Antifibrotic effects of aldehyde dextran modified superoxide dismutase in experimental silicosis]

The present paper dwells on biomedical study of aldehyde dextran modified superoxide dismutase. Pharmacokinetic data demonstrated that modification of superoxide dismutase increased its half-time. A rat model of experimental silicosis showed that aldehyde dextran modified superoxide dismutase inhibited evolving fibrosis in the lungs. The same dose of native enzyme produced no therapeutic effect. Thus, superoxide dismutase can be considered as a potential agent for treatment of fibrosis due to its modification.     

Comparative study of the properties of preparations of native and modified collagenase

Collagenase was gradually modified by aldehyde dextran and hyaluronidase. The modification increased enzyme stability and widened pH-optimum of its activity against specific and biological substrates. The modified complex with collagenolytic and hyaluronidase activity accumulated in the lungs of mice after intravenous injection. These results demonstrate its possible use for the treatment of lung disorders.     

Interrelationships among human aldo-keto reductases: immunochemical, kinetic and structural properties

We have proposed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of alpha subunits, aldehyde reductase I is a dimer of alpha, beta subunits, and aldehyde reductase II is a monomer of delta subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (alpha and beta) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (alpha subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li2SO4 (0.4 M) for the expression of maximum enzyme activity, and its Km for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li2SO4 and its Km for glucose is more than 200 mM. The beta subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The beta subunits hybridized with the alpha subunits of placenta aldehyde reductase I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristic properties of placenta aldehyde reductase I.     

Interrelationships among human aldo-keto reductase: immunochemical, kinetic and structural properties

We have propsed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α, β subunits, and aldehyde reductase II is a monomer of δ subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (α and β) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (α subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li₂SO₄(0.4 M) for the expression of maximum enzyme activity, and its K_m for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li₂SO₄ and its K_m for glucose is more than 200 mM. The β subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The β subunits hybridized with the α subunits of placenta aldehyde I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristics properties of placenta aldehyde reductase I.     

Activity of native and dextran-modified hyaluronidase in in vitro and in vivo systems

Modified hyaluronidase derivatives have been obtained. Covalent coupling of the enzyme with aldehyde dextran results in 65-85% protein binding to the carrier, residual catalytic activity accounting for 90-100% of the baseline. Modified hyaluronidase is more thermostable than the native enzyme. The data on intravenous drug distribution in the mouse organs are promising and ensure effective use of modified hyaluronidase for the treatment of pulmonary diseases.     

Formaldehyde dismutase, a novel NAD-binding oxidoreductase from Pseudomonas putida F61

A novel enzyme, formaldehyde dismutase, was purified and crystallized from the cell extract of an isolated bacterium, Pseudomonas putida F61. The enzyme catalyzes the dismutation of aldehydes and alcohol:aldehyde oxidoreduction in the absence of an exogenous electron acceptor. The enzyme is composed of four identical subunits with a Mr of 44 000. Each subunit contains 1 mol NAD(H) and 2 mol zinc/mol. The ratio of NAD+ and NADH in a crystalline preparation of the enzyme was about 7:3. The enzyme-bound coenzyme was completely reduced and oxidized on the addition of a large amount of an alcohol and an aldehyde respectively. Both the oxidized and reduced enzymes catalyzed the dismutation reaction to the same extent. Steady-state kinetics of the enzyme were investigated using an oxidoreduction reaction between an alcohol and p-nitroso-N, N-dimethylaniline. The enzyme obeys a ping-pong mechanism and is competitively inhibited by an alcoholic substrate analogue, pyrazole, but not coenzyme analogues, such as AMP, N-methylnicotinamide. These results indicate that NAD(H) binds firmly (but not covalently) at each active site. The enzyme-bound NAD(H) was reduced and oxidized only by the added second substrates, alcohol and aldehyde respectively, and not by exogenous electron acceptors [including NAD(H)].     

