用琼脂糖亲和层析一步纯化蓖麻毒蛋白

本文报道了一种单用琼脂糖(Sepharose4B)来纯化蓖麻毒蛋白的快速简便的方法。我们发现在pH5的条件下,蓖麻毒蛋白和与其密切相关的蓖麻凝集素对琼脂糖的结合能力有很大的差别。在有0.2mol/LD-半乳糖存在下可将蓖麻毒蛋白从Sepharose上洗下,同样条件下蓖麻凝集素仍牢固地结合在柱上。从而经一步柱层析便可得到电泳纯的蓖麻毒蛋白。此法不需另行合成亲和胶,适合于蓖麻毒蛋白的大规模纯化。     

A Rapid Method for the Separation of Free and Membrane-Bound Polysomes of Rat Liver by Gel Filtration on Columns of Sepharose 4B

Abstract

A rapid method is reported for the separation of free and bound polysomes from total microsomes of rat liver on columns of Sepharose 4B, in which the total procedure from sacrifice of the animal to final preparation takes less than five hours. Both forms of polysomes are active in the incorporation of amino acids into proteins, the free polysomes having approximately twice the activity of bound polysomes. Both preparations contain polysomes at least six ribosomes in length.     

Polysome Isolation by Sepharose Column Chromatography

Polysomes from the mouse myeloma MOPC-21 were purified by gel filtration on Sepharose 6B, 4B and 2B columns. All three columns eliminated nearly all intracellular material smaller than 40 S subunits. In addition, passage through 4B and 2B columns substantially reduced the amount of subunits and monosomes in the preparations. Purified polysomes retained structural integrity when stored at -85°C for at least nine weeks.     

Changes of prothrombin activity by plasmin immobilized on L-lysine Sepharose 4B]

It is demonstrated that the method of immobilization of human plasmin by its adsorption on L-lysine-Sepharose 4B can be successfully applied for bovine plasmin isolation. Proteolysis of bovine prothrombin by bovine prothrombin by bovine plasmin preparations of high caseinolytic activity results in the increase of prothrombin level. Prothrombin proteolysis by bovine plasmin preparations of high caseinolytic activity results in the decrease of prothrombin level.     

Properties of human hemoglobin immobilized on Sepharose 4B.

This paper reports the properties of human hemoglobin covalently bound to Sepharose 4B both in 'high-affinity' and 'low-affinity' conformations. The results suggest that the coupling reaction is strongly affected by the conformational changes linked to oxygenation of the protein. The rate and the extent of the reaction are different for the oxy and deoxyderivatives, probably due to the change in reactivity of the amino groups in the liganded and unliganded tetramer. The data on the equilibrium which is established between matrix-bound and soluble subunits, measured by the 'subunit-exchange chromatography', indicate that the system displays a minimal heterogeneity when hemoglobin is coupled to the gel in the deoxy state at intermediate protein concentration and pH 8. Maxtrix-bound hemoglobin is characterized by a higher oxygen affinity and by decreased homotropic and heterotropic interactions with respect to hemoglobin in solution, but the changes depend strongly on the conditions used in the coupling procedure.     

Preparation of Chlorophyll a, Chlorophyll b and Bacteriochlorophyll a by Column Chromatography with DEAE-Sepharose CL-6B and Sepharose CL-6B

Chlorophylls extracted from spinach leaves were made free fromcarotenoids, pheophytins, chlorophyllides and plastoquinonesby column chromatography with DEAE-Sepharose CL-6B. Chlorophylla and b were separated by column chromatography with SepharoseCL-6B. By a combined use of DEAE-Sepharose CL-6B and SepharoseCL-6B, pure chlorophyll a and b were prepared from the leavesin a short time. Bacteriochlorophyll a_p extracted from a photosyntheticbacterium Chromatium vinosum was made free from carotenoids,bacteriopheophytin, ubiquinone and lipids by column chromatographywith Sepharose CL-6B. (Received April 19, 1983; Accepted June 16, 1983)     

Separation of growth-stimulating activity of BSA fraction V from the bulk of albumin using Heparin Sepharose Chromatography

