenylalanine-NA and tyrosine-NA. It was inhibitedstrongly by p-chloromercuribenzoate (PCMB), iodoacetic acidand heavy metal ions, but was not affected by thiol compoundsand metal-chelating reagents. Therefore, a sulfhydryl groupcould be involved in the active site of LPAase 2. None of themetal ions tested promoted LPAase 2 activity. The propertiesof LPAase 2 were compared with those of aminopeptidases reportedfor other plants. (Received November 24, 1983; Accepted April 16, 1984)     

Purification and Properties of NADH-Dependent 5,10-Methylenetetrahydrofolate Reductase (MetF) from Escherichia coli

A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15°C and pH 7.2 in a stopped-flow spectrophotometer; the K_m for NADH is 13 μM, the K_m for CH₂-H₄folate is 0.8 μM, and the turnover number under V_max conditions estimated for the reaction is 1,800 mol of NADH oxidized min⁻¹ (mol of enzyme-bound FAD)⁻¹. NADPH also serves as a reductant, but exhibits a much higher K_m. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.     

油菜叶片硝酸还原酶同功酶的纯化与特性

通过对硝酸还原酶（ＮＲ）亲和层析洗脱过程的部分改进,从油菜叶片中分离纯化到诱导型（ｉＮＲ）及组成型（ｃＮＲ）两种硝酸还原酶同功酶。电泳分析表明两者均达到银染单带纯。ｃＮＲ分子量（ＭＷ）为４５０ｋＤ．ｉＮＲＭＷ为２２０ｋＤ;两者亚基ＭＷ均为１１０ｋＤ,但亚基数目不一样,氨基酸组成也有差异。ｉＮＲ与ｃＮＤ的等电点ｐＨ不同,分别为４．４及６．０,免疫交叉反应显示ｃＮＲ的抗原性为ｉＮＲ的７５％。     

人脑醛糖还原酶的纯化与性质

联合采用ＤＥＡＥ－纤维素层析、色谱聚焦、ＮＡＤＰ亲和层析与ＳｅｐｈａｄｅｘＧ－１００的凝胶过滤,对人脑醛糖还原酶（ＥＣ１．１．１．２１;ＡＬＲ）进行纯化．现测得该酶的等电点ｐＨ值为５．８５．经聚丙烯酰胺凝胶盘状电泳和Ｗｅｓｔｅｒｎ印迹证实,获得了满意的酶纯度．同葡萄糖,葡糖－６－磷酸与ＮＡＤＰＨ保温后,人脑ＡＬＲ纯品的活性与对照酶组相似,且不被糖酵解途径的一些磷酸化中间产物抑制．苯基硼酸琼脂糖柱层析洗脱谱峰和氢硼化钠还原反应提示,当同葡萄糖保温时,人脑ＡＬＲ（特别是其均一态）可能未被进一步糖基化．在糖尿病并发症和按结构完成药物设计的研究工作中,纯品ＡＬＲ的应用可发挥重要作用     

Purification and Properties of Sweet Potato NADPH-Cytochrome c (P-450) Reductase

NADPH-cytochrome c reductase, strictly NADPH-cytochrome P-450reductase, was purified by chromatography through DEAE-cellulose,2',5'-ADP-Sepharose, and Sephadex G-100 columns after solubilizationfrom microsomes from Ceratocystis fimbriata-infected sweet potatoroot tissue with Emulgen 913. The enzyme existed in three formsafter solubilization which migrated to positions correspondingto molecular weights of 81,000, 75,000 and 72,000 on an SDS-polyacrylamidegel. Trypsin treatment of the enzyme species with the largestpolypeptide yielded the species with the smallest one. Aftersucrose density gradient centrifugation of the pellet fractionobtained by centrifugation at 100,000?g of the crude extract,the enzyme species with the largest polypeptide was presentin the particulate fractions, whereas that with the smallestone was only found at the top of the gradient. We conclude thatthe enzyme species with the largest polypeptide is in an intact,amphipathic form, whereas that with the smallest one, and probablyalso the other species, is its hydrophilic domain produced byan endogenous protease(s). The K_m values of the enzyme in theintact form for NADPH and cytochrome c were 7.7 and 2.3 µM,respectively. ¹ Present address: Laboratory of Food Hygienics, Faculty ofAgriculture, Kagawa University, Miki-cho, Kida-gun, Kagawa 761-07,Japan. (Received September 6, 1984; Accepted December 27, 1984)     

