On the oxidase function of plant peroxidase.
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Oxidoreductases↗
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Isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes, exemplified by purification of glucoamylase.
A highly purified preparation of glucoamylase G1 from Aspergillus niger was found, by isoelectric focusing in immobilized pH gradients, to contain a major form with a pI of 3.50 and a number of minor, more acidic and more basic contaminants. The major isoform was purified to homogeneity by recycling isoelectric focusing in a multicompartment electrolyzer, by confining this form in between two zwitterionic membranes, with pI 3.49 at the anodic side and pI 3.52 at the cathodic side. Recoveries were high (90%) and, notwithstanding the rather low operational pH, the electrosmotic flow was minimal and no protein precipitation occurred up to concentrations of 2.5 mg/ml (at the isoelectric point). The forms resolved in an analytical focusing gel were subjected to two types of in situ enzyme detections, by the glucose oxidase peroxidase (GOP) test and by the starch-iodine test. By both criteria all resolved zones exhibited enzyme activity, the GOP assay, however, following more closely the Coomassie blue stained protein profile. By computer modelling, it is shown that it is impossible to obtain linear pH gradients at such low pH values (pH 2.5-4.5 intervals) when the mixture has a low buffering power (beta = 2.0 mequiv.l-1 pH-1). When the beta power was gradually raised (beta = 4, beta = 6, beta = 8) the pH gradient became progressively linear until, in a recipe with beta = 10 mequiv.l-1 pH-1 full linearity of the pH gradient could be obtained. This is shown to be due to the substantial buffering power of bulk water in the pH 2.5-3.5 region.
Aspergillus niger↗
Study of carbon nanotube modified biosensor for monitoring total cholesterol in blood.
A carbon nanotube modified biosensor for monitoring total cholesterol in blood was studied. This sensor consists of a carbon working electrode and a reference electrode screen-printed on a polycarbonate substrate. Cholesterol esterase, cholesterol oxidase, peroxidase and potassium ferrocyanide were immobilized on the screen-printed carbon electrodes. Multi-walled carbon nanotubes (MWCN) were added to prompt electron transfer. Experimental results show that the carbon nanotube modified biosensor offers a reliable calibration profile and stable electrochemical properties.
Biosensing Techniques↗
Enzyme-substrate reactions in very high magnetic fields. I.
The availability of very high magnetic fields of up to 170,000 gauss made it worthwhile to pursue the search for a critical change in the rate of four enzyme substrate reactions. The four enzymes were ribonuclease, polyphenol oxidase, peroxidase, and aldolase. The experiments showed that, to within +/-3%, no detectable change was observable in the rate of reaction of any of the systems for periods of exposure to the magnetic field of up to 20 min.
Alcohol Oxidoreductases↗
Cytochemical characterization of goldfish (Carassius auratus L.) dermis with special reference to the pigment cells.
The dermal cells in grey, xanthic, and white goldfish integuments were cytochemically characterized for the following enzymatic activities: tyrosinase, DOPA-oxidase, cytochrome oxidase, monoamine oxidase, peroxidase, non-specific esterase, cholinesterase, NAD-diaphorase, NADP-diaphorase, aryl sulfatase, nucleotide phosphodiesterase, beta-glucuronidase, acid phosphatase, alkaline phosphatase, adenosine triphosphatase, thiamine pyrophosphatase, glucose-6-phosphatase, aldolase, as well as succinate, malate, isocitrate, glutamate, glucose-6-phosphate, 6-phosphogluconate, alpha-glycerophosphate, alcohol, lactate, and beta-hydroxybutyrate dehydrogenases. It was found that the epidermis was a significant barrier to the access of cytochemical reaction substrates. Removal of the epidermal barrier provided dermal cell localizations of enzymatic activities which were reproducible. Further, alterations in reaction times and temperatures from the mammalian methodology provided conditions fe various integumental cells were compared for possible interrelationships. The basic foundations for future work with the dermis of poikilothermic vertebrates on an experimental basis were established. In addition, a previously undescribed non-pigmented dermal cell, the "x"-cell, was found to have enzymatic characteristics similar to both melanophores and lipophores. The "x"-cell may be the common precursor of both types of pigment cells.
Animals↗
Sucrase-isomaltase and other brush border glycosidases in colorectal tumors.
