亲和层析法分离胆固醇氧化酶

利用胆固醇氧化酶和底物胆固醇之间的亲和力,以胆固醇为吸附剂构成底物亲和层析柱.发酵液经盐析、透析后直接进行亲和层析.研究了洗脱液A和B的流速、洗脱液B中表面活性剂的浓度,确定了适合的层析条件,使比酶活从0.45U/mg提高到15.5U/mg.     

Purification of porcine enterokinase by affinity chromatography.

A method is described for the purification of porcine enterokinase by affinity chromatography with p-aminobenzamidine as the ligand. Purification was completed by immunoadsorption with antisera raised to components binding non-biologically to the gel. The final enterokinase preparation was 2.3 times more active than the most active preparation previously described.     

Purification of pigeon liver malic enzyme by affinity chromatography

A simple and rapid method for the purification of malic enzyme (EC 1.1.1.40) from pigeon liver is described. Malic enzyme in the crude tissue extract was partially purified by heat treatment, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Final purification was achieved by affinity chromatography on immobilized N⁶-(6-aminohexyl)-adenosine 2′,5′-bisphosphate. Apparently homogeneous enzyme was obtained in 2 days with 54% yield.     

Purification of human liver glycoprotein sialyltransferase by affinity chromatography

A simple procedure has been developed for purifying solubilized human liver glycoprotein sialyltransferase (EC 2.4.99.1) 16 000-fold in 4–5% yield. The procedure involves two centrifugation steps, affinity chromatography of the ultrasupernatant fluid on cytidine diphosphate-hexanolamine-agarose followed by gel filtration on Sephadex G-150. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the purified sialyltransferase preparation contains approximately equivalent amounts of three protein bands (with apparent molecular weights of 61 000, 63 000 and 70 000) and is highly purified if not homogeneous.     

Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.     

Purification of rat liver phenylalanine hydroxylase by affinity chromatography

Rat liver phenylalanine hydroxylase has been purified to homogeneity on a totally synthesized affinity matrix. The affinity matrix consisted of a succinylated diaminodipropylamine arm linked to Sepharose-4B, to which the cofactor, 6,7-dimethyl-5,6,7,8-tetrahydropterin, was covalently linked. The pure enzyme was eluted with buffered 50% ethylene glycol, 1 m KCl in one step after the 50% ammonium sulfate fraction of the rat liver homogenate was applied to the affinity column. Specific activities ranging from 1.4 to 3.0 units/mg of protein were obtained. The enzyme has been shown to be homogeneous by: (i) discontinuous gel electrophoresis, and (ii) sodium dodecyl sulfate gel electrophoresis. The subunit molecular weight was determined by the same technique and was calculated to be between 51,000 and 55,000.     

Purification of nucleosidediphosphatase of rat liver by metal-chelate affinity chromatography

A procedure is presented for the purification of nucleosidediphosphatase (nucleosidediphosphate phosphohydrolase, EC 3.6.1.6) of rat liver by affinity chromatography using metal conjugated to epoxy-activated Sepharose 6B. The enzyme is eluted from the conjugate by a solution of L-histidine. The enzyme, when bound to metal-chelate gel, is active in a suspended form, suggesting that the catalytic site is different from the site that binds to the metal-chelate gels. Substrate specificity and Km value of the enzyme obtained are similar to those of the enzyme obtained from the same sources by a conventional procedure, indicating that the metal-chelate affinity chromatography does not bring about any substantial change in the catalytic properties.     

Purification of galactose oxidase from Dactylium dendroides by affinity chromatography on melibiose-polyacrylamide

Galactose oxidase is a fungal enzyme which is known to oxidize the C-6 hydroxymethyl of galactose and galactosamine to an aldehyde group. It has been widely used in glycoconjugate research, for example in the labeling of asialoglycoproteins. We have developed a simple affinity purification for galactose oxidase using melibiose-polyacrylamide. This affinity procedure was used to purify the enzyme from ammonium sulfate precipitates of culture filtrates of Dactylium dendroides. The material containing proteases and other contaminants is eluted in the buffer wash. The galactose oxidase is then specifically eluted from the column with buffer containing 0.1 M D-fucose or D-galactose. Using this procedure, the enzyme was also purified from commercial samples of galactose oxidase which contain high proteolytic activity.     

