Purification of isoleucyl-tRNA synthetase from Methanobacterium thermoautotrophicum by pseudomonic acid affinity chromatography

The isoleucyl-tRNA synthetase of the archaebacterium Methanobacterium thermoautotrophicum was purified 1500-fold to electrophoretic homogeneity by a procedure based on affinity chromatography on Sepharose-bound pseudomonic acid, a strong competitive inhibitor of this enzyme. The purified enzyme is a monomer with a molecular mass of 120 kDa. In this respect and in its Km values for the PPi-ATP exchange, and aminoacylation reactions, it resembles the isoleucyl-tRNA synthetases from eubacterial and eukaryotic sources. Its aminoacylation activity is optimal at pH 8.0 and at 55 degrees C. Pseudomonic acid is a strong competitive inhibitor of the aminoacylation reaction with respect to both L-isoleucine (KiIle 10 nM) and ATP (KiATP 20 nM).     

Purification of rat liver asparagine synthetase by affinity chromatography on reactive blue 2-agarose

Studies on analysis of free animo acids using a support-coated, open-tube capillary column, and electron-capture detection or selective ion monitoring have been performed on samples from biological microenvironments. For most amino acids the detection limit was found to be less than 1 pg. The preparation of the support-coated open-tube capillary column is described as well as the gas-chromatographic conditions for direct injection and temperature-programmed separation of the N-heptafluorobutyryl iso butyl ester derivatives. Electron-capture detection and selected ion monitoring are compared with respect to linearity and sensitivity and the bases for the greater sensitivity of electron-capture detection compared with flame-ionization detection using halogenated derivatives is discussed. Applications of the gas-chromatographic method for analysis of free amino acids in environments deliberately chosen very small are demonstrated.     

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     

Neurospora crassa glutamine synthetase. Purification by affinity chromatography and characterization of subunit structure.

Neurospora crassa glutamine synthetase was purified to homogeneity by a procedure based on affinity chromatography. The enzyme is adsorbed to a matrix of anthranilic acid bound to Sepharose and eluted with AMP. Different experimental approaches indicate that the enzyme has an octameric structure formed by subunits of identical molecular weight.     

Purification of L-glutamate decarboxylase by affinity chromatography

L-Glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15) from rat brain synaptosomal extract was partially purified by affinity chromatography. On further purification by DEAE-Sephadex A 50 and Sephadex G-200, L-glutamate decarboxylase was purified to greater extent. It was found that a single affinity chromatography by appropriate elution gave a highly purified protein giving a single band of high specific activity on polyacrylamide gradient gel slab electrophoresis with minimal contamination. Substrate specificity of the purified enzyme was modified in the presence of 6-azauracil or phenylalanine resulting in decreased specificity to L-glutamate and increased specificity to L-aspartate.     

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.