Sucrase-isomaltase (SI), trehalase (T) and lactase-beta-glucosidase (LG) activities were assessed histochemically in samples of colorectal adenomas (11 tubular, 12 tubulovillous, 10 villous) and 30 adenocarcinomas obtained by biopsy during colonoscopy or from specimens removed by surgical intervention. Small samples of tumor tissue, tissue of the transitional zone and of macroscopically normal mucosa were quenched in heptan cooled in an acetone-dry ice mixture. Cryostat sections, transferred to non-precooled slides and in some cases to semipermeable membranes, were dried and subjected to the histochemical reactions for SI, T and LG. Sucrose, 2-naphthyl, 6-Br-2-naphthyl, and 5-Br-4-Cl-3-indoxyl alpha-D-glucosides, trehalose, and 5-Br-4-Cl-3-indoxyl-beta-D-fucoside were used as substrates. Sections of jejunal biopsies with normal activities of brush border glycosidases were used as controls. From samples of 5 adenomas, 5 adenocarcinomas and collected rests of jejunal biopsies with a normal finding 10% (w/vol) homogenates in 2% Triton X-100 were prepared. Homogenates were frozen and thawed 3 times and their supernatants subjected to isoelectric focusing on polyacrylamide gel plates. Zymograms were developed with the same methods as for the detection of alpha-glucosidases in sections. In no colorectal tumor LG was present. SI was found in 70% adenocarcinomas, 50% villous, 25% tubulovillous and 19% tubular adenomas when the method with sucrose, glucose oxidase-peroxidase and 3,3'-diaminobenzidine was used. Hardly discernible traces of activity were found in tumors with azo-coupling reactions applied at pH 5, 6 and 6.5. No reaction was detected with the indigogenic method applied at pH above 6.0. However, jejunal biopsies displayed very strong reactions confined to the brush border of enterocytes under the same conditions. A strongly positive reaction was seen in 7 of 12 tumors investigated recently when the indigogenic reaction was applied at pH below 6.0 (particularly at pH 5.0). In this case the deposition of indigo was due to membrane and lysosomal alpha-glucosidases of the tumor cells and lysosomal alpha-glucosidase of macrophages and leukocytes. These findings were corroborated by zymograms. T was detected in the same tumors as SI; its activity was lower, however. SI activity in colorectal tumors is a useful, but not general marker of these tumors.
Colorectal Neoplasms↗
Tissue printing for myrosinase activity in roots of turnip and Japanese radish and horseradish: a technique for localizing myrosinases.
A method for analyzing the tissue distribution of myrosinase activity in Brassicaceous plants was developed. This technique is based on 'tissue printing' to visualize enzyme activity. The freshly-cut surface (transverse direction) of the root of three species, Japanese radish (Raphanus sativus), turnip (Brassica campestris) and Japanese horseradish (wasabi, Wasabia japonica), was pressed onto a polyvinylidene difluoride (PVDF) filter to immobilize the proteins onto the membrane. The sites of myrosinase activity on the membranes were visualized by the sinigrin-glucose oxidase-peroxidase system. Signals for myrosinase activity were observed in both the epidermis and vascular cambium of the root of the Japanese radish, turnip and wasabi. Measurement of myrosinase activity in protein extracts indicated that the level of myrosinase activity in the peeling, which consisted of the epidermis, cortex and vascular cambium, was much higher than that in the peeled root of the three species. These results support the image that myrosinase activity, obtained in tissue printing, corresponded well with the tissue distribution of myrosinase activity.
Journal Article↗
New trends in cryoenzymology: probing the functional role of protein dynamics by single-step kinetics.
Cryoenzymology was initially used to slow down enzyme-catalyzed reactions so as to stabilize intermediates for further study. During the course of this early work, it became clear that cryoenzymology could be extended to other ends and some of these are described. First, the use of a cryosolvent on its own (or together with temperature) as a perturbant has allowed a resolution of the substrate binding steps of certain enzymes (myosin, D-amino acid oxidase, peroxidase and cytochrome P450). Second, by the use of cryosolvent and temperature, coupled with the classical physico-chemical perturbants, one can selectively modulate the various steps of an enzyme pathway. This approach can lead to an understanding of the mechanism of enzyme regulation. Finally, by carrying out experiments over a wide range of temperatures (-30 degrees C- +30 degrees C) and pressure (up to several kbars) in specially constructed fast reaction equipment, one can study the thermodynamic properties of the individual rate constants describing the interconversions of reaction intermediates. Experiments with creatine kinase, cytochrome P450 and peroxidase are described. The thermodynamic parameters delta H, delta G, delta S and delta V are thus measured and when this is done under different solvent conditions one can, at least within the theories available, attempt an approach to the problem of protein dynamics.
