Isolation and characterization of glutamine synthetase from the marine diatom Skeletonema costatum.

Two peaks of glutamine synthetase (GS) activity were resolved by anion-exchange chromatography from the marine diatom Skeletonema costatum Grev. The second peak of activity accounted for greater than 93% of total enzyme activity, and this isoform was purified over 200-fold. Results from denaturing gel electrophoresis and gel-filtration chromatography suggest that six 70-kD subunits constitute the 400-kD native enzyme. The structure of the diatom GS, therefore, appears more similar to that of a type found in bacteria than to the type common among other eukaryotes. Apparent Michaelis constant values were 0.7 mM for NH4(+), 5.7 mM for glutamic acid, and 0.5 mM for ATP. Enzyme activity was inhibited by serine, alanine, glycine, phosphinothricin, and methionine sulfoximine. Polyclonal antiserum raised against the purified enzyme localized a single polypeptide on western blots of S. costatum cell lysates and recognized the denatured, native enzyme. Western analysis of the two peak fractions derived from anion-exchange chromatography demonstrated that the 70-kD protein was present only in the later eluting peak of enzyme activity. This form of GS does not appear to be unique to S. costatum, since the antiserum recognized a similar-sized protein in cell lysates of other chromophytic algae.     

北京棒状杆菌AS1.299谷氨酰胺合成酶的提纯和性质

用热变性、硫酸铵分段沉淀和DEAE纤维素柱层析部分纯化了北京棒状杆菌AS1.299(Corynebacterium pekinense)谷氨酰胺合成酶,并用垂直平板电泳对酶进一步精制。酶对谷氨酰胺的Km值为16.4mM,对羟胺的Km值为43.5mM;酶的沉降系数为21S,SDS凝胶电泳测得酶的亚基分子量为56,000。化学修饰表明,该酶活力与硫氢基、色氨酸、精氨酸残基无关;酪氨酸修饰剂的作用引起酶活力下降,初步证明,组氨酸残基是该酶活力的必需基因。     

Apoplastic β-1, 3-Glucanases in Leaves of Tomato Systematically Infected by TMV

Apoplastic β-1, 3-glucanase was purified from leaves of tomato (Lycopersicon esculentum Mill. ) which were systematically infected by TMV (tobacco mosaic virus). The enzyme obtained through -20℃ acetone precipitation, CM-Sephadex C-25 ion exchange chromatography, DEAE-Sephadex A-25 ion exchange chromatography and Sephadex G-75 gel filtration, showed homogeneity in PAGE, and SDS-PAGE which had two isoenzymes of 27 kD and 36 kD. The enzyme hydrolysed laminarin at an optimum pH of 4.8--5.2 and was stable between pH 4--8 and at an optimum temperature between 30--40℃, and stable at 40℃ after 1 hour of incubation, It had a Km of 9. 2 mg/mL. SDS-PAGE profiles of the proteins in the tomato leaf intercellular fluid had the bands of 22 kD, 27 kD and 36 kD that were β-1, 3-glucanases.     

Purification and Characterization of a Soluble Phosphatidylinositol 4-Kinase from Carrot Suspension Culture Cells

Previously we reported the presence of a soluble phosphatidylinositol 4-kinase (PI 4-Kinase) in carrot (Daucus carota L.) suspension culture cells (C.M. Okpodu, W. Gross, W.F. Boss [1990] Plant Physiol 93: S-63). We have purified the enzyme over 1000-fold using Q-Sepharose ion exchange, hydroxylapatite, and G-100 gel filtration column chromatography. The Mr of the enzyme was estimated to be 83,000 by gel filtration. PI 4-kinase activity was recovered after renaturation of the 80-kD region of polyacrylamide gels, and an 80-kD peptide cross-reacted with antibodies to the yeast 55-kD membrane-associated PI 4-kinase on western blots. The isolated lipid kinase phosphorylated PI but not lysophosphatidylinositol or phosphatidylinositol monophosphate. Maximal PI kinase activity occurred when the substrate was added as Triton X-100/PI mixed micelles at pH 8. The enzyme required divalent cations. At low concentrations (1-5 mM), Mn2+ was more effective than Mg2+ in increasing enzyme activity; however, maximal activity occurred at 25 to 40 mM Mg2+. Calcium from 0.01 [mu]M to 1 mM had no effect on the enzyme activity. The Km of the enzyme for ATP was estimated to be between 400 and 463 [mu]M. The enzyme was inhibited by adenosine (100 [mu]M); however, ADP (up to 100 [mu]M) had no effect on the activity. The biochemical characteristics of the carrot soluble PI 4-kinase are compared with the previously reported PI 4-kinases from animals and yeast.     