Purification and Properties of a Diacetyl Reductase from Escherichia coli

A reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase with the ability to reduce diacetyl has been isolated from Escherichia coli and has been purified 800-fold to near homogeneity. The product of the reduction of diacetyl was shown to be acetoin. The enzyme proved to catalyze the oxidation of NADPH in the presence of both uncharged α- and β-dicarbonyl compounds. Even monocarbonyl compounds showed slight activity with the enzyme. On the basis of its substrate specificity, it is suggested that the enzyme functions as a diacetyl reductase. In contrast to other diacetyl reductases, the one reported here is specific for NADPH and does not possess acetoin reductase activity. The pH optimum of this enzyme was found to be between 6 and 7. The maximal velocity for the NADPH-dependent reduction of diacetyl was determined to be 9.5 μmol per min per mg of protein and the K_m values for diacetyl and NADPH were found to be 4.44 mM and 0.02 mM, respectively. The molecular weight was estimated by gel filtration on Sephadex G-100 to be approximately 10,000.     

Purification and Properties of 5-Ketogluconate Reductase from Gluconobacter liquefaciens

5-Ketogluconate reductase (5KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was partially purified about 120-fold by a procedure employing ammonium sulfate fractionation, and DEAE-cellulose-, hydroxylapatite- and DEAE-Sephadex A-50-column chromatographies. NADP was specifically required for the oxidative reaction of gluconic acid. The optimum pH for the oxidation of gluconic acid (GA) to 5-ketogluconic acid (5KGA) by the enzyme was 10.0 and for the reduction of 5KGA was 7.5. The optimum temperature of the enzyme was 50°C for both reactions of oxidation and reduction. The enzyme was considerably unstable and lost all of its activity within 3 days. The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ion, but remarkably stimulated by EDTA (1 × 10^?3m). Apparent Km values were 1.8 × 10^?2m for GA, 0.9 × 10^?3m for 5KGA, 1.6 × 10^?5 m for NADP, and 1.1 × 10^?5 m for NADPH₂.     

Identification of Pig Brain Aldehyde Reductases with the High-Km Aldehyde Reductase, the Low-Km Aldehyde Reductase and Aldose Reductase, Carbonyl Reductase, and Succinic Semialdehyde Reductase

Four NADPH-dependent aldehyde reductases (ALRs) isolated from pig brain have been characterized with respect to substrate specificity, inhibition by drugs, and immunological criteria. The major enzyme, ALR1, is identical in these respects with the high-Km aldehyde reductase, glucuronate reductase, and tissue-specific, e.g., pig kidney aldehyde reductase. A second enzyme, ALR2, is identical with the low-Km aldehyde reductase and aldose reductase. The third enzyme, ALR3, is carbonyl reductase and has several features in common with prostaglandin-9-ketoreductase and xenobiotic ketoreductase. The fourth enzyme, unlike the other three which are monomeric, is a dimeric succinic semialdehyde reductase. All four of these enzymes are capable of reducing aldehydes derived from the biogenic amines. However, from a consideration of their substrate specificities and the relevant Km and Vmax values, it is likely that it is ALR2 which plays a primary role in biogenic aldehyde metabolism. Both ALR1 and ALR2 may be involved in the reduction of isocorticosteroids. Despite its capacity to reduce ketones, ALR3 is primarily an aldehyde reductase, but clues as to its physiological role in brain cannot be discerned from its substrate specificity. The capacity of succinic semialdehyde reductase to reduce succinic semialdehyde better than any other substrate shows that this reductase is aptly named and suggests that its primary role is the maintenance in brain of physiological levels of gamma-hydroxybutyrate.     

大豆叶片尿囊素酶的纯化和性质

被纯化的 L—ALNase比活是 819 nkat/mg,酶的纯度提高了738倍,得率为27％。最适pH 7.4,K_m为13.7 mmol/L ALN,对还原剂和巯基试剂敏感,但5 mmol/L的抗坏血酸可提高酶活26％。磷酸盐和KCN有些抑制作用,但Mn~(2+)无活化作用。7.5％ PAGE,ANP定位测定,是 R_f 0.62、0.64、0.68、0.70和 0.725个同功酶的混合物。     

Purification and Properties of 2-Ketogluconate Reductase from Gluconobacter liquefaciens.

2-Ketogluconate reductase (2KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was purified about 1000-fold by a procedure involving ammonium sulfate fractionation and column chromatographies using DEAE-cellulose, hydroxylapatite, and Sephadex gel The purified enzyme gave a single band on polyacrymamide gel electrophoresis. NADP was specifically required for the oxidation reaction of gluconic acid. Using gel filtration a molecular weight of about 110,000 was estimated for the enzyme. The pH optimum for the oxidation of gluconic acid (GA) to 2-ketogluconic acid (2KGA) by the enzyme was 10.5 and for the reduction of 2KGA was 6.5. The optimum temperature of the enzyme was 50 C for both reactions of oxidation and reduction. The enzyme was stable at pH between 5.0 and 11.0 and at temperature under 50°C, The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ions, but remarkably stimulated by manganese ions (1×10^?3 m). Km value of the enzyme for GA was 1.3×10^?2 m and for 2KGA was 6.6×10^?3 _m. Km values for NADP and NADPH₂ were 1.25×10^?5 and 1.52×10^?5 m respectively.     