Cold Temperature↗
Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78.
White rot fungi efficiently degrade lignin, a complex aromatic polymer in wood that is among the most abundant natural materials on earth. These fungi use extracellular oxidative enzymes that are also able to transform related aromatic compounds found in explosive contaminants, pesticides and toxic waste. We have sequenced the 30-million base-pair genome of Phanerochaete chrysosporium strain RP78 using a whole genome shotgun approach. The P. chrysosporium genome reveals an impressive array of genes encoding secreted oxidases, peroxidases and hydrolytic enzymes that cooperate in wood decay. Analysis of the genome data will enhance our understanding of lignocellulose degradation, a pivotal process in the global carbon cycle, and provide a framework for further development of bioprocesses for biomass utilization, organopollutant degradation and fiber bleaching. This genome provides a high quality draft sequence of a basidiomycete, a major fungal phylum that includes important plant and animal pathogens.
Base Composition↗
Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides.
Superoxide reacts rapidly with other radicals, but these reactions have received little attention in the context of oxidative stress. For tyrosyl radicals, reaction with superoxide is 3-fold faster than dimerization, and forms the addition product tyrosine hydroperoxide. We have explored structural requirements for hydroperoxide formation using tyrosine analogues and di- and tri-peptides. Superoxide and phenoxyl radicals were generated using xanthine oxidase, peroxidase and the respective tyrosine derivative, or by gamma-radiation. Peroxides were measured using FeSO4/Xylenol Orange. Tyrosine and tyramine formed stable hydroperoxides, but N-acetyltyrosine and p-hydroxyphenylacetic acid did not, demonstrating a requirement for a free amino group. Using [14C]tyrosine, the hydroperoxide and dityrosine were formed at a molar ratio of 1.8:1. Studies with pre-formed hydroperoxides, and measurements of substrate losses, indicated that, in the absence of a free amino group, reaction with superoxide resulted primarily in restitution of the parent compound. With dipeptides, hydroperoxides were formed only on N-terminal tyrosines. However, adjacent lysines promoted hydroperoxide formation, as did addition of free lysine or ethanolamine. Results are compatible with a mechanism [d'Alessandro, Bianchi, Fang, Jin, Schuchmann and von Sonntag (2000) J. Chem. Soc. Perkin Trans. II, 1862-1867] in which the phenoxyl radicals react initially with superoxide by addition, and the intermediate formed either releases oxygen to regenerate the parent compound or is converted into a hydroperoxide. Amino groups favour hydroperoxide formation through Michael addition to the tyrosyl ring. These studies indicate that tyrosyl hydroperoxides should be formed in proteins where there is a basic molecular environment. The contribution of these radical reactions to oxidative stress warrants further investigation.
Amines↗
Implications of enzyme kinetics.
Of the many examples of oscillatory kinetic behaviour known, several are briefly reviewed, including those of glycolysis, the peroxidase-oxidase reaction and oscillations in cellular calcium concentration. It is shown that simple mathematical models employing allosteric rate laws are sufficient to explain the instability of the steady state and the appearance of sustained oscillations. The cAMP-signalling systems of cellular slime moulds and the dynamics of intracellular calcium oscillations illustrate the importance of such oscillophores to inter- and intra-cellular communication and differentiation.
Animals↗
The thylakoid lumen of chloroplasts. Isolation and characterization.
The chloroplast compartment enclosed by the thylakoid membrane, the "lumen," is poorly characterized. The major aims of this work were to design a procedure for the isolation of the thylakoid lumen which could be generally used to characterize lumenal proteins. The preparation was a stepwise procedure in which thylakoid membranes were isolated from intact chloroplasts. Loosely associated thylakoid surface proteins were removed, and following Yeda press fragmentation the lumenal content was recovered in the supernatant following centrifugation. The purity and yield of lumenal proteins were determined using appropriate marker proteins specific for the different chloroplast compartments. Quantitative immunoblot analyses showed that the recovery of soluble lumenal proteins was 60-65% (as judged by the presence of plastocyanin), whereas contamination with stromal enzymes was less than 1% (ribulose-bisphosphate carboxylase) and negligible for thylakoid integral membrane proteins (D1 protein). Approximately 25 polypeptides were recovered in the lumenal fraction, of which several were identified for the first time. Enzymatic measurements and/or amino-terminal sequencing revealed the presence of proteolytic activities, violaxanthin de-epoxidase, polyphenol oxidase, peroxidase, as well as a novel prolyl cis/trans-isomerase.