Purification and characterization of nitrobenzene nitroreductase from Pseudomonas pseudoalcaligenes JS45.

Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene as a sole source of carbon, nitrogen, and energy. The catabolic pathway involves reduction to hydroxylaminobenzene followed by rearrangement to o-amino-phenol and ring fission (S. F. Nishino and J. C. Spain, Appl. Environ. Microbiol. 59:2520, 1993). A nitrobenzene-inducible, oxygen-insensitive nitroreductase was purified from extracts of JS45 by ammonium sulfate precipitation followed by anion-exchange and gel filtration chromatography. A single 33-kDa polypeptide was detected by denaturing gel electrophoresis. The size of the native protein was estimated to be 30 kDa by gel filtration. The enzyme is a flavoprotein with a tightly bound flavin mononucleotide cofactor in a ratio of 2 mol of flavin per mol of protein. The Km for nitrobenzene is 5 microM at an initial NADPH concentration of 0.5 mM. The Km for NADPH at an initial nitrobenzene concentration of 0.1 mM is 183 microM. Nitrosobenzene was not detected as an intermediate of nitrobenzene reduction, but nitrosobenzene is a substrate for the enzyme, and the specific activity for nitrosobenzene is higher than that for nitrobenzene. These results suggest that nitrosobenzene is formed but is immediately reduced to hydroxylaminobenzene. Hydroxylaminobenzene was the only product detected after incubation of the purified enzyme with nitrobenzene and NADPH. Hydroxylaminobenzene does not serve as a substrate for further reduction by this enzyme. The products and intermediates are consistent with two two-electron reductions of the parent compound. Furthermore, the low Km and the inducible control of enzyme synthesis suggest that nitrobenzene is the physiological substrate for this enzyme.     

Purification and characterization of an N-acetyl-beta-D-glucosaminidase from cortical granules of Xenopus laevis eggs

The enzyme N-acetyl-beta-D-glucosaminidase was purified from the cortical granules of Xenopus laevis eggs using affinity chromatography, gel filtration, and density gradient centrifugation. The enzyme had a molecular weight of 37,000-40,000 as determined by polyacrylamide gel electrophoresis and density gradient centrifugation, had a Km for p-nitrophenyl-beta-D-N-acetyl-glucosaminide of 0.66 mM and a Ki for glucosamine of 4.3 mM. The kinetic properties of the cortical granule enzyme were similar to the enzyme isolated from jack bean. Treatment of unfertilized eggs with the enzyme isolated from cortical granules or jack bean rendered eggs unfertilizable. Loss of fertilizability was proportional to the product of time and enzyme concentration, consistent with an enzymatic mechanism being responsible for the loss of fertilizability. The amount of enzyme present in the perivitelline space was approximately the same as that which reduced fertilizability by 50% in one hour. We suggest that the action of cortical granule N-acetyl-beta-D-glucosaminidase on egg integuments may function as a block to polyspermy at fertilization.     

Purification and characterisation of adenosine nucleosidase from Coffea arabica young leaves

An adenosine nucleosidase (ANase) (EC 3.2.2.7) was purified from young leaves of Coffea arabica L. cv. Catimor. A sequence of fractionating steps was used starting with ammonium sulphate salting-out, followed by anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme was purified 5804-fold and a specific activity of 8333 nkat mg-1 protein was measured. The native enzyme is a homodimer with an apparent molecular weight of 72 kDa estimated by gel filtration and each monomer has a molecular weight of 34.6 kDa, estimated by SDS-PAGE. The enzyme showed maximum activity at pH 6.0 in citrate-phosphate buffer (50 mM). The calculated Km is 6.3 microM and Vmax 9.8 nKat.     

