Partial purification of plant NAD kinase by calmodulin-Sepharose affinity chromatography

Calmodulin-dependent NAD kinase has been purified more than 70fold from a crude plant (zucchini squash) homogenate by calmodulin-Sepharose affinity chromatography to a specific activity of 80 munits/mg protein. The enzyme could be activated about 8fold by calmodulin. Half-maximal activation was obtained with 6 ng of purified calmodulin from bovine brain. Together with NAD kinase other soluble plant proteins were retained specifically on the column. NaDodSo₄ polyacrylamide gel electrophoresis of the proteins which were retained by the calmodulin-Sepharose column revealed at least 7 to 8 bands. Most of the intensively stained bands on the gels obtained from the crude homogenate had disappeared.     

Partial purification of rat liver glucocorticoid binding proteins by affinity chromatography

Dexamethasone (9-fluoro-16α-methyl-116,17,21-trihydroxy-1,4-pregnadiene-3, 20-dione) binding proteins from rat liver cytosol were purified approximately 6470 fold by the use of an affinity column in which deoxycorticosterone was linked to CH-Sepharose 4B through a disulfide linkage. The receptor proteins were eluted from the column by washing with β-mercaptoethanol. A preliminary Sephadex G-200 filtration step of the cytosol was necessary in order to separate the dexamethasone binding proteins from other glucocorticoid receptors.     

Partial purification of rabbit hind brain tryptophan hydroxylase by affinity chromatography.

Chromatography of crude homogenates of rabbit hind brains on ε-amino caproyl-D-tryptophan methyl ester-agarose gels provide enzyme fractions with specific activity 7–10 times higher than the starting material. The activity was found to be associated with two distinct components. While nearly forty-fold increase in specific activity can be achieved by purification of the homogenate on calcium phosphate gels prior to affinity chromatography, only a single active component was noted in such prepurified extracts.     

Partial purification of guinea pig MIF by affinity column chromatography using macrophages.

First we have confirmed the previous observation that the macrophage migration inhibitory factor (MIF) was adsorbed on normal peritoneal macrophages when they were incubated at 4 C for 60 min. It was found that macrophages fixed with 2% glutaraldehyde gave more reproducible results than viable cells in terms of "adsorption" of guinea pig MIF. The adsorption was achieved more completely at 37 C than at 4 C, indicating that this reaction is a temperature-dependent phenomenon. Using these glutaraldehyde-fixed macrophages, a kind of cell-affinity column was successfully developed. The guinea pig MIF preparation lost its activity when it was passed through this affinity column, and MIF adsorbed on the column was recovered by elution with 0.1 M (L)-fucose of 0.1 M (D)-glucose. Such MIF active eluate was found to be at least 30--40 fold more pure than the original MIF preparation which had been previously fractionated according to its molecular weight. Therefore, this type of macrophage-affinity column may be useful for the purification of MIF.     

Partial purification of the nuclear estrogen receptor from chicken liver by affinity chromatography

The crude nuclear extract from the liver of estrogenized chickens contains 0.3–1 pmol/g tissue of the estrogen receptor. The receptor has been partially purified by ammonium sulphate precipitation and affinity chromatography on 17β-estradiol-17-hemisuccinyl-ovalbumin-Sepharose 4B. A 12% pure receptor preparation (2700-fold purification) with a yield of 17% could be obtained. The partially purified receptor has retained most properties which it displayed in cruder preparations, e.g. the dissociation constant of 10^?9?10^?10 M, the hormone specificity and the sedimentation coefficient of 3.9 S. The size (Stokes radius, 2.9 nm; molecular weight, 49 000) and the asymetry (f/f₀ = 1.10) of the receptor molecule, however, appear slightly reduced after the purification.     

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.     

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.     

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.     

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.     

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.     

A two-component affinity chromatography purification of Helix pomatia arylsulfatase by tyrosine vanadate

The inhibition of Helix pomatia arylsulfatase by the synergistic combination of N-acetyl-l-tyrosine ethyl ester and vanadate has been extended to affinity chromatography for purification. In the presence of vanadate, l-tyrosine ethyl ester (TEE), immobilized on CH-Sepharose 4B retained arylsulfatase from the digestive juice or lyophilized powder of H. pomatia. No enzyme was retained without vanadate or with arsenate or phosphate. Arylsulfatase was eluted from the column matrix by removing the vanadate to less than 50 microM with buffer containing EDTA to chelate the vanadate. Escherichia coli alkaline phosphatase and potato acid phosphatase, two enzymes which are inhibited by vanadate but not by the vanadate-TEE complex, were not retained by the immobilized TEE under any conditions used. The sulfatase activity was completely separated from contaminating glucuronidase activity present in the crude enzyme extracts. The Ki for the immobilized vanadate-TEE system was found to be 5.0 x 10(-7) M with a capacity of 25 mg/ml swollen gel. A purification of greater than 40-fold from the lyophilized powder of H. pomatia (Sigma Type H-5) was achieved using this technique. The Ki/Keq of other phenols with vanadate were determined in a 96-well plate format as an example of a rapid screening technique that could be extended to other phosphoryl and sulfuryl-transfer enzyme classes.     

A single-step purification of rat pancreatic and salivary amylase by affinity chromatography

Optimal conditions for the conjugation of carboxyl groups on low molecular weight molecules to reactive amino groups on rabbit immunoglobulin G (IgG) using a modified carbodiimide reaction have been investigated. Reaction of [¹⁴C]hippuric acid with N-ethyl-N′-(dimethylaminopropyl) carbodiimide at pH 5 followed by adjustment to pH 8 and coupling with rabbit IgG resulted in the formation of hippuric acid-IgG conjugates with less than 10% intra- and intermolecular IgG crosslinking. More than 93% of the bonds linking hippuric acid to IgG were stable to hydroxylamine hydrolysis, indicating the peptide properties of these bonds. This two-step process permitted a defined number of hippuric acid moieties to be loaded onto a single IgG molecule and should provide a useful method for the conjugation of molecules containing carboxyl groups to amino groups on a variety of polypeptides.     

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).     

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.