The cellulase of Fusarium solani. Resolution of the enzyme complex

1. Culture filtrates from Fusarium solani were fractionated by ion-exchange chromatography on DEAE-Sephadex, followed by gel chromatography on Sephadex G-100, into a C(1) component, a C(x) component (CM-cellulase) and a beta-glucosidase (cellobiase) component. 2. The individual components showed little capacity for the solubilization of cotton fibre (cellulase activity), but when recombined in their original proportions 81% of the original cellulase activity was recovered. 3. The C(1) components of F. solani and Trichoderma koningii were similar in their pH optima, heat stabilities over the pH range 5-8 and elution volumes on Sephadex G-100. 4. The C(1) component of F. solani synergized with the C(x) component of T. koningii and conversely. 5. The C(1) and the beta-glucosidase components of F. solani were devoid of the swelling-factor (S-factor) activity associated with the C(x) component.     

Allergenic activity of some kinds of plant pollen

Allergenic active fractions of ragweed, wormwood, goosefoot and sunflower pollen with a molecular weight of 37 000, 19 000, 35 000 and 14 000, respectively, were isolated by chromatography on Sephadex. All studied allergens had similar properties: affinity to Sephadex which was more pronounced to Sephadex G-75 than to G-100; absorbing components had no activity; allergenic and antigenic activities were determined only in fractions eluted in the bed volume of the column. Affinity of the allergans to Sephadex did not depend on the eluent type, but decreased in potentiation of the buffer ionic strength.     

Cadmium-binding component in Escherichia coli during accommodation to low levels of this ion.

An inducible cadmium-binding protein was isolated from Escherichia coli cells accommodated to 3 X 10(-6) M Cd2+ but not from normal or unaccommodated cells. Sephadex G-100, metal chelate affinity chromatography, and disc gel electrophoresis were used in the purification procedure. The molecular weight of the Cd2+-binding protein was estimated to be about 39,000 by Sephadex G-100 chromatography, making it different from the conventional, much smaller metallothionein.     

Highly acidic proteins from human brain: purification and properties of Glu-50 protein

Three extremely acidic proteins were isolated from human brain and purified to apparent homogeneity. One of them, Glu-50 protein, contained much glutamic acid (about 50% of the total amino acids). Its purification involved ammonium sulfate fractionation, DEAE-Sephadex A-50 chromatography, and gel filtration on Sephadex G-100 and G-75. Its molecular weight was determined to be 11,000 by SDS polyacrylamide gel electrophoresis and 34,000-36,000 by gel filtration on Sephadex G-75, suggesting that it consists of three identical polypeptide chains. Its isoelectric point was pH 3.9. Its N-terminal amino acid sequence was NH2-Asp-Glu-Pro-Pro-Asp-Glu and its C-terminal amino acid was Lys. It contained no detectable carbohydrate.     

Primary amino acid sequence of follicle-stimulating hormone from human pituitary glands. I. alpha subunit.

Follicle-stimulating hormone of a high state of physicochemical and biological purity was isolated from acetone-preserved human pituitary glands. The follicle-stimulating hormone was dissociated into alpha and beta subunits by treatment with 8 M urea and the subunits were separated by ion exchange chromatography on DEAE-Sephadex A-25. The subunits were freed of undissociated or reassociated follicle-stimulating hormone by gel filtration on Sephadex G-100. For the establishment of the primary amino acid sequence, the alpha subunit was reduced and either carboxyamidomethylated or S-aminoethylated prior to a thermolytic or a tryptic digestion. Each digest was gel filtered on a column of Sephadex G-50 to separate the glycopeptides from the peptides. The glycopeptides and the peptides were purified further by sequential gel filtration on Sephadex G-25, G-15, and Bio-Gel-P-2 and were isolated by high voltage electrophoresis at pH 6, 3.5, and 2. The purity of the isolated peptides was ascertained further by amino acid analysis. The amino acid sequences of the peptides were determined by Edman degradation followed by subtractive amino acid analysis. COOH-terminal sequences were established by digestion with carboxypeptidases A and B. The primary amino acid sequence of human follicle-stimulating hormone-alpha is identical to that of human chorionic gonadotropin-alpha and differs from that of human luteinizing hormone-alpha in having the tripeptide Ala-Pro-Asx- at the NH2-terminal end.     

