Purification of L-glutamate decarboxylase from monkey brain

Glutamate decarboxylase (GAD) is an enzyme that synthesizes gamma-aminobutyrate (GABA), a major inhibitory neurotransmitter in the central nervous system. Post-translational modification of GAD, such as N-terminal blockage, phosphorylation-dephosphorylation, and palmitoylation, is an important factor in the biological activity of GAD. In order to address the significance of post-translational events on GAD, we thought it crucial to obtain a non-recombinant form of GAD. In this study, we attempted to isolate GAD protein from the monkey brain, a model animal close to the human that has not been studied. Monkey brain was homogenized, fractionated with ammonium sulphate, and applied to a series of chromatographic steps, including hydrophobic, ion-exchange, and gel filtration. Purified GAD showed a single band on SDS-PAGE, and the enzyme was found to have a molecular weight of 61,000 and exhibited 1,100 nmol/min/mg of specific activity. It had an optimal pH of 7 and optimal thermal stability at 40 degrees C.     

Purification of the sodium transport enzyme oxaloacetate decarboxylase by affinity chromatography on avidin sepharose

Aspartic acid can be covalently linked to yeast aspartyl-tRNA synthetase and to other proteins, in the absence of tRNA, under conditions where the synthetase activates the amino acid into aspartyl-adenylate, i.e., in the presence of ATP and MgCl₂. The linkage between aspartic acid and the protein is acid and alkali resistant; thus it is likely a peptide-like amide bond formed between the activated carboxylate group of aspartic acid and the primary amine function of the side chain of lysine residues.     

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     

Purification of orotate phosphoribosyltransferase and orotidylate decarboxylase by affinity chromatography on Sepharose dye derivatives.

Blue Dextran-Sepharose and Cibacron Blue F3GA-Sepharose (Blue Sepharose) were found to act as affinity adsorbents for orotate phosphoribosyltransferase (PRTase) and orotidine 5′-monophosphate (OMP) decarboxylase from bakers' yeast. Experiments with columns of Blue Dextran-Sepharose and partially purified preparations of the PRTase and decarboxylase revealed that both enzymes were selectively eluted by a low concentration (0.1–2 mm) of their respective substrate or immediate product. On the other hand, a much higher concentration (50–400 mm) of NaCl was required to displace these two enzymes from the above columns. Larger scale experiments showed that OMP decarboxylase in crude extracts was purified about 5700- and 6600-fold on Blue Sepharose using 0.5 mm OMP and 2 mm uridine 5′-monophosphate (UMP) as the eluting ligand, respectively. In contrast, orotate PRTase did not bind to Blue Sepharose unless crude extracts were first subjected to gel filtration. The resulting preparation of orotate PRTase, purified about sixfold with respect to cell-free extracts, was purified an additional 200- and 40-fold when the enzyme was eluted from Blue Sepharose with 0.5 mm OMP and 1 mm 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P), respectively. Blue Dextran-Sepharose, on the other hand, was found to provide a lower degree of enzyme purification and exhibited a lower sample-binding capacity. Samples of the PRTase and decarboxylase that had been purified about 200- and 6000-fold, respectively, on Blue Sepharose displayed a major protein band and one or more minor bands when subjected to polyacrylamide gel electrophoresis. Enzyme activity coincided with the major band in all cases.     

Purification of cobalt-activated acylase by affinity chromatography

alpha-Hydroxyisocaproyltyrosine (HyIc-Tyr-OH), a potent competitive inhibitor of the cobalt-activated acylase form 2, was synthesized. Its derivative, alpha-aminopentyl-HyIc-Tyr-OEt was coupled to cyanogen bromide-activated Sepharose 4B and was used for about 100-fold purification of the acylase from human liver by affinity chromatography. The preparation obtained did not show aminoacylase, aspartyl acylase or alanylarylamidase activities. The same chromatographic method was also applied to isolate form 2 of the serum acylase from patients with viral hepatitis and guinea pig placenta.     

Purification of thiogalactoside transacetylase by affinity chromatography

Thiogalactoside transacetylase, the product of the lacA gene of the lactose operon of Escherichia coli, has been purified by an improved procedure. The enzyme binds tightly to immobilized Cibacron Blue F3GA columns and can be eluted by potassium chloride in high concentrations. Final purification was obtained by affinity chromatography on an agarose-coenzyme A column followed by gel filtration.     

Purification of subtilisin by single-step affinity chromatography

The dye 4-(4-aminophenylazo)phenylarsonic acid was coupled to activated CH-Sepharose 4B and the resulting affinity matrix was shown to be highly efficient for the purification of subtilisin. A crystalline subtilisin was purified to homogeneity using this affinity chromatography procedure with a purification fold of 1.4 and with an enzyme activity yield of 98%. Similarly subtilisin from a crude enzyme preparation was purified to 211 fold by this single step procedure with 94% recovery of the enzyme activity. The purified enzymes were shown to be homogeneous by polyacrylamide gel electrophoresis.     

Purification of phosphotransacetylase by affinity chromatography.

Phosphotransacetylase from Clostridium kluyveri was purified using a C⁸-(6-aminohexyl)-amino-desulfo-coenzyme A-Sepharose column. The method of synthesis of the affinity matrix is described. A crude extract was treated with ammonium sulfate and chromatographed on the desulfo-coenzyme A-Sepharose column. Using this method the enzyme was purified 83-fold and was found to be 73% pure. A new method for the determination of the purity of phosphotransacetylase by activity staining of polyacrylamide gels with 5,5′-dithiobis(2-nitrobenzoic acid) is described.     

Purification of cofactor-dependent enzymes by affinity chromatography

Several 8-(6-aminohexyl)-amino adenine nucleotide derivatives, including ATP, 2′,5′-ADP, 3′,5′-ADP and desulfo-CoA (CoA, reduced coenzyme A), were prepared and immobilized on Sepharose by cyanogen bromide activation. 8-(6-Aminohexyl)-amino-ATP-Sepharose was found to exhibit good affinity for both NAD⁺-dependent dehydrogenases and kinases. Sequential biospecific elutions with NADH and ATP resulted in a good separation of dehydrogenases from kinases. As many as eight different dehydrogenases and kinases could be substantially purified from both porcine muscle and mouse kidney extracts by this new procedure. 8-(6-Aminohexyl)-amino-2′,5′-ADP- and −3′,5′-ADP-Sepharose were shown to exhibit good affinity for many NADP⁺-dependent dehydrogenases from yeast extracts and CoA-dependent enzymes, respectively. Purification of citrate synthases from pig heart and Eschericia coli extracts by means of these 8-substituted adenine nucleotide affinity columns was also presented.     

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.