Purification of Meerrettich-peroxidase by means of affinity chromatography on concanavalin A-agarose]

Crude preparations of horse-radish peroxidase were purified by means of affinity chromatography on Concanavalin A-agarose. The peroxydase was bound to Concanavalin A, whereas the majority of other proteins of the preparation pass through the column. Subsequently the peroxidase was eluted by means of 1 M sucrose with high purity. The purified enzyme is convenient for the immunoenzyme technique.     

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.     

Rapid purification by affinity chromatography of rat brain pyridoxal kinase and pyridoxamine-5-phosphate oxidase

Rat brain pyridoxal kinase and pyridoxamine 5′ phosphate oxidase have been purified to electrophoretic homogeneity using pyridoxyl Sepharose and phosphopyridoxyl Sepharose columns as the first stages in the purification procedures. These affinity supports were synthesized by two simple steps consisting of reacting pyridoxal and pyridoxal 5′ phosphate with commercially available ω-aminohexyl Sepharose and subsequent reduction of the resultant Schiff's bases with sodium borohydride. This method allows total purification of both enzymes from the same tissue source in 4–5 days.     

Use of dye affinity chromatography for the purification of Aerococcus viridans lactate oxidase

Lactate oxidase was purified from Aerococcus viridans (A. viridans) by dye affinity chromatography and FPLC ion exchange chromatography. The lactate oxidase could be purified by comparatively simple procedures, the purification achieved from a crude extract of A. viridans was 41-fold with a specific activity of 143 units/(mg of protein). The purified enzyme was a L-lactate oxidase, which catalyses the conversion of L-lactate in the presence of molecular oxygen to pyruvate and H(2)O(2). This purified lactate oxidase showed an apparent molecular mass of 48,200 in SDS-PAGE and the native molecular weight, as estimated by FPLC gel filtration, was 187,300. This molecular weight indicates that lactate oxidase exists in tetrameric form after gel filtration. To differing degrees, all the triazine dyes tested were inhibitors of lactate oxidase, solutions of free triazine dyes showing an inhibition mechanism which was both time- and pH-dependent.     

The purification of Aeromonas aminopeptidase by affinity chromatography

A competitive inhibitor for Aeromonas aminopeptidase has been prepared from the bromomethyl ketone derived from t-butyloxycarbonyl-l-leucine and successfully coupled to aminomethyl cellulose to form an adsorbent for affinity chromatography. The blocked form of the inhibitor was coupled to aminomethyl cellulose and then deblocked in aqueous trifluoroacetic acid to yield an insolubilized analog of an NH₂-terminal l-leucyl residue. This material was effective in binding the aminopeptidase and separating it from a contaminating endopeptidase, which has a similar isoelectric point and size. Separation of the aminopeptidase and endopeptidase was shown to be due to the specificity of the affinity adsorbent for the aminopeptidase, inasmuch as separation of the enzymes did not occur on a typical anion exchange column or on a hydrophobic column lacking a free amino group.     

Partial purification of beta-N-acetylhexosaminidase A by affinity chromatography

A glycopeptide derived from bovine nasal septum by sequential treatment with trypsin, chymotrypsin, 0.05N HCl in dry methanol (desulfation), testicular hyaluronidase and β-glucuronidase, was coupled to Sepharose-4B in the presence of cyanogen bromide. β-$\text{N}$-Acetylhexosaminidase A was selectively retarded when crude extracts of human skin fibroblasts or liver were applied to the affinity column and was subsequently eluted with 0.1% Triton X-100 in 0.1M citrate-phosphate buffer pH 4.4, providing a simple method for purification.     

Human lysosomal beta-glucosidase: purification by affinity chromatography

Two Sepharose-bound substrate analogs, 6'-aminohexanoyl-(2-N-sphingosyl-O-beta-D-glucoside) and 6'-aminohexyl-dodecanedioyl-1-(2-N-sphingosyl-1-O-beta-D-glu coside), were synthesized and used sequentially for the affinity purification of lysosomal beta-glucosidase (N-acyl-sphingosyl-1-O-beta-D-glucoside:glucohydrolase, EC 3.2.1.45). The capacities of these nondegradable affinity supports were 0.1 and 0.15 mg enzyme/ml settled gel, respectively. The purified enzyme had a specific activity of 75 mumol min-1 mg-1. The preparation had a single protein band with a molecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, evidencing its apparent homogeneity. Isoelectric focusing on granular gels revealed four molecular forms of the enzyme with pI values of 4.0, 4.5, 4.7, and 5.8 to 6.2. The purified enzyme hydrolyzed glucosyl ceramide and 4-methylumbelliferyl-beta-D-glucoside with Km and Vmax values of 0.6 and 2.5 mM, and 101 and 26.1 mumol min-1 mg-1, respectively. The enzyme also hydrolyzed octyl beta-glucoside, a linear mixed-type inhibitor of the enzyme. Binding constants (Ki) were determined for the inhibitors, sphingosyl-1-O-beta-D-glucoside (Ki = 20 microM) and its N-hexyl derivative (Ki = 0.3 microM). The enzyme had a half-life of 65 and 30 min at 50 degrees C and pH 5.0 or 6.0, respectively. In addition, two other classes of ligands were used for the purification of lysosomal beta-glucosidase, and their capacities and specificities were compared to those of the substrate analog affinity supports. These included (i) the alkyl amine inhibitors octylamine, decylamine, and tetradecylamine; and (ii) the inhibitors, 6-aminohexanoyl-beta-glucosylamine and aminododecanoyl-1-(2-N-sphingosyl-1-O-beta-D-glucoside). Compared to these other ligand columns, the substrate analog affinity supports had about 100- to 1000-fold greater capacities or afforded 8- to 40-fold greater purification of human lysosomal beta-glucosidase.     

