Catalytic activity of administered gulonolactone oxidase polyethylene glycol conjugates

Scurvy in guinea pigs provides a convenient model of inborn metabolic disease for the investigation of enzyme therapy protocols. Gulonolactone oxidase, the enzyme in ascorbic acid biosynthesis that is missing from the scurvy-prone species, was modified by attachment of polyethylene glycol. The catalytic properties of this enzyme were affected little by the modification. Intravenous injection of this modified form of the enzyme elicited ascorbic acid synthesis in a dose-dependent manner. The modified enzyme was stabilized to incubation at 37 degrees C but was not protected from inactivation by trypsin. The circulating half-life of enzyme activity was not prolonged by this modification. Further, attachment of polyethylene glycol did neither abolish the enzyme's ability to react with preformed antibodies nor eliminate its immunogenicity.     

Studies on oxalate oxidase from beet stems upon immobilization on concanavalin A.

Oxalate oxidase (EC 1.2.3.4) was purified from beet stems and immobilized on concanavalin A. The bound enzyme showed a high resistance of denaturation and increased the storage stability at 4 degrees C. The immobilized oxidase showed a broad optimum at pH 3.5-5, compared to the free enzyme with a sharp optimum at pH 4.5. There was a 3-fold increase in the apparent Km value on immobilization. The lectin interaction also eliminated the inhibitory effect produced on the enzyme by azide, nitrate and glycollate. The stimulatory effect on the enzyme activity by the flavins was not seen with the bound enzyme. The interaction of oxidase on concanavalin A-Sepharose 4B column and its reversal with methyl alpha-D-mannoside, indicated the presence of polysaccharides. The glycoprotein nature was further confirmed by periodic acid-sciff staining procedure of the enzyme after gel electrophoresis.     

Degradation of oxalate in rats implanted with immobilized oxalate oxidase

Accumulation of oxalate leads to hyperoxaluria and calcium oxalate nephrolithiasis in man. Since oxalate is a metabolic end product in mammals, the feasibility of its enzymic degradation has been tested in vivo in rats by administering exogenous oxalate oxidase. Oxalate oxidase, isolated from banana fruit peels, in its native form was found to be non-active at the physiological pH of the recipient animal. However, its functional viability in the recipient animal was ensured by its prior binding with ethylenemaleic anhydride, thus shifting its pH activity curve towards the alkaline range. Rats implanted with dialysis membrane capsules containing such immobilized oxalate oxidase in their peritoneal cavities effectively metabolized intraperitoneally injected [14C]oxalate as well as its precursor [14C]glyoxalate. The implantation of capsules containing coentrapped multienzyme preparations of oxalate oxidase, catalase and peroxidase led to a further degradation of administered [14C]oxalate in rats.     

Radioprotective effect of polyethylene glycol

Polyethylene glycol of molecular weight 400 (PEG-400) had a radioprotective effect of about 20% against lethality when given ip 20 min prior to single or fractionated X-ray doses to the head and neck. Dose modification factors (DMF) based on LD50/15 values ranged from 1.14 to 1.24. A similar DMF of 1.12 based on LD50/30 values was obtained using single doses of whole-body X irradiation. Mice given head and neck irradiation had significantly reduced rectal temperatures (31.3 +/- 3.0 degrees C) 9 days post irradiation compared with unirradiated controls (35.4 +/- 0.6 degrees C). No such reduction was observed when PEG-400 was given with radiation (36.3 +/- 0.9 degrees C). PEG-400 also lessened, but not significantly, the frequency of shivering in irradiated animals. Histopathologic examination of the oral structures demonstrated only marginal protection by PEG-400. Estimation of the alpha/beta ratio from LD50 data on head and neck-irradiated mice yielded values of 4.4 +/- 1.9 (95% confidence limits) Gy without PEG-400 and 7.9 +/- 1.4 Gy with PEG-400. Since it is a non-thiol radioprotector, PEG-400 may be more useful when combined with more conventional thiol-containing radioprotectors.     

