Coimmobilization of Lactate Dehydrogenase and Low-Molecular-Weight Thiols on Sepharose

Lactate dehydrogenase (EC 1.1.1.27) and dithiothreitol (DTT) were coimmobilized on Sepharose activated with cyanogen bromide. It was demonstrated that the addition of 10 mM DTT (but not 2-mercaptoethanol) during immobilization increased the enzyme specific activity 1.5–5-fold depending on the initial extent of Sepharose activation by cyanogen bromide. The total activity increased two- to threefold. The lactate dehydrogenase preparations were rich in matrix-immobilized sulfhydryl groups (1.8–13.0 nmol per ml gel). The presence of DTT increased the stability of immobilized lactate dehydrogenase.     

General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide

1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD⁺-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD⁺–Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD⁺ was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP–Sepharose, N⁶-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD⁺ yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP–Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0–0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP–Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD⁺); 80 (lactate+NAD⁺); 95 (lactate+semicarbazide+NAD⁺); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP–Sepharose.     

Structural organization of glutamate dehydrogenase hexamer. 1. Immobilization on sepharose as a model for analysis of structural-functional characteristics of the enzyme

Bovine liver glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3) and its radioactive phosphopyridoxyl derivative were covalently immobilized on Sepharose CL-4B with different degrees of cyanogen bromide activation. The catalytical and regulatory properties of the immobilized samples of the enzymes were studied. It was shown that the enzymes were immobilized through a single subunit of hexamer when sepharose was activated by small amounts of cyanogen bromide (less than 5 mg per 1 ml of gel). In this case, the immobilization did not alter the catalytical and regulatory properties of glutamate dehydrogenase. The immobilized radioactive phosphopyridoxyl derivative of glutamate dehydrogenase completely imitated the immobilized native enzyme and can be used as a convenient model for structural and functional investigation of catalytically active hexamer of glutamate dehydrogenase.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. III) The primary structure of thermophilic lactate dehydrogenase from Bacillus stearothermophilus. Hydroxylamine-, o-iodosobenzoic acid- and tryptic-fragments. The complete amino-acid sequence

Based on the partial sequence of the cyanogen bromide fragments [Tratschin, J.D., Wirz, B., Frank, G. and Zuber, H. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 879-892], the amino-acid sequence of thermophilic lactate dehydrogenase from B. stearothermophilus was completed by the preparation and sequencing (sequenator, carboxypeptidase A and Y) of further overlapping fragments. Suitable peptide fragments were obtained by lactate dehydrogenase cleavage with hydroxylamine, o-iodosobenzoic acid and trypsin. The polypeptide chain of thermophilic lactate dehydrogenase from B. stearothermophilus consists of 317 amino-acid residues. While sequence homology with mesophilic lactate dehydrogenase of higher organisms reaches 35%, it is substantially higher with this mesophilic enzyme of bacillae (greater than 60%, B. megaterium, B. subtilis). The secondary structure elements and amino-acid residues of the active site of thermophilic lactate dehydrogenase deducted from primary structure data were compared with those from the mesophilic enzyme, the same was done for the internal sequence homology at the nucleotide-binding units. A comparative structure analysis (matrix system) based on the primary structure data of thermophilic enzyme should provide insight into the characteristic structure differences between thermophilic and mesophilic lactate dehydrogenase.     

A new approach (cyano-transfer) for cyanogen bromide activation of Sepharose at neutral pH,which yields activated resins,free of interfering nitrogen derivatives

A new approach for the reaction of Sepharose with cyanogen bromide is described, using triethylamine as a “cyano-transfer” reagent. An optimized procedure for activation at neutral pH was developed. This procedure requires only about 5% of the usual amount of cyanogen bromide. Activated resins are free of imidocarbonates and carbamates, containing only active cyanate esters. Extremely high coupling capacities (75 μmol ligand/g wet Sepharose 4B) can be obtained using this method.     

Radiomodfication of xanthine oxidoreductase system in the liver of mice by phenylmethylsulfonyl fluoride and dithiothreitol