Purification and properties of an extracellular beta-xylosidase from a newly isolated Fusarium proliferatum

An extracellular beta-xylosidase from a newly isolated Fusarium proliferatum (NRRL 26517) capable of utilizing corn fiber xylan as growth substrate was purified to homogeneity from the culture supernatant by DEAE-Sepharose CL-6B batch adsorption chromatography, CM Bio-Gel A column chromatography, Bio-Gel A-0.5 m gel filtration and Bio-Gel HTP Hydroxyapatite column chromatography. The purified beta-xylosidase (specific activity, 53 U/mg protein) had a molecular weight of 91,200 as estimated by SDS-PAGE. The optimum temperature and pH for the action of the enzyme were 60 degrees C and 4.5, respectively. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It had a Km value of 0.77 mM (p-nitrophenol-beta-D-xyloside, pH 4.5, 50 degrees C) and was competitively inhibited by xylose with a Ki value of 5 mM. The enzyme did not require any metal ion for activity and stability. Comparative properties of this enzyme with other fungal beta-xylosidases are presented.     

Purification and properties of Bacillus subtilis SA-22 endo-1, 4-beta-D-mannanase.

beta-mannanase (EC 3.2.1.78) from Bacillus subtilis SA-22 was purified successively by ammonium sulfate precipitation, hydroxyapatite chromatography, Sephadex G-75 gel filtration and DEAE-52 anion-exchange chromatography. Through these steps, the enzyme was concentrated 30.75-fold with a recovery rate of 23.43%, with a specific activity of 34780.56 u/mg. Molecular weight of the enzyme was determined to be 38 kD by SDS-PAGE and 34 kD by gel filtration. The results revealed that the optimal pH value for the enzyme was 6.5 and the optimal temperature was 70 degrees C. The enzyme is stable between pH 5 to 10. The enzyme remained most of its activity after a treatment of 4 h at 50 degrees C, but lost 25% of activity at 60 degrees C for 4 h, lost 50% of activity at 70 degrees C for 3 h. The enzyme activity was strongly inhibited by Hg2+. The Michaelis constants (Km) were measured as 11.30 mg/mL for locust bean gum and 4.76 mg/mL for konjac powder, while Vmax for these two polysaccharides were 188.68 (micromol x mL(-1) x min(-1)) and 114.94 (micromol x mL(-1) x min(-1)), respectively.     

NADP-dependent isocitrate dehydrogenase from bass (Dicentrarchus labrax L.) liver

The isocitrate dehydrogenase from bass liver was purified to homogeneity by gel filtration, affinity and ion exchange chromatographies. The molecular weight was estimated by gel filtration chromatography to about 120,000. Analysis of the enzyme on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed it to be a dimeric protein. The enzyme showed maximum activity in the pH range between 7.0 and 8.0 while its maximum activity was at pH 7.5. DL-Isocitrate and Mn2+ stabilized the enzyme, while NADP had the opposite effect. The Km for isocitrate was 0.31 mM and the Km for NADP was 36 microM.     

Purification and characterization of an extracellular beta-lactamase produced by Acinetobacter calcoaceticus.

A beta-lactamase was purified 430-fold from the culture supernatant of Acinetobacter calcoaceticus by ion exchange chromatography on CM-Sephadex and affinity chromatography on phenylboronic-acid-agarose. The purified enzyme was homogeneous as judged by SDS-PAGE, and was characterized with respect to molecular mass (38 and 41 kDa by gel filtration on Sephadex G-75 and SDS-PAGE, respectively), pH optimum (pH 7.0), temperature optimum (45 degrees C) and isoelectric point (9.3). The beta-lactamase showed mainly cephalosporinase activity. It was inhibited by cloxacillin, carbenicillin, penicillanic acid sulphone (sulbactam) and aztreonam. It was not inhibited by clavulanic acid up to a concentration of 0.25 mM. Neither EDTA nor p-chlormercuribenzoate, up to concentrations of 1 or 100 mM, respectively, affected activity. According to these characteristics, it is a typical CEP-N cephalosporinase.     

Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides

An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.     

Purification and properties of an NADPH-linked aldehyde reductase from rat kidney

Rat kidney was shown to contain two NADPH-linked aldehyde reductases (alcohol:NADP+) oxidoreductase, EC 1.1.1.2) with different substrate affinities. The high-Km aldehyde reductase, which was purified to apparent homogeneity, had a molecular weight of 32 000 as determined by Sephadex G-100 gel filtration, and of 37 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme reduced various aliphatic aldehydes of different carbon-chain lengths besides many chemicals containing aldehyde groups. The Km values for n-hexadecanal and n-octadecanal were 8 microM and 4 microM, respectively. Bovine serum albumin (1.8 mM) stimulated the reduction of n-hexadecanal and n-octadecanal, and increased the Vmax values by about 15-fold without changing the Km values. The kidney enzyme was not distinguishable from the brain and liver high-Km aldehyde reductases in mobility on polyacrylamide gel electrophoresis, immunological properties, peptide maps or substrate specificity.     