Amino Acid Sequence↗
A spectrophotometric assay for alpha-mannosidase activity.
A simple and versatile spectrophotometric assay for alpha-mannosidase activity, which can be used with unlabelled natural substrate or synthetic substrates, was developed. The reducing mannose released from the substrate by the enzyme is quantitated using glucose oxidase, peroxidase and o-dianisidine. Using recombinant alpha 1,2-mannosidase obtained from Saccharomyces cerevisiae and Man9, GlcNAc, the spectrophotometric assay yielded values of 0.3 mM for Km and 15 mU/microgram for V(max), which are comparable to those obtained using the traditional radiochemical assay. The assay was also used to evaluate some alternative oligosaccharides as substrates for the enzyme. Man5-O(CH2)8-COOCH3 shows potential as an alternative synthetic substrate as the enzyme retained its specificity for a single alpha 1,2-mannose residue. Kinetic results suggest that the lower 1,3 linked arm of Man9GlcNAc is more critically involved in substrate recognition than the upper 1,6 linked arm.
Carbohydrate Sequence↗
Prolongation of succinylcholine block by metoclopramide.
Laboratory and clinical evidence of the inhibition of plasma cholinesterase by metoclopramide was demonstrated. When succinylcholine is used as the substrate and the product choline assayed by choline oxidase-peroxidase-quinone dye colorimetry, the rate of the choline production as optical density change was reduced to 50% by 19.5 X 10(-6) M metoclopramide at 20 degrees C. Prolongation of neuromuscular blockade produced by concurrent administration of succinylcholine and metoclopramide was studied in 22 patients aged between 18 and 40 years undergoing elective gynecological surgery. EMG activity in the adductor pollicis muscle was recorded in response to a train-of-four (TOF) stimulus delivered every 10 s. Patients were randomly divided into two groups: A and B. In both groups, anesthesia was induced with thiopental and maintained with sufentanil and nitrous oxide. Tracheal intubation followed intravenous succinylcholine. Intraoperatively, after returning of neuromuscular function, patients in both groups received 20 mg succinylcholine for the determination of duration of neuromuscular blockade. Time from 95% suppression of baseline twitch following a 20 mg increment of succinylcholine until recovery to 25% of control activity was determined. Thereafter, in group A, patients receive metoclopramide (10 mg iv) followed by succinylcholine 20 mg iv, and patients in group B received succinylcholine 20 mg iv alone. Recovery times were again measured and found to be prolonged in patients receiving metoclopramide compared with those not receiving metoclopramide (P less than 0.05). Metoclopramide has no intrinsic neuromuscular blocking activity, but its ability to inhibit plasma cholinesterase probably is the mechanism by which it prolongs succinylcholine block. Reducing the dose of succinylcholine may be appropriate when metoclopramide is given concurrently.
Adult↗
The incidence and characteristics of some paraneoplastic syndromes of hepatocellular carcinoma in Nigerian patients.
OBJECTIVE: To determine whether paraneoplastic syndromes of hepatocellular carcinoma such as erythrocytosis, hypoglycaemia, hypercholesterolaemia and hypercalcaemia are found in Nigerian patients with this tumour, and, if present, to determine their incidence and characteristics. DESIGN: Consecutive patients with a diagnosis of hepatocellular carcinoma seen over a 10-year period at Ile-Ife, Nigeria, underwent haematocrit (100 patients), plasma glucose (65), cholesterol (65) and calcium studies (30). Shortage of laboratory reagents did not allow all the 100 patients to be tested for plasma glucose, cholesterol and calcium. METHODS: Haematocrit was determined by the microhaematocrit method, and the glucose oxidase-peroxidase, Liebermann-Burchard and O-cresolphthalein complexone methods were employed for the estimation of plasma glucose, cholesterol and calcium respectively. RESULTS: The haematocrit values ranged from 19 to 63% with a mean of 34.51% (SD 7.72). Only 1 out of 100 patients (1%) had erythrocytosis (haematocrit 63%). Sixty-five patients had their plasma glucose determined. The range of values for fasting plasma glucose (FPG) was 1-8.4 mmol/l with a mean of 4.3 (SD 1.6). Hypoglycaemia occurred in 18 patients (27.7%)(14 males, 4 females). The mean liver span for patients with FPG > 5.0 mmol/l was 16.87 cm (SD 3.03), while the value for those with FPG < 3.0 mmol/l was 22.0 cm (SD 6.14, P < 0.05). Plasma cholesterol was determined in 65 patients and the range of values was 2.1 -18.1 mmol/l with a mean of 5.65 (SD 2.42). Hypercholesterolaemia occurred in 16 patients (24.6%) (15 males, 1 female). The range of plasma calcium obtained in 30 patients was 1.6-3.15 mmol/l with a mean of 2.36 (SD 0.32). Three patients (10%) (all males) had hypercalcaemia. CONCLUSION: While erythrocytosis has a low incidence, hypoglycaemia and hypercholesterolaemia are important paraneoplastic syndromes of hepatocellular carcinoma in Nigerian patients. The study confirms that the larger the liver mass, the greater the tendency towards hypoglycaemia. Also, the paraneoplastic syndromes studied occurred predominantly in hepatocellular carcinomas with cirrhosis.