The widely distributed xanthine oxidoreductase (XOR) system has been shown to be modulated upon exposure of animals to ionizing radiation through the conversion of xanthine dehydrogenase (XDH) into xanthine oxidase (XO). In the present work, radiomodification of the XOR system by phenylmethylsulfonyl fluoride (PMSF) and dithiothreitol (DTT) was examined using female Swiss albino mice which were irradiated with gamma rays at a dose rate 0.023 Gy s(-1). PMSF, a serine protease inhibitor, and DTT, the sulfhydryl reagent, were administered intraperitoneally prior to irradiation. The specific activities of XDH and XO as well as the XDH/XO ratio and the total activity (XDH+XO) were determined in the liver of the mice. The inhibition of XO activity, restoration of XDH activity, and increase in the XDH/XO ratio upon administration of PMSF were suggestive of irreversible conversion of XDH into XO mediated through serine proteases. The biochemical events required for the conversion were probably initiated during the early phase of irradiation, as the treatment with PMSF immediately after irradiation did not have a modulatory effect. Interestingly, DTT was not effective in modulating radiation-induced changes in the XOR system or oxidative damage in the liver of mice. The DTT treatment resulted in inhibition of the release of lactate dehydrogenase. However, the protection appears to be unrelated to the formation of TBARS. On the other hand, the presence of PMSF during irradiation inhibited radiation-induced oxidative damage and radiation-induced increases in the specific activity of lactate dehydrogenase. These findings suggest that a major effect of ionizing radiation is irreversible conversion of xanthine to xanthine oxidase.     

Stability of nicotinamide adenine dinucleotide immobilized to cyanogen bromide activated agarose

The stability of NAD(H) immobilized to a crosslinked agarose support (Sepharose(R)-4B) was examined in buffer solutions at a pH of 7.0 and 8.5. Specifically, this study investigated particle attrition and ligand leakage rates from a cyanogen bromide activated agarose support. Particle attrition did not occur under the experimental conditions. Ligand leakage rates were found to be first order in immobilized ligand concentration with two labile populations of ligand. The two-population model is consistent with the cyanogen bromide coupling chemistry, which results in both an isourea and imidocarbonate ligand linkage. The rate of ligand leakage was found to occur over a time scale of days, with first order rate constants ranging from 0.007 to 0.15 d(-1), depending on solution pH. (c) 1997 John Wiley & Sons, Inc.     

Double-ternary complex affinity chromatography: preparation of alcohol dehydrogenases.

A general affinity chromatographic method for alcohol dehydrogenase purification has been developed by employing immobilized 4-substituted pyrazole derivatives that isolate the enzyme through formation of a specific ternary complex. Sepharose 4B is activated with 300 mg of cyanogen bromide/ml of packed gel and coupled to 4-[3-(N-6-aminocaproyl)aminopropyl]pyrazole. From crude liver extracts in 50 mM phosphate-0.37 mM nicotinamide adenine dinucleotide, pH 7.5, alcohol dehydrogenase is optimally bound at a capacity of 4-5 mg of enzyme/ml of gel. Addition of ethanol, propanol, or butanol, 500 mM, results in the formation of a second ternary complex, which allows the elution of bound enzyme in high yield and purity. This double-ternary complex affinity chromatography has been applied successfully to human, horse, rat, and rabbit liver extracts to isolate the respective homogeneous alcohol dehydrogenases.     

Properties of cellulase immobilized on agarose gel with spacer

Cellulase produced by fungus Trichoderma viride was immobilized on agarose beads (Sepharose 4B) activated by cyanogen bromide and also on activated agarose beads that contained spacer arm (activated CH-Sepharose 4B and Affi-Gel 15). The CMCase activity retained by immobilized cellulase on activated Sepharose containing the spacer tended to be higher than that immobilized without spacer, although the extent of protein immobilization was lower. Also, the higher substrate specificity for cellulase immobilized on beads with spacer was obtained for cellobiose, acid-swollen cellulose, or cellulose powder. The hydrolysis product from their substrates was mainly glucose.     

Properties of water-insoluble matrix-bound lactate dehydrogenase.

Lactate dehydrogenase enzyme was immobilized by binding to a cyanogen bromideactivated Sepharose 4B-200 in 0.1 m phosphate buffer, pH 8.5. The immobilized enzyme was found to have lower K_m values for its substrates. K_m values for pyruvate and lactate were 8 × 10 ^?5m and 4 × 10^?3m, respectively, an order of magnitude less than the value for the native (free) enzyme. Chicken heart (H₄) lactate dehydrogenase was found to lose nearly all its substrate inhibition characteristics as a result of immobilization. The covalently bound muscle-type subunits of lactate dehydrogenase showed more favorable interaction with the muscle type than with the heart type subunits. An increase in thermal and acid stability of the dogfish muscle (M₄) lactate dehydrogenase as well as a decrease in the percentage of inhibition of enzyme activity by rabbit antisera and in the complement fixation was observed as a result of immobilization. The changes in the properties of the enzyme as a result of immobilization may be attributable to hindrance produced by the insoluble matrix as well as conformational changes in the enzyme molecules.     