Complexes of glucose oxidase with chitosan and dextran possessing enhanced stability

Abstract

In this study, the different mole ratios of glucose oxidase/chitosan/dextran–aldehyde and glucose oxidase/chitosan/dextran–sulfate complexes were synthesized. The modification of glucose oxidase by non-covalent complexation with dextran and chitosan in different molar ratios was studied in order to increase the enzyme activity. The enzyme/polymer complexes obtained were investigated by UV spectrophotometer and dynamic light scattering. Activity determination of synthesized complexes and free enzyme were performed at a temperature range. The best results were obtained by C_chitosan/C_{dextran–aldehyde} = 10/1 ratio and C_chitosan/C_{dextran–sulfate} = 1/5 ratio that were used in thermal stability, shelf life, salt stress, and ethanol effect experiments. The results demonstrated that both complexes were thermally stable at 60?°C and had superior storage stability compared to the free glucose oxidase. Complexes showed higher enzymatic activity than free enzyme in the organic solvent environment using 10% ethanol. The complexes were resistant to salt stress containing 0.1?M NaCl or CaCl₂. The particle size distribution results of the triple complex evaluated the complexation of the chitosan, dextran derivative, and glucose oxidase. The average size of the triple complex in diameter was found to be 325.8?±?9.3?nm. Overall findings suggest that the complexes of glucose oxidase, chitosan, and dextran showed significant enhancement in the enzyme activity.     

An induced aliphatic aldehyde dehydrogenase from the bioluminescent bacterium, Beneckea harveyi. Purification and properties.

A NAD+-dependent aldehyde dehydrogenase, the activity of which induces at the same time as luceriferase, has been purified from the bioluminescent bacterium Beneckea harveyi, and its chemical and physical properties have been investigated. The purification is accomplished in three steps resulting in an enzyme preparation that gives a single protein band on three different gel electrophoresis systems. The molecular weight of the purified enzyme was estimated to be 120,000 by gel filtration. Sodium dodecyl sulfate-gel electrophoresis gave a molecular weight of 59,000 indicating that aldehyde dehydrogenase has a dimeric structure with subunits of similar molecular weight. The purified enzyme has a high specificity for long chain aliphatic aldehydes; the Michaelis constants for aldehydes decrease with increasing chain length as also observed for bacterial aldehyde dehydrogenases involved in the metabolism of hydrocarbons. The aldehyde specificity of the aldehyde dehydrogenase is similar to that of luciferase indicating that the functional role of the enzyme may be linked with the bioluminescent system.     

Resistance of Dextran-Modified Hyaluronidase to Inhibition by Heparin

Properties of native and aldehyde dextran-modified hyaluronidase (with surface amino group modification about 98%) were investigated. Optimal endoglycosidase activity of the native enzyme was observed at 0.15 M NaCl and pH 5.5 and electrostatic interactions influenced the enzyme activity. The inhibitory effect of heparin on hyaluronidase activity slightly differed at pH 5.5 (1.5-fold inhibition) and 7.5 (1.2-fold inhibition). Ionic strength of the reaction medium only slightly influenced the effect of heparin. Modification of hyaluronidase with dextran increased hydrophobic interactions and steric hindrance. Conjugation with dextran increased the resistance of hyaluronidase activity to denaturing agents (urea, guanidinium hydrobromide) and extended the optimal conditions for maximal endoglycosidase activity (pH 4.5-6.5, the range of NaCl concentration from 0.1 to 0.3 M). The conjugation also reduced electrostatic effects on the active site of hyaluronidase and efficacy of heparin inhibition. At pH 7.5 the enzyme was almost insensitive to heparin. The resistance of dextran-modified hyaluronidase to heparin points to approaches for subsequent studies of the heparin binding site of this enzyme and biomedical trial of the stabilized enzyme for the treatment of acute cardiovascular lesions.     

