Affinity chromatography of sn-glycerol-3-phosphate dehydrogenase on immobilized NAD

Three types of potential affinity chromatography columns have been examined for the purification of sn-glycerol-4-phosphate dehydrogenase (EC 1.1.1.8) from rabbit tissues. Each column contained nicotinamide adenine dinucleotide (NAD) covalently attached to an agarose matrix with a different mode of attachment for each column. The most effective column was one in which the NAD was linked to the agarose via the C-8 position of the adenine moiety. Release of the bound enzyme from this column was accomplished by elution with NADH or NAD. The enzymes from brain, heart, kidney, muscle and liver were purified using this procedure with nearly quantitative yields and up to a 90-fold purification. The binding capacity and elution profiles were dependent upon pH, ionic strength and temperature. The capacity was lowest at pH 7 and increased at higher and lower values. Increasing ionic strength and higher temperatures decreased the binding capacities.     

Affinity chromatography on immobilised nucleotides. The synthesis, specificity and applications of immobilised inosine 5''-monophosphate

In addition to the 2'-azido analogue of (I)n x (C)n, (dIn3)n x (C)n, we have found two other (I)n x (C)n analogues, (dIfl)n x (C)n and (dIcl)n x (C)n, in which the 2'-hydroxyls of the (I)n strand are replaced by either fluorine or chlorine, to be highly effective in inducing interferon. This contrasted with the lack of interferon-inducing activity noted for various other 2'-halogeno analogues of (I)n x (C)n and (A)n x (U)n, i.e. (I)n x (dCcl)n, (dAfl)n x (U)n, (dAcl)n x (U)n, (A)n x (dUfl)n and (A)n x (dUcl)n. In most assay systems, viz. primary rabbit kidney cells, human diploid fibroblasts, HeLa cells, interferon-primed mouse L-929 cells, and intact rabbits, (dIfl)n x (C)n and (dIcl)n x (C)n induced interferon levels that were comparable to those induced by (I)n x (C)n. There was one particular system (L-929 cells treated with DEAE-dextran), however, in which (dIfl)n x (C)n and (dIcl)n x (C)n, unlike (I)n x (C)n, failed to stimulate interferon production. As monitored by both radiochemical and biological means, (dIfl)n x (C)n and, to a lesser extent, (dIcl)n x (C)n were more resistant to degradation by ribonuclease A, T1 and human serum nucleases than was (I)n x (C)n. In their reactivity towards antibodies to double-stranded RNA (dIfl)n x (C)n and (dIcl)n x (C)n conformed more closely to (I)n x (C)n than did other 2'-substituted (e.g. 2'-O-methyl or 2'-O-ethyl) analogues of (I)n x (C)n. The high interferon-inducing potency of (dIfl)n x (C)n and (dIcl)n x (C)n has both theoretical and practical implications. While our findings suggest that (dIfl)n x (C)n and (dIcl)n x (C)n should be further explored for their therapeutic potentials, they also strengthen the notion that the interferon-inducing capacity, and possibly other biological functions of double-stranded RNAs is dependent on the recognition of the overall conformation of the polynucleotide rather than on the binding of specific functional groups such as the 2'-hydroxyl group.     

Affinity chromatography on immobilised triazine dyes. Studies on the interaction with multinucleotide-dependent enzymes

A systematic investigation into the interaction of several triazinyl dyes with two enzymes from purine metabolism, IMP dehydrogenase (IMP: NAD+ oxidoreductase, EC 1.2.1.14( and adenylosuccinate synthetase (IMP: L-aspartate ligase (GDP-forming), EC 6.3.4.4) has been conducted. Evidence from kinetic inhibition studies, enzyme inactivation with specific affinity labels and specific elution techniques from agarose-immobilised dyes indicate that triazine dyes such as Procion Blue H-B (Cibacron Blue F3G-A), Red HE-3B and Red H-3B are able to differentiate between the nucleotide-binding sites of these enzymes. This information has been exploited to design specific elution techniques for the purification of these enzymes by affinity chromatography.     

Affinity chromatography on immobilised nucleotides. Some applications to the purification of thermophilic dehydrogenases and kinases.

The effect of pH and temperature on the capacity and binding of Bacillus stearothermophilus, alcohol dehydrogenase and phosphofructokinase to N6-(6-aminohexyl)-5'-AMP-Sepharose has been examined. Specific elution from the substituted AMP-Sepharose was examined using a variety of cofactors, fragments of cofactors and substrates. A purification scheme for each enzyme on the substituted AMP-Sepharose using nucleotides and gradients of pH and salt is presented. Interestingly, elevated temperature increased the affinity of both enzymes for N6-(6-aminohexyl)-5'-AMP-Sepharose, however, the Michaelis constant for nucleotide determined at various temperatures remained constant. The effect of pH and salt concentration on the binding of B. stearothermophilus glyceraldehyde-3-phosphate dehydrogenase to 6-aminohexanoyl-NAD+-Sepharose was also examined; raising the pH above 7.5 lowers the capacity of the matrix and the effect of a range of ammonium sulphate concentrations on the adsorption of the enzyme was examined. A specific purification of glyceraldehyde-3-phosphate dehydrogenase from partially purified extracts of this organism was achieved.     