Interaction of lactate dehydrogenase with skeletal muscle troponin

Rabbit muscle troponin complex covalently bound to CNBr-activated Sepharose 4B was shown to interact with soluble lactate dehydrogenase with a stoichiometry of 2 mol lactate dehydrogenase/mol of troponin. The presence of Ca2+ influenced the strength of association (the KD values of 0.73 and 2.3 microM were determined in the presence of 200 microM EGTA or 100 microM Ca2+, respectively). In the absence of Ca2+, the affinity of lactate dehydrogenase to troponin was strongly pH-dependent, reaching a maximum in the region of pH 6.0-7.0. No change of catalytic activity was observed as a result of interaction between lactate dehydrogenase and troponin, the enzyme appeared capable of functioning in the bound form.     

Structural organization of glutamate dehydrogenase. 2. Inactivation and dissociation of immobilized hexamer induced by urea

The urea-induced inactivation and dissociation of catalytically active hexamer of glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3) from bovine liver were studied using radioactive phosphopyridoxyl derivative of the enzyme immobilized on cyanogen bromide-activated Sepharose CL-4B. It is shown that at neutral pH (7.0-7.8) urea causes dissociation of glutamate dehydrogenase to directly yield catalytically inactive immobilized monomers rather than hexamer's stable fragments at the same time. At pH 8.9 or 5.6 the urea-induced is accompanied by the formation of conformationally stable immobilized dimers or trimers, respectively. The trimers are catalytically active, whereas the dimers did not exhibit any enzymatic activity. The data obtained led to suggestion that the hexamer consists of three either equivalent dimers (3 alpha 2) or of two equivalent trimers (2 alpha 3).     

Purification of canine plasma renin by affinity chromatography.

An affinity column for the purfication of canine plasma renin was prepared using goat anti-renin (dog kidney) gammaG gloublins. The antiserum was prepared against a purified kidney renin preparation. The anti-renin globulins were coupled to cyanogen bromide activated Sepharose. Using the anti-renin globulin-coupled Sepharose as an immuno-adsorbant, a method was devised allowing purification of plasma renin to a 1,000-fold purity.     

Involvement of pyruvate dehydrogenase in product formation in pyruvate-limited anaerobic chemostat cultures of Enterococcus faecalis NCTC 775

Enterococcus faecalis NCTC 775 was grown anaerobically in chemostat culture with pyruvate as the energy source. At low culture pH values, high in vivo and in vitro activities were found for both pyruvate dehydrogenase and lactate dehydrogenase. At high culture pH values the carbon flux was shifted towards pyruvate formate lyase. Some mechanisms possibly involved in this metabolic switch are discussed. In particular attention is paid to the NADH/NAD ratio (redox potential) and the fructose-1,6-bisphosphate-dependent lactate dehydrogenase activity as possible regulatory factors.Abbreviations PDH pyruvate dehydrogenase complex (EC 1.2.2.2) - PFL pyruvate formate lyase (EC 2.3.1.54) - LDH lactate dehydrogenase (EC 1.1.1.27) - FBP fructose-1,6-bisphosphate - MTT 3-(4,5-dimethyl-thiazoyl-2)-2,5-diphenyltetrazolium bromide - TPP thiamine pyrophosphate     

PURIFICATION OF NERVE GROWTH FACTOR ANTIBODIES BY AFFINITY CHROMATOGRAPHY

Pure antibodies to nerve growth factor have been isolated from sheep nerve growth factorantiserum by affinity chromatography using 2.5 S nerve growth factor linked to Sepharose 4B by means of cyanogen bromide. The elution of the antibodies was accomplished either at low pH (pH 2) or by high salt concentration (4.5 wMgC12). The purity of the antibodies was established by SDS-gel electrophoresis. Their immunological activity was tested by imrnunoprecipitation and their biological activity in a tissue culture assay using embryonic chick dorsal root ganglia.     

Interaction between glyceraldehyde-3-phosphate-dehydrogenase and lactate dehydrogenase

Physical interaction between rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase was detected by means of matrix immobilization technique. Glyceraldehyde-3-phosphate dehydrogenase covalently bound to CNBr-activated Sepharose 4B was capable of forming a complex with soluble lactate dehydrogenase with a stoichiometry of 0.8 mole of lactate dehydrogenase per mole of glyceraldehyde-3-phosphate dehydrogenase and KD of 0.385 microM at pH 6.5. The bienzyme association weakened when pH changed to 7.0 (the KD increased to 1.25 microM).     

Enzyme activation by denaturants in organic solvent systems with a low water content.