Bovine serum albumin (BSA) is a potential source of biological contamination in cell culture medium. The aim of this work was to attempt to replace BSA in low serum and serum-free medium (SFM). BSA fraction V was subjected to a variety of processes in order to determine if the growth promoting activity observed for NRK cells could be extracted from the BSA molecule. These included solvent extractions, diafiltration, reverse phase HPLC and affinity chromatography using heparin sepharose. Solvent extraction and diafiltration failed to remove the activity from the BSA. Affinity chromatography using heparin sepharose indicated that all of the activity observed with BSA was retained in the 0.5 M NaCl fraction and was associated with less than 3% of the original protein. The major protein band in the 0.5 M NaCl fraction had the same apparent molecular weight as albumin (as seen by SDS-PAGE and analytical reverse phase HPLC). Unlike the untreated BSA, the 0.5 M NaCl fraction was partially susceptible to proteolytic digestion and to variations in pH.Abbreviations HS heparin sepharose - DHS donor horse serum - SFM serum free-medium     

The Purification of Soybean 11S Globulin with ConA-Sepharose 4B and Sepharose 6B

Kinetics of the acyl transfer catalyzed by Xanthomonas α-amino acid ester hydrolase was studied. The enzyme hydrolyzed d-α-phenylglycine methyl ester (d-PG-OMe) to give equimolar amounts of d-α-phenylglycine and methanol. With d-PG-OMe as an acyl donor and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) as an acyl acceptor, the enzyme transferred the acyl group from d-PG-OMe to 7-ADCA in competition with water. The addition of amine nucleophiles (7-ADCA and 6-aminopenicillanic acid) decreased the molecular activity (k_o) of the enzyme-catalyzed hydrolysis of d-PG-OMe, whereas it did not alter the Michaelis constant (K_M), and plots of l/k_o against the initial concentration of a nucleophile (n_o) gave a straight line. These results support the assumptions that the overall process for hydrolysis and acyl transfer proceeds through a common acyl-enzyme intermediate, that the acylation step of the enzyme is rate-limiting, and that the transfer competes with the hydrolysis of the acyl donor.     

Coupling of glycosaminoglycans to agarose beads (Sepharose 4B)

1. Heparin, heparan sulphate, chondroitin sulphate and dermatan sulphate were covalently attached to beads of agarose activated by cyanogen bromide. The bond is probably mediated by the amino group of a serine or peptide residue at the reducing end of the polysaccharide chain. 2. The uptake of glycosaminoglycan during the coupling procedure is about 0.9mg/ml of wet gel. However, direct analysis of washed and freeze-dried gels reveals that only about one-third of this amount is firmly attached to the gel. 3. The use of the gels for polysaccharidase analyses is exemplified by a hyaluronidase assay. Further applications, e.g. interaction studies and preparative purposes, are discussed.     

Separation of bovine brain mono- and diacylglycerol lipases by heparin Sepharose affinity chromatography

Mono- and diacylglycerol lipases are differentially inhibited by heparin. No other glycosaminoglycan resembles heparin in this respect. Mono- and diacylglycerol lipases can be separated by heparin Sepharose affinity chromatography. Diacylglycerol lipase was completely retained on a heparin--Sepharose column and was eluted with either 0.5 M NaCl or 2–5 mg/ml heparin, whereas monoacylglycerol lipase was recovered in the washings. Adenosine phosphates markedly affected the activity of diacylglycerol lipase in a concentration dependent manner. ATP was the most potent inhibitor followed by ADP. AMP had no effect and cAMP slightly stimulated the diacylglycerol lipase.     

用Blue Sepharose CL-6B快速纯化天花粉蛋白

差光谱显示染料cibacron blue F3GA与天花粉蛋白(TCS)有特异性结合,复合物在可见光部分的最大吸收波长在690 nm,摩尔消光系数ε=2.6×10^-3(mol/L)^-1·cm^-1,解离常数K_d=1.8 μmol/L,0.5 mol/L NaCl可使复合物解离.根据这一特点,用Blue-Sepharose CL-6B凝胶从栝篓块茎中亲和纯化了TCS.此法快速、简便、高效,易于大量制备.     