Purification and Properties of Methanol Dehydrogenase and Aldehyde Dehydrogenase from Methylobacillus glycogenes

Methanol dehydrogenase (MDH) and aldehyde dehydrogenase (ALDH) were purified to a homogenous state from Methylobacillus glycogenes, an obligate methylotroph. MDH (M_r 140,000) was composed of two different subunits (M_r 60,000 and 9,000) forming an α₂β₂ structure. MDH was indicated as a metalloquinoprotein containing one atom of calcium (Ca) per enzyme molecule. Binding of Ca was so tight that it was hard to remove Ca completely without denaturation of enzyme protein. A partially resolved enzyme resumed its original enzyme activity upon exogenous addition of Ca. Purified ALDH (M_r 144,000) was composed of two identical subunits of molecular mass of 72,000. ALDH was proved to be a quinoprotein in which PQQ is bound covalently.     

Purification and Properties of Cytokinin-Binding Proteins from Tobacco Leaves

A cytokinin-binding protein complex was purified 700-fold fromleaves of tobacco (Nicotiana sylvestris). The purification procedureconsisted of four chromatographic steps on columns of DEAE-cellulose,Mono Q, Phenyl Superose and Superose 12, respectively. The purifiedcytokinin-binding protein complex behaved as a 130-kDa globularprotein on gel filtration. This complex contains two proteinspecies whose molecular masses are estimated to be 57 kDa and36 kDa. Binding to benzyl[8-¹⁴C]adenine was inhibited by adenine,ATP, zeatin and cAMP but not by indoleacetic acid. Scatchardanalysis indicated the existence of at least two cytokinin-bindingsites in the purified complex. The dissociation constant forthe high-affinity site was 2.1 10^-5 M. (Received October 19, 1992; Accepted February 27, 1993)     

Purification and Properties of Adenosine Nucleosidases from Tea Leaves

Three adenosine nucleosidases (adenosine ribohydrolase, EC 3.2.2.7) with high substrate specificity were isolated from the extracts of tea leaves by a procedure including fractionation with ammonium sulfate, column chromatography on DEAE- and CM-cellulose, and gel filtration on Sephadex G-100. They were designated adenosine nucleosidase I, II and III, respectively, and their properties were characterized.

Among the naturally occurring nucleosides only adenosine and 2′-deoxyadenosine were hydrolyzed by these three enzymes and cleavage rate of the N-glycosidic bond in 2′-deoxyadenosine was three or four times greater than that in adenosine.     

Purification and Properties of Cow Splenic Biliverdin Reductase

Abstract

Biliverdin reductase was purified from cow spleen. The specific activity of the final enzyme preparation was 24.01 u/mg, representing 686-fold purification as measured with NADPH. The yield was 3 grams of enzyme per 100 grams of cow spleen. The purified enzyme was a monomeric protein with an apparent molecular weight of about 34,000 and an isoelectric point of about 6.2. The biliverdin reductase was specific for biliverdin and reduced IXα faster than the biliverdin isomers IXβ, IXr, or IXδ. The purified enzyme could utilize both NADH and NADPH, but the kinectic properties of the NADH-dependent and the NADPH-dependent enzyme activities were different: the time course of the NADPH-dependent reaction displayed a sigmoidal curve, whereas that of the NADH-dependent reaction did not. Km for biliverdin IXα was 4 × 10^?4 mM in the NADPH system, while it was 1.5 × 10^?3 mM in the NADH system. Both enzyme activities were inhibited by excess biliverdin, but the inhibition of the NADPH-dependent enzyme activity was more pronounced. The pH optimum was 7.0 with NADH, and 6.8 with NADPH.     