The effect of urea and guanidine hydrochloride (GdmCl) on the activity of heart lactate dehydrogenase, glycerol-3-phosphate dehydrogenase, hexokinase, inorganic pyrophosphatase, and glyceraldehyde-3-phosphate dehydrogenase was studied in low-water systems. Most of the experiments were made in a system formed with toluene, phospholipids, Triton X-100, and water in a range that varied over 1.0-6.5% (by vol.) [Garza-Ramos, G., Darszon, A., Tuena de Gómez-Puyou, M. & Gómez-Puyou, A. (1990) Biochemistry 29, 751-757]. In such conditions at saturating substrate concentrations, the activity of the enzymes was more than 10 times lower than in all-water media. However the activity of the first four aforementioned enzymes was increased between 4 and 20 times by the denaturants. The most marked activating effect was found with lactate dehydrogenase; with 3.8% (by vol.) water maximal activation was observed with 1.5 M GdmCl (about 20-fold); 4 M urea activated, but to a lower extent. Activation by guanidine thiocyanate was lower than with GdmCl. The activating and inactivating effects of GdmCl on lactate dehydrogenase depended on the amount of water; as the amount of water was increased from 2.0% to 6.0% (by vol.), activation and inactivation took place with progressively lower GdmCl concentrations. When activity was measured as a function of the volume of 1.5 M GdmCl solution, a bell-shaped activation curve was observed. In a low-water system formed with n-octane, hexanol, cetyltrimethylammonium bromide and 3.0% water, a similar activation of lactate dehydrogenase by GdmCl and urea was observed. The water solubility diagrams were modified by GdmCl and urea, and this could reflect on enzyme activity. However, from a comparison of denaturant concentrations on the activity of the enzymes studied, it would seem that, independently of their effect on the characteristics of the low-water systems, denaturants bring about activation through their known mechanism of action on the protein. It is suggested that the effect of denaturants is due to the release of constraints in enzyme catalysis imposed by a low-water environment.     

Continuous regeneration of NAD(P)+ by flavins covalently bound to sepharose.

Various flavins, FMN, FAD, and acriflavin, were immobilized to Sepharose using several different coupling methods. The only product stable enough to permit extended studies was acriflavin coupled to epoxy-substituted Sepharose. The nonenzymic oxidizing capacity towards NAD(P) H was investigated and a 25% specific activity, compared to that of free acriflavin, was observed. The reduced acriflavin was immediately auto-reoxidized in air and could thus be reused. It was shown that acriflavin-Sepharose preparations function as NAD(P)H oxidizing agents in a number of different dehydrogenase systems including lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), alanine dehydrogenase (alaDH), and glutamate dehydrogenase (GDH). The amount of expensive coenzyme necessary for high product formation of such systems was thereby markedly reduced.     

The synthesis of 8-(6-aminohexyl)-amino-GTP and -GDP and their application as ligands in affinity chromatography

Two guanine nucleotide analogs, 8-(6-aminohexyl)-amino-guanosine-5′-triphosphate and 8-(6-aminohexyl)-amino-guanosine-5′-diphosphate, were synthesized from the monophosphate by phosphorylation with pyrophosphate or orthophosphate. Structural assignments were made according to nuclear magnetic resonance spectra. The ability of these nucleotides to act as enzyme substrates has been determined. Both guanosine nucleotides were used as ligands for affinity chromatography by attaching them to Sepharose 4B by the cyanogen bromide method. The effectiveness of these new affinity columns in enzyme purification was investigated with polypeptide elongation factor II from rat and pig liver.     

Evaluation of CNBr, FMP and hydrazide resins for immunoaffinity purification of factor IX.

The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation Factor IX to high levels of purity for therapeutic treatment of hemophilia B. The resin currently used in this process is Sepharose CL2B, a cross-linked 2% agarose, which is activated with cyanogen bromide to immobilize an anti-Factor IX monoclonal antibody. This study evaluated two alternative resins and coupling chemistries, a synthetic polymer bead activated by 2-fluoro-1-methyl-pyridinium toluene 4-sulfonate (FMP) and a cross-linked 2% agarose bead with free hydrazide groups for site-specific coupling. The cyanogen bromide and FMP chemistries immobilize the monoclonal antibody in a random orientation. In hydrazide coupling, the monoclonal antibody is immobilized by the non-antigen-binding part of the molecule which, theoretically, should increase the amount of immobilized monoclonal antibody able to bind antigen. To examine this, the capacity of the resins to bind Factor IX and the purity and recovery of Factor IX eluted from the resins were measured. The FMP-activated resin exhibited the lowest capacity, binding only 2% of the Factor IX feed. Sepharose CL2B bound 87% of the loaded protein, while the hydrazide resin bound 43%. These results suggest that (a) hydrazide activation may be insufficient to orient monoclonal antibody and (b) other factors such as steric hindrances and diffusional resistances during immobilization may be important. Neither of the other resins tested demonstrated improved performance compared with cyanogen bromide-activated Sepharose CL2B for the immunoaffinity purification of Factor IX.