Purification of a beta-D-galactosidase from bovine liver by affinity chromatography

A beta-D-galactosidase from bovine liver was purified to apparent homogeneity. The major purification step was affinity chromatography on a column of D-galactose attached to a Sepharose support activated with divinyl sulfone. Affinity media prepared by binding ligands to Sepharose activated with cyanogen bromide were unsuitable for purification of the enzyme, even though such media have been used to purify beta-D-galactosidases from other sources. The molecular weight of the denatured enzyme was 67,000. The molecular weight of the native enzyme at pH 7.0 was 68,000, and at pH 4.5 or 5.0, was 141,000. These data suggest that the enzyme has a single, fundamental subunit with a molecular weight of 67,000, and that the enzyme exists as a monomer at pH 7.0, and a dimer at pH 4.5 or 5.0. The Vmax values of the enzyme with p-nitrophenyl beta-D-galactoside, p-nitrophenyl beta-D-fucoside, lactose, and beta-Gal-(1----4)-beta-GlcNAc-1---- OC6H4NO2 -p were 10,204, 11,550, 9,479, and 8,859 nmol/min/mg of protein, respectively, and the Km values for these substrates were 0.08, 14.9, 14.2, and 1.6mM, respectively. D-Galactose, beta-D- galactosylamine , p-aminophenyl 1-thio-beta-D-galactoside, and D- galactono -1,4-lactone were competitive inhibitors of the enzyme, with Ki values of 0.9, 0.6, 0.6, and 0.8mM, respectively. The enzyme catalyzed the transfer of the D-galactosyl group from p-nitrophenyl beta-D-galactoside to D-glucose. The pH optimum of the enzyme was 4.5, and the pI was 4.7.     

Purification of cytoplasmic actin by affinity chromatography using the C-terminal half of gelsolin

A new rapid method of the cytoplasmic actin purification, not requiring the use of denaturants or high concentrations of salt, was developed, based on the affinity chromatography using the C-terminal half of gelsolin (G4-6), an actin filament severing and capping protein. When G4-6 expressed in Escherichia coli was added to the lysate of HeLa cells or insect cells infected with a baculovirus encoding the beta-actin gene, in the presence of Ca²⁺ and incubated overnight at 4 °C, actin and G4-6 were both detected in the supernatant. Following the addition of Ni-Sepharose beads to the mixture, only actin was eluted from the Ni-NTA column by a Ca²⁺-chelating solution. The functionality of the cytoplasmic actins thus purified was confirmed by measuring the rate of actin polymerization, the gliding velocity of actin filaments in an in vitro motility assay on myosin V-HMM, and the ability to activate the ATPase activity of myosin V-S1.     

Purification of flavin-containing liver polyamine oxidase and luminescent properties of the ferment

A flavin-containing polyamine oxidase was isolated in an electrophoretically homogeneous state from cattle liver cytosol. The molecular mass, subunit composition and flavin content of the enzyme were determined; flavin, was shown to be covalently bound to the protein fragment of the polyamine oxidase molecule. Some optical and luminescent properties of the native and denatured enzyme were investigated. Denaturation and quenching were found to affect the luminescent properties of polyamine oxidase.     

Purification and characterization of mouse liver xanthine oxidase

Xanthine oxidase (EC 1.1.3.22) is purified to homogeneity from mouse liver after induction with bacterial lipopolysaccharide. The enzyme has an apparent molecular weight of 300,000 in its native state and it is suggested to be constituted of two identical subunits of Mr 150,000 each. The isoelectric point is 6.7 and the apparent Km value for xanthine is 3.4 microM. The amino acid composition of mouse xanthine oxidase is quite similar to that of Drosophila xanthine dehydrogenase.     

Purification of ficin by affinity chromatography

The sulfhydryl proteinase ficin (EC 3.4.4.12) was purified by chromatography on an agarose-mercurial column. Two separate protein fractions were eluted, ficin and mercurificin, both exhibiting enzymatic activity upon activation by excess thiol.     

Purification of antibodies by affinity chromatography

This review focusses on affinity purification of immunoglobulins, a methodology which is a powerful tool to obtain pure and intact antibodies. Affinity techniques allow antibody purification both in a single step chromatographic procedure as well as in complex purification protocols depending on the intention to use the target antibody. The purification strategies for antibodies by interaction with affinity ligands such as antibodies and Fe receptors or low molecular weight compounds are described.     

Purification of alternanase by affinity chromatography

The enzyme alternanase, produced by Bacillus sp. NRRL B-21195, hydrolyzes alternan, a polysaccharide produced by certain strains of Leuconostoc mesenteroides that consists of glucose linked by alternating α(1→6), α(1→3) linkages. The main product of enzymatic hydrolysis by alternanase is a novel cyclic tetrasaccharide of glucose that also has alternating linkages between the glucose moieties. An improved purification scheme for alternanase has been developed that incorporates the use of isomaltosyl units linked to agarose for selectively binding the alternanase enzyme. Bound enzyme was eluted with 0.5 M sodium chloride and was nearly pure after this procedure. When followed by preparative isoelectric focusing, a single band of 117 kDa was measured when the purified protein was analyzed by HPLC size-exclusion chromatography/multiangle light scattering. The purification procedure can be scaled to permit large quantities of enzyme to be purified in high (36%) yield. Electronic Publication