Inducible or constitutive polyethylene glycol dehydrogenase involved in the aerobic metabolism of polyethylene glycol

2, 6-Dichlorophenolindophenol (DCIP)-dependent polyethylene glycol (PEG) dehydrogenase activity was found in the particulate fractions of cell-free extracts prepared from PEG-utilizing bacteria (Pseudomonas and Flavobacterium species). This result suggested that PEG dehydrogenase is linked to the respiratory chain of each bacterium and that the enzyme plays a major role in the aerobic metabolism of PEG. Enzyme activities were strongly inhibited by 1, 4-benzoquinone. No metal ion was indispensable for the enzyme activities. Enzyme activities of PEG-utilizing bacteria were induced by PEG except for the activity of PEG 4000-utilizing Flavobacterium sp. no. 203 which had a constitutive enzyme. Although PEG-utilizing bacteria had different growth substrate specificities toward PEGs 200–20,000, their PEG dehydrogenases oxidized the same molecular wt. range of PEGs (dimer-20,000). Cell-free extracts of PEG 400-, 1000- or 4000-utilizing bacteria oxidized PEG 6000 and 20,000 though these bigger PEGs could not be utilized as the sole carbon and energy sources by the bacteria. Methanol, ethylene glycol and glycerol were not or only barely dehydrogenated by all the enzyme preparations.     

Spectrophotometric determination of oxalate in urine and plasma with oxalate oxidase

In order to establish a standard procedure for the spectrophotometric determination of urinary and plasma oxalate with oxalate oxidase (Laker, M.F., et al. (1980) Clin. Chem. 26, 827-830; Sugiura, M., et al. (1980) Clin. Chim. Acta 105, 393-399) and to define the limitations of the method, the procedures and reactions involved in the assay have been examined. Among the chromogenic hydrogen donors for peroxidase tested, a combination of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and sodium N-sulfopropylaniline (HALPS) was found to be best for the oxalate determination under the conditions used. Urine contained substance(s) which were inhibitory to the measurement of hydrogen peroxide by the peroxidase-catalyzed oxidative condensation of MBTH and HALPS, but they were largely removed by charcoal treatment at pH 5.6 without significant loss of oxalate. Deproteinization of plasma was carried out by ultrafiltration through a membrane cone (Centriflo CF-25) at neutral pH. The plasma oxalate ultrafiltrability under the conditions employed was calculated to be approximately 95%. A standard assay system for oxalate in these urine and plasma samples was then set up based on a series of studies on the reactions involved in the assay. In the case of normal plasma, however, the absorbance change was very small due to the low concentration of oxalate, and in addition, pretreatment of plasma with excess oxalate decarboxylase followed by the ultrafiltration and oxalate determination did not abolish completely the oxalate oxidase-dependent absorbance increase. It was concluded that the enzymic method was useful for the assay of urinary oxalate and in detecting elevated levels of plasma oxalate such as those in hemodialysis patients but was not sensitive enough to determine accurately the normal or decreased level of oxalate in plasma. The apparent concentration of oxalate in normal human plasma was measured in this work as 3.5 +/- 0.8 microM (mean +/- S.D., n = 8), and this result was interpreted to mean that the concentration of plasma oxalate was less than approximately 3.5 microM, as estimated by the present method.     

Cultivar-specific seedling vigor and expression of a putative oxalate oxidase germin-like protein in sugar beet (Beta vulgaris L.)

For genetic screening and breeding purposes, an in vitro germination system that reflects relative field emergence potential was used to screen for germination-enhancing and stress-induced genes from germinating seedlings from two varieties of sugar beet. Three full-length germin-like protein (GLP) gene classes were recovered from stress-germinated seedlings of a superior emerging variety. GLP gene expression, oxalate oxidase protein activity, the H(2)O(2) content of stressed seedlings, but not catalase activity, were induced by stress germination conditions (e.g. excess water, NaCl, mannitol, or oxalate) in a good emerging hybrid and were not induced in a poor emerging variety. Only one of the three germin-like protein genes ( BvGer165) was differentially regulated, and was induced only in the good emerger. Hydrogen peroxide promoted germination and partially compensated solute-depressed germination percentages. Unlike other solute recovery by hydrogen peroxide regimes, recovery in oxalic acid plus H(2)O(2) was cultivar-independent. A block in oxalate metabolism is postulated to contribute to lower germination under stress in the lower emerging variety. Selection for stress-induced germin expression, or for down-stream targets, presents the first direct target to enable breeding for improved field emergence of sugar beet.     

Purification and characterization of polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene glycol

Polyethylene glycol (PEG) dehydrogenase in crude extracts of a PEG 20,000-utilizing mixed culture was purified 24 times by precipitation with ammonium sulfate, solubilization with laurylbetaine, and chromatography with diethylamino-ethyl-cellulose, hydroxylapatite, and Sephadex G-200. The purified enzyme was confirmed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme, which appeared to consist of four identical subunits, was 2.4 X 10(5). The enzyme was stable below 35 degrees C and in the pH range of 7.5 to 9.0. The optimum pH and temperature of the activity were around 8.0 and 60 degrees C, respectively. The enzyme did not require any metal ions for activity and oxidized various kinds of PEGs, among which PEG 6,000 was the most active substrate. The apparent Km values for tetraethylene glycol and PEG 6,000 were about 10.0 and 3.0 mM, respectively.     