Purification and Properties of ent-Kaurene Synthase B from Immature Seeds of Pumpkin

ent-Kaurene synthase B (KSB) was purified 291-fold from a crude enzyme preparation from endosperm of pumpkin (Cucurbita maxima L.). Separation of ent-kaurene synthase A and KSB was achieved by hydrophobic interaction chromatography. The fractions containing KSB activity were further purified by diethylaminoethyl, phenyl, and hydroxyapatite column chromatography. Using sodium dodecyl phosphate-polyacrylamide gel electrophoresis, the purest enzyme preparation showed a major band at an apparent molecular mass of 81 kD. The amount of protein in this band was correlated with KSB activity after diethylaminoethyl and hydroxyapatite chromatography. The N terminus of the 81-kD protein was blocked. Therefore, the protein was partially digested with protease and the amino acid sequences of the resulting major peptide fragments were analyzed. A polyclonal antibody was raised against a synthetic peptide based on the longest peptide fragment combined with a keyhole limpet hemocyanin. The antibody recognized only the 81-kD denatured protein and not the native KSB. The properties of KSB were examined using the phenyl-purified enzyme preparation. The Km value for copalyl pyrophosphate was 0.35 [mu]M, and the optimal pH was 6.8 to 7.5. The KSB activity required divalent cations such as Mg2+, Mn2+, and Co2+, whereas Cu2+, Ca2+, and Ba2+ inhibited the activity.     

蓖麻β-半乳糖苷酶的纯化及其基本性质

蓖麻籽黄化苗中存在高活性β-半乳糖苷酶。经硫酸铵分级分离、DEAE-纤维素离子交換层析、Sephadex G-100、CM-Sephadex和DEAE-Sephadex层析纯化。活性收率为6.4％,纯化倍数达107倍。纯化了的酶经聚丙烯酰胺凝胶电泳显示单一蛋白带,SDS-PAGE显示两条蛋白带,其相应分子量分别为3.25×10~4和2.94×10~4。用Sephadex G-200分子筛层析法测得分子量为6.7×10~4。综合上述结果推测该酶是由两个不同的亚基构成。以邻硝基苯酚-β-半乳糖苷为底物测得该酶的表观Km为5.9×10~(-3)mol/L。最适pH和最适温度分别为4.5和50℃。酸碱稳定区域在pH4.6—7.5之间。不同浓度缓冲液以及不同种类缓冲液、不同金属离子对酶活性影响均进行了讨论。     

3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).

3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.     

Functional characterization of D-galacturonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38-39 kDa, as judged by SDS-PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5+/-4.5 microM and uronic acids, such as D-galacturonic acid (Km=3.79+/-0.5 mM) and D-glucuronic acid (Km=4.67+/-0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP(+). The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H(2)O(2), suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

人胃腺苷脱氨酶的分离纯化及性质研究

用硫酸铵分级沉淀、ＤＥＡＥ－纤维素离子交换层析、免疫亲和层析、ＳｅｐｈａｄｅｘＧ１００凝胶柱层析从人胃组织中提取出腺苷脱氨酶,酶纯化１９３２４倍,比活力为５７９７Ｕ／ｍｇ蛋白．提取酶液经ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和等电聚焦只呈现一条区带。测得该酶的分子量为４１．２ｋＤ,等电点为ｐＨ４．８．氨基酸组成分析表明该酶由３８８个氨基酸残基组成,Ｎ端氨基酸为精氨酸。酶的最适ｐＨ为６．５,ｐＨ小于５．０或大于９．０时不稳定;最适温度为３７℃,对热不太稳定,以腺苷及２－脱氧腺苷作为底物,其Ｋｍ分别为８７μｍｏｌ／Ｌ和４１μｍｏｌ／Ｌ。     

Purification and characterization of glucose 6-phosphate dehydrogenase from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: homogenate preparation and 2', 5'-ADP Sepharose 4B affinity gel chromatography, which took 7-8 hours. Thanks to the two consecutive procedures, the enzyme, having specific activity of 38 EU/mg protein, was purified with a yield of 44.39% and 1310 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. Optimal pH, stable pH, optimal temperature, Km and, Vmax values for NADP+ and glucose 6-phosphate (G6P) were also determined for the enzyme. In addition, molecular weight and subunit molecular weights were found by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography respectively.