Immobilization of cycloisomaltooligosaccharide glucanotransferase for the production of cycloisomaltooligosaccharides from dextran

Immobilization of cycloisomaltooligosaccharide glucanotransferase (CITase) and its application in the production of cycloisomaltooligosaccharides (CIs) from dextran were studied. Among various carrier materials examined, the enzyme adsorbed physically on Chitopearl BCW-3505 showed the highest activity (1.75 U/ml carrier). The activity remaining was 35%. The maximum CI yield in batch reactions at 0.2, 2 and 10% dextran was 28, 24 and 12%, respectively. The maximum CI yield at 2% dextran (24%) was slightly less than that with the free enzyme under the same conditions (26%). The concentration of linear oligosaccharides, the byproducts in the reaction mixture, was greater with the immobilized CITase than the free enzyme. The immobilized CITase was less thermostable than the free enzyme by about 10 degrees C. The pattern of influence of Ca(2+) concentration on the thermostability differed between the free and immobilized CITase. A Ca(2+) concentration of 50-100 mM was optimum for the thermostability of the immobilized CITase, 10-50 mM for the free enzyme. CIs were produced continuously by a column system packed with the immobilized enzyme at 40 degrees C with a space velocity (SV) of 6 h(-1). The three quarters life time was 4 weeks. We think that relatively long life time at fast SV was accomplished and CI production cost by this method should be lower than the batch reaction. This is the first report on immobilization of CITase.     

High stabilization of immobilized Rhizomucor miehei lipase by additional coating with hydrophilic crosslinked polymers: Poly-allylamine/Aldehyde–dextran

Immobilized enzymes have a very large surface region which is not in contact with the support surface and, thus, have potential as a target for novel stabilization strategies. In this paper, coating the surfaces of such enzymes with a highly hydrophilic and compact cross-linked poly-aminated polymer as a strategy to increase the thermal stability of the immobilized enzymes is proposed. In particular, Rhizomucor miehei lipase (RML) was immobilized by interfacial adsorption onto octyl-agarose and further coated with poly-allylamine (PAA), a polymer that is very rich in primary amino groups. Cross-linking of the PAA layer to coat the immobilized enzyme was carried out, in situ, by reaction with freshly oxidized dextran (aldehyde–dextran). The PAA layer only exerted moderate stabilizing effects (around 4-fold), but further cross-linking with aldehyde–dextran highly increased the stabilizing effects; the new derivative was 440-fold more stable than uncoated derivative at 55 °C and pH 7 and exhibited 6-fold more catalytic activity compared to the soluble enzyme used for immobilization. We hypothesize that the hydrophilicity of PAA reduces the exposure of internal hydrophobic pockets to the enzyme surface at high temperatures. Besides, the compactness of the polymer may reduce distortion of the enzyme surface during inactivation.     

A new ether bond-splitting enzyme found in Gram-positive polyethylene glycol 6000-utilizing bacterium, <Emphasis Type="Italic">Pseudonocardia</Emphasis> sp. strain K1

Pseudonocardia sp. strain K1 is the only Gram-positive bacterium among the bacteria aerobically metabolizing polyethylene glycol (PEG). Generally, PEG is metabolized by an oxidative pathway in which a terminal alcohol group of PEG is oxidized to aldehyde and to carboxylic acid and then an ether bond is oxidatively cleaved. As the cell-free extract of Pseudonocardia sp. strain K1 has PEG dehydrogenase, PEG aldehyde dehydrogenase and diglycolic acid (DGA) dehydrogenase (DGADH) activities, all of which are constitutively formed, the strain has a metabolic pathway similar to that so far known. We purified an ether bond-splitting enzyme as DGADH. The molecular mass of the enzyme was estimated to be 55 kDa; and it consisted of two identical subunits. The enzyme oxidatively cleaved both an ether bond of PEG 3000 dicarboxylic acid and DGA. The N-terminal amino acid sequence of the purified enzyme had high homology with various superoxide dismutases and the enzyme had also superoxide dismutase activity. The atomic absorption spectrum showed that approximately one atom of Fe was included in each subunit of the enzyme. DGADH activity increased in the cells grown in a PEG medium supplemented with FeCl₃. Thus, we concluded that the enzyme purified from Pseudonocardia sp. strain K1 is a new ether bond-splitting enzyme.     