Enhanced resistance to sclerotinia stem rot in transgenic soybean that overexpresses a wheat oxalate oxidase

Sclerotinia stem rot (SSR), caused by the oxalate-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, is one of the devastating diseases that causes significant yield loss in soybean (Glycine max). Until now, effective control of the pathogen is greatly limited by a lack of strong resistance in available commercial soybean cultivars. In this study, transgenic soybean plants overexpressing an oxalic acid (OA)-degrading oxalate oxidase gene OXO from wheat were generated and evaluated for their resistance to S. sclerotiorum. Integration and expression of the transgene were confirmed by Southern and western blot analyses. As compared with non-transformed (NT) control plants, the transgenic lines with increased oxalate oxidase activity displayed significantly reduced lesion sizes, i.e., by 58.71–82.73% reduction of lesion length in a detached stem assay (T₃ and T₄ generations) and 76.67–82.0% reduction of lesion area in a detached leaf assay (T₄ generation). The transgenic plants also showed increased tolerance to the externally applied OA (60 mM) relative to the NT controls. Consecutive resistance evaluation further confirmed an enhanced and stable resistance to S. sclerotiorum in the T₃ and T₄ transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (H₂O₂) levels were also observed in the transgenic leaves after S. sclerotiorum inoculation. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of OXO reached a peak at 1 h and 4 h after inoculation with S. sclerotiorum. In parallel, a significant up-regulation of the hypersensitive response-related genes GmNPR1-1, GmNPR1-2, GmSGT1, and GmRAR occurred, eventually induced by increased release of H₂O₂ at the infection sites. Interestingly, other defense-related genes such as salicylic acid-dependent genes (GmPR1, GmPR2, GmPR3, GmPR5, GmPR12 and GmPAL), and ethylene/jasmonic acid-dependent genes (GmAOS, GmPPO) also exhibited higher expression levels in the transgenic plants than in the NT controls. Our results demonstrated that overexpression of OXO enhances SSR resistance by degrading OA secreted by S. sclerotiorum and increasing H₂O₂ levels, and eliciting defense responses mediated by multiple signaling pathways.

     

Determination of oxalate in plant tissues with oxalate oxidase prepared from wheat

A method for determination of oxalate with oxalate oxidase (OxO, EC 1.2.3.4) prepared from wheat bran, is based on specific oxidation of oxalate to produce H₂O₂. The H₂O₂ formed was colorimetrically determined using horseradish peroxidase-catalyzed oxidation of 4-aminoantipyrine and N,N-dimethylaniline by H₂O₂. The new method was tested on rice, buckwheat, soybean and oxalis leaves, showing it is precise, sensitive, inexpensive, highly reproducible and simple to perform. Good agreement could be obtained between this method and the HPLC.     

Improved determination of urinary oxalate with alkylamine glass bound barley oxalate oxidase

The measurement of oxalate in urine was improved by employing barley oxalate oxidase immobilized on alkylamine glass beads affixed in a glass beaker. The minimum detection limit was 3.6 mg l(-1) urine. The recovery of added oxalate was 88.9+/-9.2%. Within- and between-assay coefficients of variation (CV) were <4.0 and <9.4%, respectively. The urinary oxalate values were obtained by a commercial kit method and the present method showed a good correlation (0.999). The method is free from tedious handling of glass beads and Cl- interference.     

Purification and characterization of polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene glycol.

Polyethylene glycol (PEG) dehydrogenase in crude extracts of a PEG 20,000-utilizing mixed culture was purified 24 times by precipitation with ammonium sulfate, solubilization with laurylbetaine, and chromatography with diethylamino-ethyl-cellulose, hydroxylapatite, and Sephadex G-200. The purified enzyme was confirmed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme, which appeared to consist of four identical subunits, was 2.4 X 10(5). The enzyme was stable below 35 degrees C and in the pH range of 7.5 to 9.0. The optimum pH and temperature of the activity were around 8.0 and 60 degrees C, respectively. The enzyme did not require any metal ions for activity and oxidized various kinds of PEGs, among which PEG 6,000 was the most active substrate. The apparent Km values for tetraethylene glycol and PEG 6,000 were about 10.0 and 3.0 mM, respectively.     

