A novel affinity column for purification of glycerol phosphate dehydrogenase

An affinity column was synthesized and utilized to partially purify glycerolphosphate dehydrogenase (L-glycerol-3-phosphate: NAD⁺ oxidoreductase, E.C.1.1.1.8) from rat skeletal muscle. The novelty of the column resides in the fact that the ligand used, 6-phosphogluconic acid, is neither an inhibitor nor a substrate of the enzyme when free in solution but when immobilized on an agarose matrix, glycerol phosphate dehydrogenase binds to it with a high degree of specificity. The bound enzyme could be eluted by either increasing the ionic strength or by addition of its natural substrate, α-glycerol phosphate. Using a combination of these methods and ammonium sulfate precipitation GPDH was purified about 250 fold with a 75% yield within 24 hours.     

Mode of action of endopolygalacturonase immobilized by adsorption and by covalent binding via amino groups

Action pattern of endopolygalacturonase (E.C.3.2.1.15) immobilized by adsorption on porous powdered poly(ethyleneterephthalate) and covalently bound via amino groups on poly(2, 6-dimethyl-p-phenyleneoxide) and poly(6-caprolactame), respectively, were investigated in suspension and packed columns using polymeric and oligomeric D-galactosiduronates as substrates. The covalent binding invariably led to a lowering of randomness of degradation of high-molecular substrates and loss of specificity of (3 + 1) splitting of tetra(galactosiduronic acid), typical of the free enzyme. In the adsorbed endopolygalacturonase the degree of randomness of degradation of D-galacturonan and K(m,app) value were dependent on the substrate transfer; the former parameter increased, the later decreased with increasing flow-rate of the substrate through the immobilized enzyme bed. The action pattern on low-molecular substrates was not altered. The changes in action pattern of the covalently immobilized endopolygalacturonase are ascribed to sterical limitations resulting from a binding of the enzyme molecule in the proximity of its active site. In endopolygalacturonase bound to the support by hydrophobic interactions external diffusion effects are regarded the factors governing the enzyme action.     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

Affinity chromatography of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase.

Purified glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis bound to affinity adsorbents containing immobilized adenine nucleotides. Although the enzyme probably bound via an allosteric site at which AMP acts most effectively, 50 times more enzyme was bound by N⁶-(aminohexyl)-ATP-agarose than by N⁶-(aminohexyl)-AMP-agarose. The enzyme could be efficiently and specifically eluted from N⁶-(aminohexyl)-ATP-agarose with the substrate phosphoribosylpyrophosphate, which antagonizes AMP inhibition in kinetic experiments. Elution could also be effected by 0.5 m KCl or by chelation of Mg²⁺ ions. The usefulness of these techniques in purification of partially purified amidotransferase was demonstrated.     

Regulation of bound peroxidase by polyamines and guanidines in maize scutellum

Peroxidase bound to the membrane either ionically or covalently, but not the free enzyme, is inhibited by polyamines and activated by guanidines. The ionically bound peroxidase detached from the membrane by Ca²⁺, or the peroxidase present in the cytosolic fraction, can be associated with the membrane fraction from which the ionically bound enzyme is removed, by Ca²⁺. The reconstituted membrane fraction, either with the enzyme solubilized by Ca²⁺, or with the cytosolic enzyme, can again be modulated by these compounds by changing the affinity of the enzyme for its substrate.     

Cobra venom phospholipase A2 immobilized to porocus glass beads.

