Purification of tryptophan oxygenase and its interaction with cadmium

Purification of tryptophan oxygenase (L-tryptophan oxidoreductase EC 1.13.1.12) from $\text{Pseudomonos acidovorans}$ is described. When chromatographed on Sephadex G-200 or on DEAE Sephadex A-50, the enzyme was eluted in two distinct bands. Over the concentration range 10^?6 – 10^?4 M, cadmium stimulated the activity of the enzyme but inhibited it non-competitively at higher concentrations. A mechanism is suggested for the behaviour of the enzyme towards cadmium.     

Isolation and purification of NADP-dependent "L-malate"-enzyme from corn leaves]

Isolation and purification of "malic-enzyme" NADP was done using fractionation by ammonium sulfate, anion-exchange chromatography on DEAE cellulose, gel-filtration through Sephadex G-200 and purification on DEAE Sephadex A-50. The isoenzyme isolated had a specific activity of 40-50 mkM/mg protein per min (approximately 80-fold purification) and contained negligible admixtures.     

Purification and Some Properties of a Fungal Lipase Preparation Used for Milk Flavouring

Intracellular lipase of a strain of Rhizopus fungus which is effective for producing a milk flavor was purified and fractionated into two components, I and II, by DEAE Sephadex A-50 column chromatography. They both proved homogeneous by electrophoresis and ultracentrifugal analysis. The sedimentation coefficient was respectively calculated to be 5.8×10^?13 for lipase I, and to be 2.2×10^?13 for lipase II. From substrate specificity, it was found that lipase I was an ordinary lipase hydrolyzing olive oil and tributyrin favourably, while, II, rather, a special lipase having a high affinity towards tricaprylin. They, also, respectively had an apparent phospholipase activity on soy-lecithin and, clearing activity on chylomicron prepared from olive oil and human serum. Their mode of action, and the effect of metals and emulsifying agents on their activity are also presented.     

Insecticidal toxin fromXenorhabdus nematophilus, symbiotic bacterium associated with entomopathogenic nematodeSteinernema glaseri

Entomopathogenic nematodes are being used for insect control. We purified a toxin secreted by the insect-pathogenic bacterium,Xenorhabdus nematophilus, which lives in the gut of entomopathogenic nematodes. Culture broth ofX. nematophilus was separated by centrifugation and concentrated by ultrafiltration. The concentrated culture broth was applied to a DEAE Sephadex A-50 column, and proteins were eluted stepwise with increasing concentrations of KCl. Fractions with insect toxicity were further concentrated and then applied to a HPLC with a gel filtration column. The molecular weight of purified toxin was 39 kDa on SDS-PAGE, and Fourier transformed infrared (FTIR) spectroscopy indicated that this toxin could be a new protein exhibiting the characteristics of C=O stretching peak near 1650 cm⁻¹.     

Production of a homogeneous Cl. botulinum type B neurotoxin

The method for obtaining the neurotoxin, or alpha-fraction of the toxin, of Cl. botulinum, type B, is described. In accordance with this method, the toxin was precipitated three times with hydrochloric acid in the isoelectric zone with subsequent extraction with phosphate (pH 6.8) and citrate-phosphate (pH 5.6) buffers, then fractionated in columns with DEAE cellulose (pH 5.6), DEAE Sephadex A-50 (pH 7.2) and Sephadex G-200 (pH 7.2). The homogeneous neurotoxin preparations with molecular weights ranging from 145,000 to 160,000 and having the isoelectric point at pH 5.5 and toxicity 5.0--10.0 x 10(7) Dlm per 1 mg protein were obtained.     