Water potential of aqueous polyethylene glycol

Water potential (Ψω) values were determined for aqueous colloids of four molecular sizes of polyethylene glycol (PEG) using freezing-point depression and vapor-pressure deficit methods. A significant third-order interaction exists between the method used to determine Ψω, PEG molecular size, and concentration. At low PEG concentrations, freezing-point depression measurements result in higher (less negative) values for Ψω than do vapor-pressure deficit measurements. The reverse is true at high concentrations. PEG in water does not behave according to van't Hoff's law. Ψω is related to molality for a given PEG but not linearly. Moreover, Ψω varies with the molecular size of the PEG. It is suggested that the Ψω of PEG in water may be controlled primarily by the matric forces of ethylene oxide subunits of the PEG polymer. The term matricum is proposed for PEG in soil-plant-water relation studies.     

Bacterial oxidation of polyethylene glycol.

The metabolism of polyethylene glycol (PEG) was investigated with a synergistic, mixed culture of Flavobacterium and Pseudomonas species, which are individually unable to utilize PEGs. The PEG dehydrogenase linked with 2,6-dichlorophenolindophenol was found in the particulate fraction of sonic extracts and catalyzed the formation of a 2,4-dinitrophenylhydrazine-positive compound, possibly an an aldehyde. The enzyme has a wide substrate specificity towards PEGs: from diethylene glycol to PEG 20,000 Km values for tetraethylene glycol (TEG), PEG 400, and PEG 6,000 were 11, 1.7, and 15 mM, respectively. The metabolic products formed from TEG by intact cells were isolated and identified by combined gas chromatography-mass spectrometry as triethylene glycol and TEG-monocarboxylic acid plus small amounts of TEG-dicarboxylic acid, diethylene glycol, and ethylene glycol. From these enzymatic and analytical data, the following metabolic pathway was proposed for PEG: HO(CH2CH2O)nCH2CH2OH leads to HO(CH2CH2O)nCH2CHO leads to HO(CH2CH2O)nCH2COOH leads to HO(CH2CH2O)n-1CH2CH2OH.     

Determination of urinary oxalate with alkylamine glass-bound sorghum oxalate oxidase and horseradish peroxidase

Oxalate in urine was analyzed using sorghum oxalate oxidase and horseradish peroxidase immobilized on alkylamine glass through glutaraldehyde. The minimum detection limit was 0.46 g/0.1 ml urine. The recovery of added oxalate was 97.5%. Within and between assay coefficients of variation were <3.5% and <6.5% respectively. A good correlation (r=0.9234) was found between oxalate values obtained by a commercial kit method and the present method. The method is unaffected by Cl– and NO3– found in urine.     

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Arginine-specific modification of proteins with polyethylene glycol

In this study, the residue-selective modification of proteins with polymers at arginine residues is reported. The difficulty in modifying arginine residues lies in the fact that they are less reactive than lysine residues. Consequently, typical chemo-selective reactions which employ "kinetic" selectivity (active esters, Michael addition, etc.) cannot be used to target these residues. The chemistry exploited herein relies on "thermodynamic" selectivity to achieve selective modification of arginine residues. ω-Methoxy poly(ethylene glycol) bearing an α-oxo-aldehyde group was synthesized and used to demonstrate the selective modification of lysozyme at arginine residues. In addition, the optimization of reaction conditions for coupling as well as the stability of the formed adduct toward dilution, toward a nucleophilic buffer, and toward acidification are reported. It was concluded that this approach is a convenient, mild, selective, and catalyst-free method for protein modification.     

The osmotic potential of polyethylene glycol 6000

Osmotic potential (ψ_s) of aqueous solutions of polyethylene glycol 6000 (PEG-6000) was curvilinearly related to concentration. At given concentrations, ψ_s increased linearly with temperature. The effects of concentration and temperature on ψ_s of PEG-6000 solutions differ from those for most salts and sugars and apparently are related to structural changes in the PEG polymer. Measurements of ψ_s with thermocouple psychrometers are more negative than those with a vapor pressure osmometer, with the psychrometer probably giving the more nearly correct ψ_s for bulk solutions. An empirical equation permits calculation of ψ_s from known concentrations of PEG-6000 over a temperature range of 15 to 35 C. Viscometery and gravimetric analysis are convenient methods by which the concentrations of PEG-6000 solutions may be measured.