Phospholipase A₂ (EC 3.1.1.4) from cobra venom (Naja naja naja) has been covalently immobilized to aryl amine porous glass beads by diazo coupling. The attachment of the enzyme to the glass beads is apparently through tyrosine. The activity of the immobilized enzyme toward phospholipid substrate has been monitored using the Triton X-100/phospholipid mixed micelle assay system. The activity of the immobilized phospholipase A₂ toward phosphatidylcholine is about 160 μmol min^?1 ml^?1 of glass beads, and the specific activity is about 13 μmol min^?1 mg^?1 of protein in this assay system. The pH rate profile and apparent pK_a in 10 mm Ca²⁺ of the immobilized enzyme parallels that of the soluble enzyme. The substrate specificity of the immobilized enzyme toward individual phospholipid species in mixed micelles is phosphatidylcholine ? phosphatidylethanolamine. In binary lipid mixtures in mixed micelles containing phosphatidylcholine and phosphatidylethanolamine together, a reversal in specificity is observed, and phosphatidylethanolamine is the preferred substrate. This unusual specificity reversal in binary mixtures is also observed for the soluble enzyme. The activity of the immobilized enzyme toward phospholipid inserted in mixed micelles is the same as toward a synthetic phospholipid which forms monomers, while a 20-fold decrease in activity toward monomeric substrate is observed for the soluble enzyme. The immobilized enzyme is stable at temperatures of 90 °C as is the soluble enzyme. However, p-bromphenacyl bromide, a reagent which inactivates the soluble enzyme, does not inactivate the immobilized enzyme. The immobilized enzyme can be stored frozen for several months and is reusable. The mechanism of action of immobilized phospholipase A₂ from cobra venom and the potential usefullness of the bound enzyme as a probe for phospholipids in surfaces of membranes is considered.     

Immobilized isocrite dehydrogenase: some aspects of its catalytic, chemical and immunological properties

Isocitrate dehydrogenase from Azotobacter vinelandii has been immobilized on Sepharose 4B with an efficiency of between 60 and 75%. The immobilized enzyme is assayed by a flow technique which monitors a final steady state level of product formation. By the assay system described it is estimated that the immobilized enzyme retains between 30 and 40% of the catalytic activity of the free enzyme. Studies have been carried out on the substrate dependence of the enzyme. The enzyme requires magnesium ions with optimal concentrations of 10⁻³m and above. The dependence on isocitrate and TPN⁺ concentrations was determined and analyzed by double-reciprocal plots. The immobilized enzyme is inactivated by DTNB [5,5′-dithiobis(2-nitrobenzoic acid)] and reactivated by DTT (dithiothreitol). The DTNB-modified enzyme can be reactivated by potassium cyanide. Comparison of these reactions with those of the free enzyme suggest that the steric environment of the active site was not grossly altered by immobilization. Some supporting evidence is derived from the identity of the energies of activation, 16,600 cal/mole, of free and immobilized enzyme catalyzed oxidation of isocitrate. Furthermore, the immobilized enzyme is inactivated by antibody prepared against the free enzyme. The covalently attached enzyme is resistant to tryptic digestion except in the presence of 2 m urea. This suggests that exposed lysyl residues which may be the primary site of attack by trypsin are utilized in immobilization. Treatment of the enzyme with 2 m urea unfolds the enzyme to a conformation which has very little activity but which recovers full activity upon removal of the urea. Interaction of the enzyme with antibody suggest that the antibody reacts univalently. The second valence can be satisfied by addition of free enzyme. The free enzyme bound to the immobilized enzyme-antibody complex is active. Preliminary attempts to dissociate the enzyme-antibody complexes have been unsuccessful.     

Studies on Horseradish Peroxidase Immobilized onto Arylamine and Alkylamine Glass

Peroxidase from horseradish has been immobilized onto zirconia coated arylamine and alkylamine glass through the process of diazotization and glutaraldehyde coupling, respectively. Arylamine glass bound enzyme retained 77% of the initial activity with a conjugation yield of 18 mg g^-1 support, while alkylamine glass bound enzyme retained 38% of the initial activity with a conjugation yield of 16 mg g^-1 support. The immobilized enzyme showed an increase in optimum pH, temperature for maximum activity, energy of activation (Ea), and thermal stability but decrease in time for linearity and Km for H₂O₂. Vmax value of arylamlne conjugated enzyme decreased but Vmax of alkylamine conjugated enzyme was unaltered compared to free enzyme. Both arylamine and alkylamine bound enzyme showed higher stability in cold compared to that of free enzyme. The application of glass bound peroxidase in discrete analysis of serum urate is demonstrated.     