Isolation and properties of extracellular cellulase-hemicellulase complex of Phoma hibernica

A cellulase-hemicellulase complex was obtained from the culture supernatant of Phoma hibernica. It was purified by ammonium sulfate precipitation, column chromatography on diethylaminoethyl-Sephadex A-50 and Sephadex G-100. The preparation was capable of degrading carboxymethyl-cellulose, insoluble cellulose, xylan, galacto-, gluco-, and galactogluco-mannan. The distinct protein band obtained after isoelectrofocusing also showed activities towards these substrates. Optimum pH for cellulase and galactomannase activities was 4.5 and for xylanase activity 4.5–5.5. Tetranitromethane, urea and Fe³⁺ inhibited all the enzymatic activities of the complex. The preparation attacked carbohydrate polymers in different manners depending on the substrate. Cellulose was attacked in an exo-wise, xylan in an endowise manner. Nitrophenyl derivatives of carbohydrates were hydrolyzed slowly. It is suggested that the purified enzyme preparation is a complex most probably composed of subunits of different enzymatic activities.Abbreviations Used CM carboxymethyl - DEAE diethylaminoethyl - CMC carboxymethylcellulose     

Isolation of a non-thionein copper-binding protein from liver of copper-injected rats

1. The characterization of a low molecular weight, non-thionein, Cubinding protein isolated from rat liver is reported. The protein was isolated following chronic administration of Cu(NO₃)₂ using a combination of Sephadex G-75 and Sephadex DEAE A-25 chromatography. The protein did not bind to fully equilibrated Sephadex DEAE which formed the basis of the isolation procedure. 2. The final protein pereparation was found to be homogeneous by a variety of electrophoretic techniques and was distinguished from metallothionein on the basis of its behaviour on ion exchange and electrophoretic systems, spectral properties, and amino acid composition and metal content. It contains 6.8% cysteine and was found to bind Cu in a ratio of 1.5:1 based on a molecular weight of 11 000. 3. These results confirm the necessity to use techniques other than gel filtration alone to obtain adequate separation of low molecular weight metal-binding protein fractions.     

荔枝果皮过氧化酶的纯化与性质研究(英)

荔枝果皮采后褐变是影响这一重要热带水果经济价值的主要问题,酚类物质的酶促氧化一直被认为是造成植物组织褐变的关键因素,其中多酚氧化酶被研究得最多.过氧化物酶在植物体中分布很广,能够氧化多种底物,在荔枝果皮中的含量也很高.非结合性过氧化物酶已经被证明在果实的采后成熟与老化过程中参与多种过程.在这项研究中,用磷酸缓冲液提取荔枝果皮的非结合性过氧化物酶,并通过硫酸铵沉淀,DEAE Sephadex A-50离子交换柱层析以及Sephadex G-100凝胶过滤进行纯化.对得到的酶溶液进行了酶学性质的研究,发现荔枝果皮过氧化物酶具有较高的热稳定性和高的最适反应pH值（6.8）,能够氧化许多底物尤其是单酚和各种多酚类物质,反应抑制剂专一性与其他植物来源的过氧化物酶略有不同.显示了过氧化物酶参与荔枝果皮褐变过程的可能性,并为提高荔枝采后贮藏性提供了新的思路.     

rNM23-H1/NDPK-A中试纯化工艺研究

为比较3种DEAE填料对rNM23-H1/NDPK-A的纯化效果,利用同一批中试发酵样品在相同的条件下进行离子交换层析,分别收集P0．2和P1．0两个洗脱峰。通过对洗脱峰中蛋白质含量、目标蛋白相对含量以及酶比活的测定,计算得出Matrex Cellufine A-200,DEAE Sephadex A-25,Macro-Prep DEAE Support三种填料相对应的NDPK－A得率及纯化倍数分别为74．5％、40．8％、92．6％、2．4、1．9、3．1倍。综合分析表明Macro-Prep DEAE Support填料对rNM23-H1/NDPK-A的纯化效果最好。     

Immunological Studies on Antisera of Mice Immunized with Dried or DEPC-treated Ehrlich Ascites Tumor Cells