One-Step Purification of a Lipolytic Enzyme from Bacillus pumilus by Mild Hydrophobic Interaction Chromatography on Polyoxyalkylene Glycol-Bound Sepharose 6B

An esterase from Bacillus pumilus was obtained in a highly purified form, starting from a crude extract, by a new chromatographic technique requiring no detergent during the elution step. The stationary phase involved was mildly hydrophobic and was prepared by covalent immobilization of a polyoxyalkylene glycol onto Sepharose 6B.     

Use of Sepharose 4B for Preparative Scale Fractionation of Eukaryotic Messenger RNA's

A new technique using Sepharose 4B column chromatography for the partial purification of the total messenger RNA population of several animal tissues is described. The column when eluted with 0.1M sodium acetate, pH 5.0, containing. 001M EDTA, resolves a total nucleic acid extract into four major peaks. DNA is eluted at the void volume, followed successively by peaks of 18S ribosomal RNA, 4S transfer RNA and 28S ribosomal RNA. Ribonucleic acid containing mRNA activities is eluted after the DNA peak but immediately before the 18S rRNA peak. Hence the column enables quantitative removal of DNA, 5S RNA, tRNA and 28S rRNA from the majority of total cellular mRNA's. Partial segregation of mRNA's in the column is also demonstrated. The method does not require the isolation of polysomes as the initial procedure in mRNA isolation and is readily adaptable to large scale preparations. One hundred mg of total nucleic acid extracted from whole tissues can be fractionated on a 5 × 100 cm Sepharose 4B column. Recovery of total mRNA activities ranges between 60–70% and purification with respect to the total cell extract is 7 to 8 fold.     

Sepharose 4B一步法对金环蛇蛇毒磷脂酶A2的分离纯化

利用经酸处理的Sepharose 4B为层析介质,以含0.2mol/L半乳糖,pH7.4台氏液作为洗脱液,从广西产金环蛇(Bungarus fasciatus)蛇毒中一步分离得到一种磷脂酶A2.用SDS-聚丙烯酰胺凝胶电泳测定其分子量为14 kDa.N端部分序列测定表明,所分离得到的磷脂酶A2其N端16个氨基酸残基序列与已报道的金环蛇蛇毒磷脂酶A2同功酶Ⅵ(Lu & Lo,1978)一致.该酶糖含量较高,为13.4%;具有弱的磷脂酶A2活性,无毒,也无溶血和出血毒活性.     

Selective Fractionation of Maize Sperm Cells by RCA I Sepharose 6MB Affinity Chromatography

RCA I-Sepharose 6MB was employed for affinity chromatography of maize ( Zea mays L. ) sperm cells. The result indicates that the loading capacity of 0.1 mL RCA I-6MB is about 1.5 × 10 . If the amount of sperm cells is less than this capacity, about 70% of the sperm cells specifically bind to RCA I-6MB. 42.1% of the absorbed sperm cells can be released from RCA I-6MB in 0.2 mol/L galactose and recovered with less impurities and with some viability, which will facilitate further study on plant sperm cells. It remains to be elucidated whether there are two types of maize sperm cells different from each other in monosaccharide residues on their plasma membranes.     

Specific binding of sulfated proteoglycans to concanavalin A - Sepharose 4B.

More than 90 % of [³⁵S]proteoglycans isolated from the secretions of human skin fibroblasts bind to Concanavalin A-Sepharose 4B (Con A-Sepharose) in the presence of 1 M NaCl. Above pH 5.0 1 M concentrations of methyl-α-D-mannoside and other haptenic inhibitors for Con A-sugar interaction prevent binding of [³⁵S]proteoglycans, whereas equimolar concentrations of non-haptenic carbohydrates do not effect binding. Below pH 5.0 [³⁵S]proteoglycans bind to Con A-Sepharose in the presence of both methyl-α-D-mannoside and galactose. About 60 % of the proteoglycans bound at pH 4.0 are eluted at pH 7.5 in the presence of 1 M methyl-α-D-mannoside. [³⁵S] Glycosaminoglycans prepared from [³⁵S] proteoglycans do not bind to Con A-Sepharose in the presence of 1 M NaCl.These results indicate a [³⁵S]proteoglycan-Con A interaction via the protein core of the proteoglycan and the sugar binding sites of Con A.