Inversion of sucrose with β-d-fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β-d-fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min^?1 g^?1. The inversion of sucrose with immobilized β-d-fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

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 ox heart mitochondrial F1-ATPase with immobilized ADP and ATP.

The reaction of mitochondrial F1-ATPase with immobilized substrate was studied by using columns of agarose-hexane-ATP. Mg2+ was required for binding of the enzyme to the column matrix. The column-bound enzyme could be eluted fully by ATP and other nucleoside triphosphates. Nucleoside di- and mono-phosphates were less effective. At a fixed concentration of nucleotide the effectiveness of elution was proportional to the charge on the eluting molecule. The ATP of the column matrix was hydrolysed by the bound F1-ATPase to release phosphate, probably by a uni-site reaction mechanism. Thus the F1-ATPase was bound to the immobilized ATP by a catalytic site. Treatment of the bound F1-ATPase with 4-chloro-7-nitrobenzofurazan prevented complete release of the enzyme by ATP. Only one-third of the bound enzyme was now eluted by the nucleotide. The inhibition of release could be due either to the inhibitor blocking co-operative interactions between sites or to its increasing the tightness of binding of immobilized ADP at the catalytic site.     

Some factors affecting the stability of glucoamylase immobilized on hornblende and on other inorganic supports

Glucoamylase from four different companies was studied: three had similar stability (half-life at 50°C about 140 hr); the fourth was less stable (half-life at 50°C about 20 hr). The immobilized enzymes were all less stable than their soluble counterparts: immobilized enzyme stability depended on the soluble enzyme used, the support, and method of immobilization. Thus enzyme bound to Enzacryl-TIO was less stable than enzyme bound to hornblende (metal-link method); this, in turn, was less stable than enzyme bound to hornblende by a silane–glutaraldehyde process. Bound enzyme stability was also improved by the presence of substrate or product (starch maltose or glucose). After 110 hr at 50°C in the presence of maltose (10% (w/v)) one preparation (a more stable soluble enzyme boul1d to hornblende by a silane–glutaraldehyde process) retained over 95% of its activity: activity loss was too low to permit the estimation of a half-life.     

Covalent linkage of CYP101 with the electrode enhances the electrocatalytic activity of the enzyme: Vectorial electron transport from the electrode

Direct electrochemical transfer of electrons to the enzyme provides an excellent method of driving the catalytic reactions of cytochrome P450 enzymes that form a superfamily of vital heme enzymes involved in biological monooxygenation reactions. Covalent attachment of N-(1-pyrenyl) maleimide (pyrene maleimide) to the bacterial cytochrome P450, CYP101 has been carried out and the conjugated enzyme was shown to be specifically immobilized onto the glassy carbon electrode through the pyrene group. The electrode immobilized pyrene-conjugated enzyme showed quasi-reversible electrochemistry with a midpoint potential at −330 ± 10 mV versus Ag/AgCl. The unconjugated enzyme that did not have specific linkage with the pyrene maleimide was non-specifically adsorbed on the electrode surface and the electrochemical response was much weaker than that observed in case of the conjugated enzyme, though the midpoint potential was almost unchanged. The pyrene maleimide bound CYP101 was found to have surface coverage of 1.35 ± 0.3 × 10⁻¹⁰ mol/cm² and the heterogeneous rate of electron transfer was found to be 0.21 ± 0.02 s⁻¹, which is larger than that for the unconjugated enzyme. The pyrene maleimide linked immobilized enzyme was oriented to the electrode so that efficient electron transfer takes place from the electrode to the immobilized enzyme. The oxygenase activity of the immobilized conjugated enzyme was assayed from the enhancement of catalytic current in presence of oxygen and the natural substrate camphor. Mass spectrometric studies also showed enhanced formation of hydroxycamphor by electrochemically driven catalysis in the pyrene maleimide linked immobilized CYP101.     