Intracellular arylsulfatases from Klebsiella aerogenes W70 cells grown in methionine medium (M enzyme) and inorganic sulfate medium containing tyramine (T enzyme) were purified respectively by fractionation with (NH₄)₂SO₄, followed by successive chromatographies on DEAE cellulose, hydroxylapatite, Sephadex G-100 and DEAE Sephadex A-25. On polyacrylamide gel electrophoresis, the two enzymes gave single bands with the same mobilities. Molecular weights of both, determined by SDS gel electrophoresis and by Sephadex G-100 chromatography, were 47,000 and 45,000, respectively. Their activities were maximal at pH 7.5. The affinities of the enzymes (M and T enzymes) for their substrate (Km) and the maximum velocity of hydrolysis (V_max) were enhanced by addition of electron withdrawing substituents. The enzymes were inhibited by inorganic phosphate, cyanide, hydroxylamine and tyramine. The inhibition by tyramine was competitive (Ki = 1.0 × 10^?4 m). These results show that the two enzymes were identical. This was confirmed by the fact that mutant strains, which were unable to synthesize arylsulfatase when grown with methionine, could also not synthesize the enzyme when grown with tyramine.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

Degradation of the four monoiodoinsulin isomers by the insulin protease of rat liver

The cleavage of insulin by the partially purified insulin protease was studied using the four [¹²⁵I]tyrosine-monoiodoinsulins (tyrosine A-14 and A-19 of the A-chain; tyrosine B-16 and B-26 of the B-chain). The rates of conversion of the four isomers to trichloroacetic acid-soluble form was in the order B-26 > A-14 > A-19 > B-16. The following was observed in experiments which gave 19/14/5/3 percent conversion to trichloroacetic acid-soluble products: the loss of ability to bind to IM-9 lymphocytes was approx. 55% for all four isomers. About 70% of the radioactivity was in the ‘insulin’ peak, and about 30% was in peptides smaller than insulin as judged by gel filtration on Sephadex G-50. The descending limb of the ‘insulin’ peak contained significant amounts of radioactive material not binding to IM-9 lymphocytes. This material showed multiple peaks when applied to high performance liquid chromatography. Other experiments were designed to cause an almost complete degradation of the isomers. Under these conditions, the radioactivity eluted on Sephadex G-50 largely as iodotyrosine (and some small peptides) using the A-14, B-16 and B-26 isomers, whereas iodotyrosine was absent using the A-19 isomer. Thus, the insulin protease appears to first degrade insulin to multiple products with molecular sizes slightly smaller than insulin and subsequently to small peptides (e.g. containing tyrosine A-19) and amino acids (e.g. tyrosine A-14, B-16 and B-26).     

Detection of multiple forms of polyphosphate phosphohydrolase from Endomyces magnusii]

Labile polyphosphate phosphohydrolase from Endomyces magnusii is 27-fold purified by means of fractionation with ammonium sulphate, gel filtration on Sephadex G-75 and Biogel P-60 and chromatography on DEAE cellulose. Chromatography on DEAE Sephadex A-50, isoelelctric focusing and polyacrylamide gel electrophoresis of the enzyme preparation revealed 3 different fractions with polyphosphate phosphohydrolase activity (PPPH1, PPPH2 and PPPH3). Relative content of these fractions in E. magnusii cells is 30%, 55% and 15% respectively. Isoelectric points are: PPPH1--pH 5.1--5.2; PPPH2--pH 6.0--6.1; PPPH3--pH 6.3--6.4. PPPH1 and PPPH2 are found to be the most labile. PPPH3 is more stable under isolation procedure and storage. The fractions have similar molecular weight (48 000 +/- 3000).     

赤霉菌超氧化物歧化酶的纯化及部分理化性质

采用加热、Sephadex G—100凝胶过滤及DEAE-Sephadex A-50柱层析的方法,提纯了赤霉菌的超氧化物歧化酶(SOD),纯酶比活力为2640U/mg蛋白,最大紫外吸收峰为276nm,为Mn-SOD,由二个亚基组成,亚基分子量为14.5kD。此外还报道了该酶的氨基酸组成。     

Purification and immunological characterization of human myocardial MB creatine kinase