Antioxidant Properties of the Peptides Isolated From Ganoderma lucidum Fruiting Body

Ganoderma lucidum is widely used as traditional medicine for centuries particularly in China, Japan and Korea. Many bioactive metabolites isolated from G. lucidum were therapeutically active against various diseases. The peptide isolated from water extract of G. lucidum was purified by employing Sephadex G-25, Sephadex G-50 and reverse phase HPLC column chromatography. The antioxidant property of the peptide fractions was determined by various in vitro methods. All fractions obtained from Sephadex G-25 and fraction G from Sephadex G-50 are effective antioxidants and comparably fraction C has the highest antioxidant activity. The molecular weight of purified peptide determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration chromatography and Matrix-assisted laser desorption ionization time-of-flight-mass spectrometer was found to be 2.8, 3.34 and 3.35?kDa respectively. The amino acid composition of the peptide was rich in phenylalanine, aspartic acid, proline, histidine and isoleucine. Peptide isolated in the present investigation suggests that has beneficial antioxidant properties may be due to its low molecular weight and specific amino acid composition.     

Isolation of an inhibitor of ß-glucuronidase from porcine sublingual gland

An inhibitor of ß-glucuronidase was isolated from porcine sublingual gland by successive fractionation of trypsin extracts of the latter on Sephadex G-100, DEAE-cellulose, Sephadex G-200, and DEAE-cellulose. Its purity and homogeneity were established by DEAE-cellulose column chromatography, ultracentrifugation, and electrophoresis on cellulose-acetate membrane. The sedimentation coefficient of the purified ß-glucuronidase inhibitor was 3.75 S (S₂₀⁰, w), and the molecular weight was determined to be 340 000 from Sephadex G-200 column chromatography. The inhibitor contained 17.5% protein, 20.8% total hexoses, 19.9% hexosamine, 21.8% N-acetylneuraminic acid, and 9.6% fucose. The inhibition was non-competitive, and it was completely suppressed by the addition of NaCl, KCl, Na₂SO₄, or CaCl₂, respectively.     

Purification of the messenger ribonucleic acid for the lipoprotein of the Escherichia coli outer membrane.

The mRNA for the lipoprotein of the Escherichia coli outer membrane has been purified to 85% homogeneity. The purification procedure involved phenol extraction, NaCl extraction, gel filtration on Sephadex G-100 and Sephadex G-200, and reversed-phase column chromatography on RPC-5. The purity of the final product was estimated to be 85% by analysis of the ribonuclease T1 fingerprint of the mRNA. The purified mRNA was able to direct the synthesis of cross-reactive material with antilipoprotein serum in both the E. coli and the wheat germ cell-free protein-synthesizing systems. The size of the mRNA was determined to be 8.2 S from its mobility in polyacrylamide--agrose gels. During the purification, two other RNA species, similar in size to the lipoprotein mRNA, were also isolated. Their sizes were determined to be 8.7 and 9.1 S. They both were inactive in an E. coli cell-free protein-synthesizing system.     

In Vitro Stability of Nitrate Reductase from Wheat Leaves: III. Isolation and Partial Characterization of a Nitrate Reductase-inactivating Factor

A nitrate reductase (EC 1.6.6.1)-inactivating factor has been isolated from 8-day-old wheat leaves. The purification schedule involved ammonium sulfate precipitation, Sephadex G-100 filtration, DEAE-cellulose chromatography, and Sephadex G-150 filtration. No accurate assessment could be made as to the degree of purification relative to crude extract as the inactivating factor could not be detected in crude extract. However a 2,446-fold purification was achieved from the ammonium sulfate fraction to the pooled enzyme from the Sephadex G-150 step.     

Properties and substrate specificity of proteinase from buckwheat seeds]

A thiol proteinase was isolated from buckwheat seeds and purified 300-fold, using ammonium sulfate, acetone fractionation ion-exchange chromatography on Sephadex CM-50 and electrofocussing. The proteinase preparation obtained was found homogenous after polyacrylamide gel electrophoresis at pH 4.5. The molecular weight of the enzyme (75.000) was determined by gel-filtration through Sephadex G-100. The activation of proteinase by cysteine, 2-mercaptoethanol and dithiothreitol, its inhibition by p-chloromercurybenzoate and the absence of inhibition by diisopropyl fluorophosphate and EDTA suggest that the enzyme isolated is a thiol proteinase. The enzyme hydrolyzed many peptide bonds in the B-chain of insulin, showing high substrate specificity. The glutelin and globulin fractions of buckwheat seed proteins were hydrolyzed by the enzyme. It is assumed that the hydrolysis of reserve proteins of buckwheat seeds is the main function of the proteinase isolated.     