Isolation and purification of morphine receptor by affinity chromatography

Brain membranes were solubilized by sonication and Triton X-100 extraction and applied to an affinity column consisting of a 6-succinyl morphine derivative of Affi Gel-102. A fraction exhibiting high opiate binding was eluted by tris-buffer containing naloxone, CHAPS and NaCl. This fraction consisted of both proteins and acidic lipids. The opiate binding properties of this purified material exhibited many properties similar to those of membrane bound receptors of the u-type, including high affinity, stereospecificity, Na-effect and rank order in affinity for opiates. This opiate binding material was highly sensitive to both trypsin and N-ethylmaleimide. Based on the protein content of the isolated membrane receptor, a 3200-fold purification over the original brain P2 fraction was achieved.     

The purification of beta-galactosidase-specific polysomes by affinity chromatography.

Affinity chromatography on a β-galactosidase substrate analog-Sepharose column was used to purify β-galactosidase-specific polysomes from E. coli. The purification was monitored by hybridization of [³H]uridine pulse-labeled RNA extracted from polysomes to p lac 5 DNA. A purification of at least 12-fold was obtained. Binding of lac polysomes to the column required the presence of Sepharose-bound substrate analog; salt and pH conditions favorable to β-galactosidase binding; and intact polyribosomes. It was calculated that 40–50% of the labeled mRNA recovered was lac RNA.     

Rapid partial purification of S-adenosyl-L-methionine decarboxylase by affinity chromatography

A Sepharose-ethylenediamine-PCMB column can be used to obtain a rapid purification of S-adenosyl-L-methionine decarboxylase. PCMB-affinity fractions from both rat liver and sea urchin eggs have high specific activity, particularly the latter. The activity of the purified rat liver enzyme is stimulated by the addition of either putrescine or spermidine, whereas the purified enzyme fraction from sea urchin eggs has no measurable activity without the addition of either putrescine or spermidine. In both preparations there is a stoichiometric relationship between the release of ¹⁴CO2 from S-adenosyl-L-carboxyl-¹⁴C-methionine and the formation of spermidine.     

Trans-N-deoxyribosylase: purification by affinity chromatography and characterization.

trans-N-Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans-N-deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively: trans-N-deoxyribosylase-I, the other catalyzes the transfer to and from pyrimidine and purine bases: trans-N-deoxyribosylase-II. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine-specific enzyme is a polymeric enzyme of molecular weight 86 000 (+/- 4000).     

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.     

Rapid purification of dehydrogenases by affinity chromatography with ternary complexes

The rapid purification of dehydrogenases by a modification of affinity chromatography was investigated. A ternary complex enzyme-NAD(H)-inhibitor (E-NADH-I) was formed by the addition of coenzyme and a substrate-competitive inhibitor to the dehydrogenases initially separated from nondehydrogenases by an NAD-affinity column. The enzyme in the ternary complex cannot rebind to the NAD-agarose column in the presence of inhibitor. As all other dehydrogenases do, this yields a highly purified enzyme-inhibitor complex. Aldehyde dehydrogenases in the presence of chloral hydrate and alcohol dehydrogenase with pyrazole were purified as their E-NAD⁺-I ternary complexes, while lactic dehydrogenase in the presence of oxamate was purified as the E-NADH-I complex. This technique allows for the rapid separation of a specific dehydrogenase from other dehydrogenases. The technique should be applicable to the purification of other enzymes exhibiting ordered sequential binding.     

Enhanced purification of Mullerian inhibiting substance by lectin affinity chromatography

Mullerian inhibiting substance (MIS), a secreted testicular product responsible for regression of the Mullerian ducts in the male mammalian embryo, was purified 7000 fold, exploiting the glycoprotein nature of this important fetal regressor to achieve enhanced purification. The present procedure employs media incubation of newborn calf testis, passage through DEAE Bio-Gel A and CM Bio-Gel A and sequential lectin affinity chromatography on wheat germ lectin (WGL)-Sepharose 6MB and concanavalin A (Con A)-Sepharose 4B. Strongly bioactive MIS was released from both lectin columns in the bound glycoprotein fraction only after elution with lectin-specific sugar. Carbohydrate analysis of the highly purified glycoprotein fraction eluted from Con A indicated the presence of both N-acetyl glucosamine and mannose, as would be expected from its sequential lectin affinity, as well as of galactose, galactosamine and N-acetyl neuraminic acid. Electrophoresis of this fraction on polyacrylamide-SDS gels showed an identical band pattern after staining with either Coomassie blue or periodic acid-Schiff reagent, further indicating that MIS is a glycoprotein.