Elevated plasma MB creatine kinase (CK) is considered the most sensitive and specific diagnostic indicator of myocardial infarction. However, attempts to purify human MB CK have been unsuccessful. The need for purified human MB CK was further enhanced with the development of a radioimmunoassay for CK isoenzymes which would provide more prompt and specific detection of myocardial infarction. The major protein contaminant of MB CK is albumin which has been difficult to separate due to their similar electrophoretic mobility. Human hearts were obtained within 2 h postmortem and the tissue homogenized in 50 mm Tris-HCl (pH 7.4), 2 mm mercaptoethanol. The CK was recovered from the supernatant (31,000g) by ethanol extraction (50–70%). The resuspended pellet was fractionated on DEAE Sephadex A-50 with a salt gradient (50–500 mm, pH 8.0). The MB fraction contained about 90% albumin. The preparation was bound to an Affigel blue column and contaminating proteins other than albumin were eluted with 50 mm Tris-HCl (pH 8.0), 2 mm mercaptoethanol. MB CK was eluted with 250 mm NaCl, but the albumin remained bound. The MB fraction with a specific activity of 453 IU/mg represented an 80-fold increase in purity and exhibited a single protein band on polyacrylamide gels. Purified MB CK labeled with ¹²⁵I exhibited no binding to human albumin antiserum, but bound to MB CK antiserum, and unlabeled MB CK competitively inhibited binding of ¹²⁵I-MB CK in the radioimmunoassay system exhibiting a sensitivity for detection of plasma MB CK at the nanogram level.     

Differences in the binding of 3-methylcholanthrene and phenobarbital to rat liver cytosolic and nuclear protein fractions

The inducers of cytochrome P-450c and P-450b, 3-methylcholanthrene and phenobarbital, respectively, have been studied in their interaction with subcellular fractions from rat liver. 3-Methylcholanthrene bound to both nuclear and cytoplasmic components as demonstrated by DNA-cellulose chromatography. The binding of 3-methylcholanthrene to cytosolic proteins, on DNA-cellulose, was approximately 27 fmol/mg of applied protein, whereas the binding to nuclear proteins was 250–570 fmol/mg applied protein. Phenobarbital did not bind to proteins of rat serum, rat liver cytosol, or rat liver nuclei which could bind to DNA-cellulose. Further examination of the potential interaction of phenobarbital to rat liver cytosolic proteins was carried out using either DEAE A-50 Sephadex chromatography, charcoal dextran analysis, or sucrose density gradients. No binding of phenobarbital to rat liver cytosolic proteins was observed under these experimental conditions. In contrast, the binding of 3-methylcholanthrene to cytosolic proteins showed four peaks of radioactivity after DEAE A-50 Sephadex chromatography, two peaks by sucrose density gradient analysis, and specific binding (0.13 pmol/mg protein) was observed using the charcoal dextran technique. One of the peaks on sucrose gradients was labile in the presence of salt. The uptake and intranuclear distribution of 3-methylcholanthrene and phenobarbital were markedly different after incubation with whole nuclei: 64% of the available 3-methylcholanthrene but only 3% of the available phenobarbital radioactivity became associated with nuclei. Of this radioactivity, the highest specific activity of the 3-methylcholanthrene radioactivity was associated with the 2 m KCl-resistant nuclear pellet and the highest specific activity of the phenobarbital radioactivity was associated with the nuclear fraction soluble in the absence of salt. These results are interpreted in regard to the induction of cytochrome P-450c.     

Utility of solid-phase spectrophotometry to determine trace amounts of zinc in environmental and biological samples

A solid-phase spectrophotometric analysis has been proposed for preconcentration and determination of Zn(II) in real samples. The procedure is based on sorption of zinc(II) as 5-(2-benzothiazolylazo)-8-hydroxyquinoline (BTAHQ) complex on dextran-type anion-exchange gel (Sephadex DEAE A-25). The influences of the analytical parameters, including pH of the aqueous solution, amounts of BTAHQ, and sample volume, were investigated. The absorbance of the gel at 675 and 750 nm, packed in a 1.0-mm cell, was measured directly. The molar absorptivities were found to be 2.50 × 10⁷ and 9.55 × 10⁷ L mol⁻¹ cm⁻¹ for 500 and 1000 ml, respectively. Calibration was linear over the range of 0.05–1.10 μg L⁻¹ with a relative standard deviation of less than 1.60% (n = 10). The detection and quantification limits of the 500-ml sample method were 12 and 40 ng L⁻¹ on using 50 mg. For the 1000-ml sample, the detection and quantification limits were 7.5 and 25 ng L⁻¹ using a 50-mg exchanger. Increasing the sample volume can enhance sensitivity. No considerable interferences were observed from other investigated anions and cations on the Zn(II) determination. The proposed method was applied to determine zinc in environmental samples, including natural water, food, certified reference materials, meat, and biological samples, comparing the results simultaneously with those obtained using a flame atomic absorption spectrophotometer, whereby the validity of the method was tested.     