海枣曲霉β-半乳糖苷酶的提纯与性质

 通过过聚乙二醇6000-磷酸钾缓冲液双相分离、Sephadex G-100凝胶过滤、DEAE-Sephadex A-50离子交换层析、羟基磷灰石层析及SephadexG-100凝胶过滤等提纯步骤,从海枣曲霉(Aspergillus phoenicis)麦麸培养物抽提液中提纯得到凝胶电泳均一的β-半乳糖苷酶。该酶的最适pH为3.5—4.0,最适温度为60℃(反应15分钟),在pH5.0—8.5之间及60℃以下稳定。在65℃和70℃保温时失活50％的时间分别为27和2分钟。用SDS凝胶电泳法和梯度凝胶电泳法分别测得该酶的分子量为115,000和118,000。薄层凝胶等电聚焦法测得其等电点为pH4.6。     

Monkey pepsinogens and pepsins. III. Carbohydrate moiety of Japanese monkey pepsinogens and the amino acid sequence around the site of its attachment to protein.

Purified Japanese monkey pepsinogens I and II contain carbohydrate as a part of the enzyme molecule. By gel filtration on Sephadex G-100, chromatography on DE-32 cellulose, and polyacrylamide disc gel electrophoresis, the carbohydrate moiety could not be separated from the enzyme protein, and the content did not decrease on repeated chromatography. Glycopeptides were obtained by successive digestion of pepsinogens with thermolysin and aminopeptidases and isolated by chromatography on Sephadex G-25 and G-50. Identification and determination of carbohydrate components was performed by paper and gas-liquid chromatographies. The presence of 4 glucosamines, 6 galactoses, 6--8 mannoses, and 8--11 fucoses per molecule of the glycopeptide of both pepsinogens was observed, of which the high content of fucose is especially unique. The molecular weight of the carbohydrate chains should be around 4,000--5,000. The amino acid sequence of a major glycopeptide was deduced to be Ile-Gly-Ile-Gly-Thr-Pro-Gln-Ala-Asn, in which the asparagine residue is the site of attachment of the carbohydrate chain.     

The cellulase of Trichoderma viride: Separation of the components involved in the solubilization of cotton

1. Culture filtrates from Trichoderma viride have been fractionated by gel filtration on Sephadex G-75 followed by ion-exchange chromatography on DEAE- and SE-Sephadex. 2. The components essential for attack on cotton are a carboxymethylcellulase, a cellobiase and a third (C₁) component which has no action on CM-cellulose, cellobiose or cotton. 3. These components, which together can completely convert cotton into water-soluble products, lose this ability when separated and regain it quantitatively when recombined in their original proportions.     

Analysis of chemical components of shiitake polysaccharides and its anti-fatigue effect under vibration

The shiitake polysaccharides were obtained from shiitake mushroom. Four fractions were isolated from the polysaccharides using a Sephadex G-100 gel column. Chemical components of the two main fractions were determined by thin layer chromatography (TLC), and high performance liquid chromatography (HPLC). F1 was composed of rhamnose, glucose, and mannose. F3 was composed of xylose, mannose, arabinose and galactose. The obtained results still showed that administration of shiitake polysaccharides could improve muscle's comfortability of animals under a long period of vibration. The above findings might be applicable to studies of vibration ergonomics.     

Isopentenyl pyrophosphate isomerase and prenyltransferase from tomato fruit plastids