Affinity chromatography of lactate dehydrogenase on immobilized nucleotides

The interaction of two isoenzymes of lactate dehydrogenase from pig heart muscle (H(4)) and rabbit skeletal muscle (M(4)), with immobilized nucleotides was examined: the effects of pH and temperature on the binding of lactate dehydrogenase were studied with immobilized NAD(+) matrices. The influence of substrate, product and sulphite on the binding of heart muscle lactate dehydrogenase to immobilized NAD(+) was investigated. The interaction of both lactate dehydrogenase isoenzymes with immobilized pyridine and adenine nucleotides and their derivatives were measured. The effects of these parameters on the interaction of lactate dehydrogenase with immobilized nucleotides were correlated with the known kinetic and molecular properties of the enzymes in free solution.     

Affinity chromatography of β-hydroxybutyrate dehydrogenase on NAD and hydrophobic chain derivatives of sepharose

Partially purified beef heart apo-β-hydroxybutyrate dehydrogenase, which requires phospholipids for catalytic activity, binds to NAD covalently linked to Sepharose through 6-aminocaproic acid. The apodehydrogenase is denatured by free carboxyl groups covalently linked to Sepharose so that it is not possible to ascertain if the apoenzyme binds specifically to affinity columns made from Sepharose derivatized with β-hydroxybutyrate analogs. The apoenzyme, because of its hydrophobicity, is retarded on Sepharose columns to which various aminoacylaminoalkanes have been covalently attached. Affinity chromatography of the apodehydrogenase on NAD-Sepahrose and a combination of NAD-Sepharose and hydrophobic chain-Sepharose have been utilized to purify the enzyme.     

Affinity chromatographic separation of plant lactate dehydrogenase

Lactate dehydrogenase from potato tubers was purified by the use of several standard purification procedures as well as by affinity chromatography on C     

Affinity chromatography purification of Pseudomonas aeruginosa exotoxin A on specifically linked NAD agarose.

The specific binding of P. aeruginosa exotoxin A to NAD was exploited for the rapid purification of the toxin. Affinity chromatography on a column of agarose-N6-(aminohexyl)carbamoylmethyl-NAD resulted in an enzymatically, biologically, and immunologically active purified toxin preparation. Other NAD-agarose resins were not efficient substrates for toxin purification.     

Affinity chromatography of succinate dehydrogenase from the membranes of Micrococcus lysodeikticus.

Isolated plasma membranes of Micrococcus lysodeikticus were subjected to extraction with n-butanol in a two-phase system. Succinate dehydrogenase obtained in the soluble aqueous phase after high-speed centrifugation was resolved by separation on calcium phosphate gel and affinity chromatography. The affinity ligand used was oxaloacetate and elution from the column was achieved with 0.5 M succinate. In the final product there was an eleven-fold reduction in the 32P-lipid to protein ratio and a fourteen-fold increase in specific activity relative to the high speed supernatant fraction following n-butanol extraction.     

Immobilised lipoamide dehydrogenase. 3. Preparation and properties of an immobilised polythiolated enzyme.

After consideration of its electrophoretic behaviour, amino acid composition and phosphate content, bovine alpha s0 casein has been shown to differ from alpha s1 casein only in respect of its phosphate content. The presence in alpha s0 casein of one phosphate residue more than occurs in alpha s1 casein was confirmed by comparative degradative studies performed on both proteins. From these it was concluded that alpha s0 casein may be considered as being alpha s1 casein which has been modified by phosphorylation of the seryl residue located at position 41.     

Affinity chromatography of malic enzyme from grape berries

NADP-dependent malic enzyme from grape berries is associated with NAD-dependent malate dehydrogenase. A two step procedure, involving affinity chromatography on 2′,5′-ADP-Sepharose 4B, followed by gel- permeation on Bio-Gel A- 1.5 m, was used to separate malic enzyme from malate dehydrogenase and other proteins. The yield was ca 60% Malic enzyme and malate dehydrogenase migrated respectively as three bands and one band during disc electrophoresis in polyacrylamide gel. The MW resulting from gel-permeation was 220 000 for malic enzyme and 53 000 for malate dehydrogenase.     

Affinity isolation of a cold-adapted enzyme: lactate dehydrogenase from Bacillus psychrosaccharolyticus

A simple, economical and rapid affinity chromatography procedure with dyes as the ligand has been described for the one-step purification of a cold-adapted lactate dehydrogenase. Non-specific elution of Procion blue H-ERD-modified Sepharose yielded homogeneous preparations of lactate dehydrogenase both in column based procedures and in batch wise operations. Low operational temperatures resulted in the enhanced binding of the enzyme to the blue dye. The dissociation constants of the enzyme-dye complexes were 7.2 +/- 0.2 microM and 11.2 +/- 0.2 microM at 5 degrees C and 20 degrees C respectively.     

Affinity isolation of a cold-adapted enzyme: lactate dehydrogenase from Bacillus psychrosaccharolyticus

A simple, economical and rapid affinity chromatography procedure with dyes as the ligand has been described for the one-step purification of a cold-adapted lactate dehydrogenase. Non-specific elution of Procion blue H-ERD-modified Sepharose yielded homogeneous preparations of lactate dehydrogenase both in column based procedures and in batch wise operations. Low operational temperatures resulted in the enhanced binding of the enzyme to the blue dye. The dissociation constants of the enzyme-dye complexes were 7.2±0.2 M and 11.2±0.2 M at 5 °C and 20°C respectively.This revised version was published online in October 2005 with corrections to the Cover Date.