Properties of a 5'-AMP specific nucleotidase which accumulates in one cell type during development of Dictyostelium discoideum

5′-AMP nucleotidase activity accumulates during the culmination stage of development in a thin layer of cells at the prestalk-prespore interface of Dictyostelium discoideum. In this report we characterize a highly purified preparation of this enzyme in an attempt to determine the physiological significance of the accumulation and localization of the activity during cellular differentiation. A pH optimum of 9.5 was determined using nine different buffer systems tested over a range of pH from 3 to 13.5. The Michaelis constants for p-nitrophenylphosphate (NPP) and 5′-AMP were 1.8 and 1.2 mm, respectively. Substrate concentrations of 5′-AMP in excess of 2.5 mm were found to inhibit the activity. Little or no effect on the activity of the enzyme was observed in the presence of EDTA, Mg²⁺, Mn²⁺, Ca²⁺, Fe²⁺, or Zn²⁺ ions. However, the enzyme appears to be a zinc metalloprotein as evidenced by its inhibition with 1,10-phenanthroline and recovery of activity in the presence of zinc. Other inhibitors of enzymatic activity include dithiothreitol and imidazole. The enzyme was bound by calcium phosphate, but could not be immobilized on matricies containing other substrate or product analogs, including 5′-AMP, cyclic AMP, ATP, phenylalanine, blue dextran, and Procion Red HE3B. The hydrophobicity of 5′-AMP nucleotidase was demonstrated by its strong affinity for immobilized alkyl and ω-amino alkyl ligands, as well as phenyl Sepharose. Isoelectric focusing of the enzyme in granulated gel required both the presence of detergent to prevent aggregate formation and precipitation of the enzyme, and the addition of zinc after focusing to reverse Ampholine inhibition. Apparently, Ampholine chelates zinc away from the enzyme much like 1,10-phenanthroline. Using this method, the isoelectric point of 5′-AMP nucleotidase was found to be 4.5–4.9, with a 30% recovery of the applied activity.     

The binding of the retro-analogue of glutathione disulfide to glutathione reductase

The retro-analogue of glutathione disulfide was bound to the GSSG binding site of crystalline glutathione reductase. The binding mode revealed why the analogue is a very poor substrate in enzyme catalysis. The observed binding mode difference between natural substrate and retro-analogue is explained.     

Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody

A procedure is described which allows the characterization of enzyme by a hybrid approach using an enzyme and an antibody. The presented method is related to the affinity determination of antibodies by the 'affinity in solution' procedure for BlAcore. The antibody is used as an indicator for the concentration of substrate, which is also the antigen. A mixture of enzyme, substrate and antibody is incubated, and an aliquot of this solution is injected periodically into a flowcell containing immobilized substrate, which is bound by the antibody, but not cleaved by the enzyme. The chosen initial concentration of substrate inhibits the binding of antibody to the immobilized substrate by 90%. During the enzymatic reaction, increased amounts of antibody bind to the surface, as the substrate concentration is decreased. With this method, the cleavage of creatinine with creatinine iminohydrolase (6 mU/ml) was monitored for up to 11 h. A recently developed monoclonal antibody against creatinine was used as the indicating protein. For the calculation of enzyme activity, the signals were compared with a calibration curve for inhibition of antibody binding to the chip by creatinine in solution.     

Inversion of sucrose with β- -fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β- -fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min⁻¹ g⁻¹. The inversion of sucrose with immobilized β- -fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

The stabilization by a coenzyme analog of a conformational change induced by substrate in 6-phosphogluconate dehydrogenase.

The reaction of NADP⁺ with periodate yields a coenzyme analog that can be bound to the NADP⁺ binding site of 6-phosphogluconate dehydrogenase from Candida utilis. This coenzyme analog can be irreversibly bound to the enzyme by reduction with sodium borohydride. The binding of one molecule of inhibitor to only one of the two subunits of the enzyme causes the inactivation of this subunit but does not alter the catalytic activity of the other subunit. Thus the two subunits do not have apparent catalytic interactions. When the reaction between the enzyme and the coenzyme analog is carried out in the presence of the substrate, the covalent modification of only one subunit causes the inactivation of both subunits. In this case the two subunits show an extreme negative cooperativity. It is suggested that the binding of the substrate induces in the enzyme molecule a conformational change that is stabilized by the irreversible binding of the coenzyme analog.