Biochemical analysis of a fibrinolytic enzyme purified from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain A1

A fibrinolytic enzyme from Bacillus subtilis strain Al was purified by chromatographic methods, including DEAE Sephadex A-50 column chromatography and Sephadex G-50 column gel filtration. The purified enzyme consisted of a monomeric subunit and was estimated to be approximately 28 kDa in size by SDS-PAGE. The specific activity of the fibrinolytic enzyme was 1632-fold higher than that of the crude enzyme extract. The fibrinolytic activity of the purified enzyme was approximately 0.62 and 1.33 U/ml in plasminogen-free and plasminogen-rich fibrin plates, respectively. Protease inhibitors PMSF, DIFP, chymostatin, and TPCK reduced the fibrinolytic activity of the enzyme to 13.7, 35.7, 15.7, and 23.3%, respectively. This result suggests that the enzyme purified from B. subtilis strain Al was a chymotrypsin-like serine protease. In addition, the optimum temperature and pH range of the fibrinolytic enzyme were 50°C and 6.0–10.0, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as Q-T-G-G-S-I-I-D-P-I-N-G-Y-N, which was highly distinguished from other known fibrinolytic enzymes. Thus, these results suggest a fibrinolytic enzyme as a novel thrombolytic agent from B. subtilis strain Al.     

Purification, Properties, and Localization of Two Carbonic Anhydrases from Amaranthus cruentus Leaves

The method has been developed for obtaining two purified forms of carbonic anhydrase (CA, A and B forms) from amaranth (Amaranthus cruentus L.) leaves. The method includes precipitation with ammonium sulfate, fractionation by ion-exchange chromatography on DEAE Sephadex A-50, gel filtration on AcA-34 ultragel, and ion-exchange chromatography on DEAE cellulose. The molecular weights of A and B forms were different and equaled to 151 and 251 kD, respectively. The results suggest that SH groups and zinc play important roles in the catalytic activity of both CA forms. Both forms exhibited a high hydratase activity and did not represent allosteric enzymes. However, the catalytic properties of A form, evaluated from the pH dependence of kinetic parameters, differed from those of B form, which was apparently caused by dissimilar structures of these forms. Furthermore, the A form was localized in chloroplast membranes of bundle sheath cells, whereas B form was a soluble enzyme located in the cytoplasm of mesophyll cells.     

Simultaneous microdetermination of homovanillic acid and 5-hydroxyindoleacetic acid in brain tissue using Sephadex G-10 and QAE-Sephadex A-25

Homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in animal brains were simultaneously purified by two steps of column chromatography on Sephadex G-10 and QAE-Sephadex A-25. Perchloric acid extracts of brain tissue were directly passed through a column of Sephadex G-10. The gel retained both HVA and 5-HIAA, thereby separating them from Cl0^?₄ which interferes with subsequent purification process and from endogenous substances which give blank fluorescence. HVA was loosely adsorbed on the gel and was easily desorbed with dilute acetic acid. This effluent was successively passed onto a column of QAE-Sephadex A-25 placed beneath the G-10 column and the adsorbed HVA was eluted with 0.1 M Na₂HPO₄. The 5-HIAA remaining on the Sephadex G-10 without being desorbed by acetic acid was eluted with dilute ammonia. The recovery of both acid metabolites by this column procedure was more than 90%. Thus, it is possible to determine the levels of HVA and 5-HIAA in single brains of small rodents.