Isopentenyl pyrophosphate isomerase has been isolated from an extract of tomato fruit plastids and purified 245-fold by fractionation with ammonium sulfate, gel filtration on Bio-Gel A 1.5m, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and chromatofocusing. Gel filtration on Sephadex G-100 separated the isopentenyl pyrophosphate isomerase from a prenyltransferase fraction that catalyzed the conversion of isopentenyl pyrophosphate to acid-labile compounds in the presence of dimethylallyl, geranyl, or farnesyl pyrophosphates. The molecular weights of the isopentenyl pyrophosphate isomerase and prenyltransferase were determined to be 34,000 and 64,000, respectively, by gel filtration on Sephadex G-100. The only cofactor required by either the isomerase or the prenyltransferase was a divalent cation, either Mg²⁺ or Mn²⁺. Isopentenyl pyrophosphate isomerase could also be totally inactivated by 1 × 10^?3m iodoacetamide, and this property was utilized in the assay of prenyltransferase activity in the presence of contaminating isomerase. The inactivation of isomerase by iodoacetamide is consistent with the stabilization of isopentenyl pyrophosphate isomerase by dithiothreitol. The K_m of isopentenyl pyrophosphate isomerase for isopentenyl pyrophosphate was found to be 5.7 × 10^?6.     

The purification and properties of microsomal palmitoyl-coenzyme A synthetase

1. Cell-free culture filtrates of the fungus Fusarium solani were examined for homogeneity with respect to beta-d-glucosidase and C(x) activities. 2. o-Nitrophenyl beta-d-glucoside and cellobiose were both used as substrates for beta-d-glucosidase activity. 3. No evidence for the non-identity of nitrophenyl beta-d-glucosidase and cellobiase activities could be found, either by heat treatment, gel filtration on Sephadex G-100 or by isoelectric focusing. 4. The beta-d-glucosidase component was also a feeble exo-beta-glucanase: it had a molecular weight of approx. 400000. 5. The fall in viscosity of a solution of CM-cellulose, the formation of reducing sugars in a solution of CM-cellulose and the solubilization of phosphoric acid-swollen cellulose (Walseth cellulose), were all used for the measurement of C(x) activity. 6. The ratio of the two types of CM-cellulase activity was not changed after gel filtration on Sephadex G-100 or after chromatography on DEAE-Sephadex. 7. Three peaks of C(x) activity were obtained after electrofocusing, but all three possessed the same ratio of the two types of CM-cellulase activity as well as the same CM-cellulase/Walseth activity ratio, as the unfractionated enzyme; all three isoenzymes (isoelectric points, 4.75, 4.80-4.85 and 5.15) acted in synergism with a mixture of the C(1) and the beta-d-glucosidase components to the same extent in the solubilization of cotton fibre. 8. The molecular weight of the C(x) component was approx. 37000.     

The isolation and characterization of pepsinogens from the proventriculus of the ostrich Struthio camelus

Three pepsinogens were isolated and purified from the proventriculus of the ostrich Struthio camelus, by a combination of chromatography steps on DEAE-cellulose, Sephadex G-100 and Hydroxylapatite. The purified pepsinogens manifested peptic activity towards haemoglobin as substrate after activation, but resembled chicken pepsinogens in that they appeared to lose their potential peptic activities during storage. All three pepsinogens contained glycine as N-terminal amino acid, but differed in their overall amino acid compositions. The pH and temperature optima of the activated pepsinogens were determined, as well as their molecular weights.     

江珧活性蛋白的分离及抗氧化作用研究

采用匀浆、硫酸铵分级沉淀和Sephadex G-100柱层析的方法从江珧的闭壳肌及内脏团分别分离到3个和2个活性蛋白峰,经化学发光法进行体外抗氧化测定,这些活性蛋白都具有清除超氧阴离子自由基、羟自由基、过氧化氢和抑制脂质过氧化作用,其中闭壳肌第三个蛋白峰(PB3)与内脏团第二个蛋白峰(PN2)抑制能力最强。     

Purification and characterization of alpha-galactosidase from watermelon

An alpha-galactosidase [EC 3.2.1.22] was isolated from the fruit of the watermelon, Citrullus battich. The enzyme was purified by procedures including extraction, ammonium sulfate precipitation, and chromatographies on DEAE-Sephadex, CM-Sephadex and Sephadex G-100. The final preparation was found to be fairly homogeneous on disc and SDS-polyacrylamide gel electrophoresis, and sufficiently free from other glycosidase activities. The molecular weight of the enzyme was estimated to be 45,000 by Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 4.5 for natural substrates and at 5.9 for artificial substrates. The enzyme liberates the alpha-galactose units from oligosaccharides of the raffinose series and ceramide trihexoside, and the hemagglutination-inhibiting activities of human ovarian cyst B-glycoprotein and blood group B-type ghosts were abolished by the enzyme.     

Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.