Purification and Properties of Flavin- and Molybdenum-Containing Aldehyde Oxidase from Coleoptiles of Maize

Aldehyde oxidase (AO; EC 1.2.3.1) that could oxidize indole-3-acetaldehyde into indole-3-acetic acid was purified approximately 2000-fold from coleoptiles of 3-d-old maize (Zea mays L.) seedlings. The apparent molecular mass of the native enzyme was about 300 kD as estimated by gel-filtration column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was composed of 150-kD subunits. It contained flavin adenine dinucleotide, iron, and molybdenum as prosthetic groups and had absorption peaks in the visible region (300-600 nm). To our knowledge, this is the first demonstration of the presence of flavin adenine dinucleotide and metals in plant AO. Other aromatic aldehydes such as indole-3-aldehyde and benzaldehyde also served as good substrates, but N-methylnicotinamide, a good substrate for animal AO, was not oxidized. 2-Mercaptoethanol, p-chloromercu-ribenzoate, and iodoacetate partially inhibited the activity, but well-known inhibitors of animal AO, such as menadione and estradiol, caused no reduction in activity. These results indicate that, although maize AO is similar to animal enzymes in molecular mass and cofactor components, it differs in substrate specificity and susceptibility to inhibitors. Immunoblotting analysis with mouse polyclonal antibodies raised against the purified maize AO showed that the enzyme was relatively rich in the apical region of maize coleoptiles. The possible role of this enzyme is discussed in relation to phytohormone biosynthesis in plants.     

Characterization and Partial Purification of Aldose-6-phosphate Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves

The Pereskia are morphologically primitive, leafed members of the Cactaceae. Gas exchange characteristics using a dual isotope porometer to monitor ¹⁴CO₂ and tritiated water uptake, diurnal malic acid fluctuations, phosphoenolpyruvate carboxylase, and malate dehydrogenase activities were examined in two species of the genus Pereskia, Pereskia grandifolia and Pereskia aculeata. Investigations were done on well watered (control) and water-stressed plants. Nonstressed plants showed a CO₂ uptake pattern indicating C₃ carbon metabolism. However, diurnal fluctuations in titratable acidity were observed similar to Crassulacean acid metabolism. Plants exposed to 10 days of water stress exhibited stomatal opening only during an early morning period. Titratable acidity, phosphoenolpyruvate carboxylase activity, and malate dehydrogenase activity fluctuations were magnified in the stressed plants, but showed the same diurnal pattern as controls. Water stress causes these cacti to shift to an internal CO₂ recycling (“idling”) that has all attributes of Crassulacean acid metabolism except nocturnal stomata opening and CO₂ uptake. The consequences of this shift, which has been observed in other succulents, are unknown, and some possibilities are suggested.     

Partial Purification and Properties of an Alkaline alpha-Galactosidase from Mature Leaves of Cucurbita pepo

A fourth molecular from of α-galactosidase, designated L_IV, an alkaline α-galactosidase, was isolated from leaves of Cucurbita pepo and purified 165-fold. It was active over a narrow pH range with optimal hydrolysis of p-nitrophenyl-α-d-galactoside and stachyose at pH 7.5. The rate of stachyose hydrolysis was 10 times that of raffinose. K_m determinations in McIlvaine buffer (200 millimolar Na₂-phosphate, 100 millimolar citric acid, pH 7.5) for p-nitrophenyl-α-d-galactoside, stachyose, and raffinose were 1.40, 4.5, and 36.4 millimolar, respectively. L_IV was partially inhibited by Ca²⁺, Mg²⁺, and Mn²⁺, more so by Ni²⁺, Zn²⁺, and Co²⁺, and highly so by Cu²⁺, Ag²⁺, Hg²⁺ and by p-chloromercuribenzoate. It was not inhibited by high concentrations of the substrate p-nitrophenyl-α-d-galactoside or by myo-inositol, but α-d-galactose was a strong inhibitor. As observed for most other forms of α-galactosidase, L_IV only catalyzed the hydrolysis of glycosides possessing the α-d-galactose configuration at C₁, C₂, and C₄, and did not hydrolyze p-nitrophenyl-α-d-fucoside (α-d-galactose substituted at C₆). The enzyme was highly sensitive to buffers and chelating agents. Maximum hydrolytic activity for p-nitrophenyl-α-d-galactoside was obtained in McIlvaine buffer (pH 7.5). In 10 millimolar triethanolaminehydrochloride-NaOH (pH 7.5) or 10 millimolar Hepes-NaOH (pH 7.5), hydrolytic activity was virtually eliminated, but the addition of low concentrations of either ethylenediaminetetraacetate or citrate to these buffers restored activity almost completely. Partial restoration of activity was also observed, but at higher concentrations, with pyruvate and malate. Similar effects were found for stachyose hydrolysis, but in addition some inhibition of L_IV in McIlvaine buffer, possibly due to the high phosphate concentration, was observed with this substrate. It is questionable whether the organic acid anions possess any regulatory control of L_IVin vivo. It was possible that the results reflected the ability of these anions, and ethylene-diaminetetraacetate, to restore L_IV activity through coordination with some toxic cation introduced as a buffer contaminant.