Studies on the oligomeric structure of yeast aldehyde dehydrogenase by cross-linking with bifunctional reagents.

The molecular w:ight of yeast aldehyde dehydrogenase determined by sucrose density gradient centrifugation was 207,000 +/- 13,000. The enzyme activity was proportional to the enzyme concentration in the range of 2 X 10(-11) M to 1 X 10(-7) M. Cross-linking patterns obtained with yeast aldehyde dehydrogenase after treatment with a series of diimidoesters of increasing chain lengths with different reaction times resulted in the appearance of tetramers as the largest cross-linked product of the enzyme subunits. The molecular weights of its monomer, dimer, trimer, and tetramer were, 57,000, 114,000, 171,000, and 228,000, respectively, as estimated from their mobilities on SDS-electrophoresis. In tetramers monomers are probably assembled in a heterologous square arrangement.     

Studies on cyclic GMP-dependent protein kinase properties by blue dextran-sepharose chromatography.

Cyclic GMP-dependent protein kinase prepared from calf lung was studied for its binding properties with blue dextran-Sepharose affinity column chromatography. Blue dextran competitively inhibited [³H]cGMP binding to the enzyme. ATP + Mg⁺⁺ did not prevent cGMP-kinase binding to blue dextran, nor did it facilitate the liberation of blue dextranbound enzyme. Substrate proteins such as histone and protamine dissociated the native enzyme into subunits. Considering all these results, cGMP-kinase seemed to conform with the “dissociation model” proposed for cAMP-kinase but with peculiarities of binding to blue dextran.     

Preparation and characterization of a dextran-amylase conjugate

Bacillus amyloliquefaciens alpha-amylase was attached to dextran after activation of the polysaccharide by using a modification of the cyanogen bromide method. The soluble dextran-amylase conjugate was purified by molecular-sieve chromatography. The conjugated enzyme has greater stability than the unmodified enzyme at low pH values, during heat treatment, and on removal of calcium ions with a chelating agent. Attachment of dextran to alpha-amylase did not alter the Michaelis constant of the enzyme acting on starch. The polysaccharide-enzyme conjugate probably consists of a cross-linked aggregate of many dextran and many enzyme molecules, in which a proportion of the enzyme molecules, although not inactivated, are unable to express their activity, except after dextranase treatment.     

Isolation and characterization of an aldehyde dehydrogenase encoded by the aldB gene of Escherichia coli

An aldehyde dehydrogenase was detected in crude cell extracts of Escherichia coli DH5alpha. Growth studies indicated that the aldehyde dehydrogenase activity was growth phase dependent and increased in cells grown with ethanol. The N-terminal amino acid sequence of the purified enzyme identified the latter as an aldehyde dehydrogenase encoded by aldB, which was thought to play a role in the removal of aldehydes and alcohols in cells that were under stress. The purified enzyme showed an estimated molecular mass of 220 +/- 8 kDa, consisting of four identical subunits, and preferred to use NADP and acetaldehyde. MgCl2 increased the activity of the NADP-dependent enzyme with various substrates. A comparison of the effect of Mg2+ ions on the bacterial enzyme with the effect of Mg2+ ions on human liver mitochondrial aldehyde dehydrogenase revealed that the bacterial enzyme shared kinetic properties with the mammalian enzyme. An R197E mutant of the bacterial enzyme appeared to retain very little NADP-dependent activity on acetaldehyde.     