Calcium↗
Automatic network coupling analysis for dynamical systems based on detailed kinetic models.
We introduce a numerical complexity reduction method for the automatic identification and analysis of dynamic network decompositions in (bio)chemical kinetics based on error-controlled computation of a minimal model dimension represented by the number of (locally) active dynamical modes. Our algorithm exploits a generalized sensitivity analysis along state trajectories and subsequent singular value decomposition of sensitivity matrices for the identification of these dominant dynamical modes. It allows for a dynamic coupling analysis of (bio)chemical species in kinetic models that can be exploited for the piecewise computation of a minimal model on small time intervals and offers valuable functional insight into highly nonlinear reaction mechanisms and network dynamics. We present results for the identification of network decompositions in a simple oscillatory chemical reaction, time scale separation based model reduction in a Michaelis-Menten enzyme system and network decomposition of a detailed model for the oscillatory peroxidase-oxidase enzyme system.
Algorithms↗
Reaction Properties of the Ascorbic Acid Oxidase from Myrothecium verrucaria.
Ascorbic acid oxidase activity in Myrothecium verrucaria extracts resulted in O(2) uptake exceeding 0.5 mole per mole of ascorbic acid and in CO(2) evolution. Measurement of oxidized ascorbic acid at completion of the reaction demonstrated that an average of 10% of the oxidized product disappeared. A comparison of the gas exchange data with the amount of ascorbic acid not accounted for indicated that the reaction could not be explained by independent oxidase and oxygenase systems. Chromatographic examination of the reaction mixtures identified l-threonic acid. Experiments with ascorbic acid-1-(14)C showed that C-1 was partially decarboxylated during the oxidation. Test of the fungal extracts for enzymes that might explain the deviation from expected stoichiometry showed that phenolase, glutathione reductase, cytochrome oxidase, peroxidase and oxalic decarboxylase were not involved. Addition of azide in concentrations sufficient to block catalase increased excess O(2) consumption about 65%. No enzymes were found that could directly attack oxidized ascorbic acid. H(2)O(2) accumulated during oxidation in azide-blocked systems.The O(2) excess could be explained by assuming the enzyme had peroxidative capacity on a reductant other than ascorbic acid. An intermediate of ascorbic acid oxidation appeared to function as the substrate yielding CO(2) and l-threonic acid on degradation. The increase in excess O(2) utilized in azide-blocked systems and the H(2)O(2) accumulation also were explained by the proposed scheme.Another interpretation would involve production of free radicals during ascorbic acid oxidation. Evidence for this was the ability of extracts to oxidize DPNH in the presence of ascorbic acid. Oxygen radicals formed in such reactions were considered possible agents of degradation of ascorbic acid.
Journal Article↗
Optimization of apple allergen preparation for in vivo and in vitro diagnostics.
The aim of our investigation was to obtain a well-characterized active apple extract suitable for both in vivo and in vitro diagnostics by a technically simple method. For this purpose, apple extracts were prepared by homogenization in potassium phosphate buffer or by precipitation in organic solvents and resolubilization in potassium phosphate buffer in the presence or in the absence of enzyme inhibitors. These extracts were comparatively investigated by means of SDS-PAGE, two-dimensional electrophoresis, immunoblotting, RAST inhibition, and prick test. The in vitro investigations indicated that extracts prepared by precipitation in organic solvents (diacetone alcohol) at -20 degrees C have a higher allergen activity than those prepared by extraction in aqueous solutions. From the in vivo tests (prick test), it was concluded that application of inhibitors of cytoplasmic enzymes (phenol oxidases, peroxidases, proteases) already during extraction is an essential precondition for active prick test solutions. Correspondingly, the extract obtained by solvent precipitation in the presence of enzyme inhibitors appeared to be most suitable for clinical application.
Allergens↗