Effect of chemical modification on thermal stability of horseradish peroxidase]

Soluble preparations of horse radish peroxidase are obtained by means of its amino groups modification with glutaric aldehyde, maleic anhydride and inert proteins including albumin. The enzyme activity is found to decrease under the modification with glutaric aldehyde and to be unchanged at all other cases. Thermal stability of the enzyme preparations obtained is studied within the temperature range from 56 to 80 degrees C. Thermostability of glutaric aldehyde-modified peroxidase is approximately 2.5-fold decreased at 56 degrees C. Thermostability of other preparations exceeds the stability of native peroxidase in 25--90 times at 56 degrees C. Thermodynamic parameters of activation for the process of irreversible thermoinactivation of native and modified enzyme are calculated. A strong compensation effect between activation enthalpy and entropy values is observed, which were changed in 1.5--2 times, while the free activation energy is changed by 2--3 kcal/mol only. Possible mechanism of the change of the enzyme thermal stability under its chemical modification is discussed.     

Purification and properties of betaine aldehyde dehydrogenase from Xanthomonas translucens

Betaine aldehyde dehydrogenase from Xanthomonas translucens was purified to apparent homogeneity by ammonium sulfate fractionation, followed by ion-exchange, butyl-Toyopearl and gel filtration chromatography. The amino acid composition and the N-terminal sequence of 35 amino acid residues were determined. The enzyme was found to be a tetramer with identical 50 kDa subunits. Both NAD and NADP could be used as a cofactor for the enzyme and K_m values for NAD and NADP were 70 μM and 50 μM, respectively. The enzyme was highly specific for betaine aldehyde and the K_m value for betaine aldehyde was 0.19 mM.     

DNA-methyltransferase SsoII as a bifunctional protein: Features of the interaction with the promoter region of SsoII restriction-modification genes

DNA duplexes bearing an aldehyde group at the 2'-position of the sugar moiety were used for affinity modification of (cytosine-5)-DNA methyltransferase SsoII. It is shown that lysine residues of M.SsoII N-terminal region are located in proximity to DNA sugar-phosphate backbone of a regulatory sequence of promoter region of SsoII restriction-modification enzyme coding genes. The ability of the two M.SsoII subunits to interact with DNA regulatory sequence has been demonstrated by affinity modification using DNA duplexes with two 2'-aldehyde groups. Changes in nucleotide sequence of one half of the regulatory region prevented cross-linking of the second M.SsoII subunit. The results on sequential affinity modification of M.SsoII by two types of modified DNA ligands (i.e. by 2'-aldehyde-containing and phosphoryldisulfide-containing) have demonstrated the possibility of covalent attachment of the protein to two different DNA recognition sites: regulatory sequence and methylation site.     

NAD(P)+-independent aldehyde dehydrogenase from Pseudomonas testosteroni. A novel type of molybdenum-containing hydroxylase

Aldehyde dehydrogenase from Pseudomonas testosteroni was purified to homogeneity. The enzyme has a pH optimum of 8.2, uses a wide range of aldehydes as substrates and cationic dyes (Wurster's blue, phenazine methosulphate and thionine), but not anionic dyes (ferricyanide and 2.6-dichloroindophenol), NAD(P)+ or O2, as electron acceptors. Haem c and pyrroloquinoline quinone appeared to be absent but the common cofactors of molybdenum hydroxylases were present. Xanthine was not a substrate and allopurinol was not an inhibitor. Alcohols were inhibitors only when turnover of the enzyme occurred in aldehyde conversion. The enzyme has a relative molecular mass of 186,000, consists of two subunits of equal size (Mr 92,000), and 1 enzyme molecule contains 1 FAD, 1 molybdopterin cofactor, 4 Fe and 4 S. It is a novel type of NAD(P)+-independent aldehyde dehydrogenase since its catalytic and physicochemical properties are quite different from those reported for already known aldehyde-converting enzymes like haemoprotein aldehyde dehydrogenase (EC 1.2.99.3), quino-protein alcohol dehydrogenases (EC 1.1.99.8) and molybdenum hydroxylases.