Studies on kinetic parameters and stability of aminoacylase in non-conventional media.

Catalytic properties and conformational stability of aminoacylase (N-acylamino acid amidohydrolase, EC 3.5.1.14) were studied in water-N,N-dimethylformamide (DMF) and water-dioxane solvent mixtures. Beside the prompt inhibition the solvents caused further inactivation during incubations. In the presence of 5% DMF content the inactivation proceeds with a well-measurable rate (t1/2 39 min), while in the case of 20% DMF the enzyme practically lost its starting activity during 50 min incubation (t1/2 13 min). The K(m) value of the enzyme increased about three times with increasing DMF concentrations up to about 2.6 M DMF, while the Vmax value decreased practically to zero in the same concentration range.

The effect of environmental pollution on the function of enzymes of active skeleton muscle. Part 1: An adequate method to study the effect of metal ions on the activity of phosphofructokinase.

The possibilities of exact measuring the effect of metal ions on the activity of phosphofructokinase (PFK) by means of both a heat inactivation and an auxiliary enzyme system were studied. It was found that both methods are suitable for measuring the inhibition of phosphofructokinase by metal ions. It was further found that metal ion concentrations causing 50% deactivation of PFK could not considerably influence the measuring capacity of the auxiliary enzyme system.

Factors influencing the operation of a vertical bioreactor segmented with perforated plates.

Factors influencing the operation of a vertical bioreactor segmented with perforated plates supporting immobilized yeast cells were studied. It was found that the most important factors are the length-diameter (L/D) ratio of the reactor and the dilution rate. It was supposed that the optimal L/D ratio is about 1. The operation of the reactor was more favourable at a low liquid phase-solid phase ratio. The spatial distribution of the biocatalyst had only a slight, if any effect. The periodically changed direction of feed flow has no improving effect on the fermentation process.

Effects of paraquat treatment on fish transaminase molecular subforms.

The effects of paraquat (PQ; 1,1'-dimethyl-4,4'-bipyridylium dichloride) treatment were investigated in carp, silver carp and wels. The serum glutamic-oxalacetic transaminase (GOT; L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1.) level was enhanced by 50% at 1 ppm exposure and by 100% at 10 ppm exposure in all species, and there was a change in the distribution of the molecular subforms of GOT in the liver and heart. The activities of the individual subforms decreased with increasing PQ concentration or after a longer exposure. In some cases, one of the subforms was no longer present in the liver. An increased serum GOT activity, a decreased enzyme activity in different organs and the disappearance of molecular subform indicate tissue damage.

Immobilization of pig muscle aldolase on a silica-based support.

Pig muscle aldolase was covalently attached to a silica-based support possessing aldehyde functional groups. The activity of the immobilized enzyme was 37 U/g solid, and the specific activity calculated on a bound protein basis was 1.9 U/mg protein. The optimum pH for the catalytic activity was pH 7.5. The apparent optimum temperature was found to be 45 degrees C. The Km app value of the immobilized aldolase with D-fructose 1,6-diphosphate as substrate was 1.25 X 10(-4) M. The conformational stability was improved by the immobilization. The immobilized aldolase was used for the continuous splitting of D-fructose 1,6-diphosphate.

[Immobilization of glucose oxidase on silica-based supports]. / Immobilizatsiia gliukozooksidazy na nositeliakh s silikatnymi osnovami.

Glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) was covalently coupled to silica-based supports containing aldehyde functional groups. The activity of the immobilized enzyme was about 1000 U/g support. The optimum pH of the catalytic activity was 5.5 for the soluble enzyme and 6.0 for the immobilized enzyme. With glucose as a substrate the Km value of the immobilized enzyme was higher than in case of the soluble enzyme. The immobilized enzyme was found to be more thermostable than the soluble one. The immobilization did not affect the stability of glucose oxidase against the denaturing effect of urea.

Determination of glycolytic intermediates in a flow injection system using immobilized enzymes.

Some glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, enolase and phosphoglyceromutase) were immobilized on a polyacrylamide-type bead polymer containing carboxylic functional groups activated by water-soluble carbodiimide. The immobilized enzymes were used for the determination of pyruvic acid, phosphoenolpyruvic acid, 2-phosphoglyceric acid and 3-phosphoglyceric acid in a flow injection system. The immobilized lactate dehydrogenase column was repeatedly employed for the determination of pyruvic acid in clinical samples. The results of the flow injection method accorded well in accuracy, sensitivity and reproducibility with those of soluble enzyme analysis.

Immobilization of lactate dehydrogenase on polyacrylamide beads.

Pig muscle lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) was covalently immobilized on polyacrylamide beads containing carboxylic functional groups activated by water-soluble carbodiimide. The effects of immobilization on the catalytic properties and stability of the lactate dehydrogenase were studied. There was no shift in the pH optimum of the immobilized enzyme compared to that of the soluble one. The apparent optimum temperature of the soluble enzyme was 65 degrees C, while that of the immobilized enzyme was between 50 and 65 degrees C. The apparent Km values of the immobilized enzyme with pyruvate and NADH substrates were higher than those of the soluble enzyme. As a result of immobilization, enhanced stabilities were found against heat treatment, changes in pH, and urea denaturation.

Enzymatic determination of isocitrate by amperometric monitoring of the rate of oxygen consumption.

A coupled enzymatic method elaborated for NAD+-dependent dehydrogenases has been adapted for NADP+-dependent isocitrate dehydrogenase, in combination with amperometric measurements. The isocitrate dehydrogenase activity dependent linearly on the isocitrate concentration in the range 0-2 X 10(-4) M. Application of this method affords a sensitive estimation of isocitrate even in turbid liquids such as fermentation broths.

Comparative studies on soluble and immobilized rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase was immobilized by covalent attachment to a polyacrylamide support (Akrilex C) containing carboxylic functional groups. As a result of immobilization, the pH optimum for catalytic activity shifted into a more alkaline direction. The apparent Km value with phosphoenolpyruvate increased, and that with ADP slightly decreased. With respect to the stability against urea and thermal inactivation, the immobilized pyruvate kinase seemed to be the more stable at lower urea concentrations and between 45 and 55 degrees C. At 1.5 and 2.5M urea and at higher temperature, there were no marked differences between the soluble and the immobilized enzyme.

Characterization and comparison of soluble and immobilized pig muscle aldolases.

Pig muscle aldolase was insolubilized by covalent attachment to a polyacrylamide matrix containing carboxylic functional groups. The catalytic activity of the Akrilex C-aldolase was 2014 units/g solid, i.e., an activity loss of only about 5% relative to the initial activity. The pH optimum for catalytic activity shifted form 7.25 to 7.5 and the apparent temperature optimum from 313 to 318 K. The Michaelis constant of the insolubilized enzyme was significantly higher than that of the soluble aldolase. Heat- and urea-inactivation experiments revealed that the immobilization increased the stability of the enzyme.

Studies on the properties of glucose-6-phosphatase from carp liver microsomes (Cyprinus carpio L.).

The effects of temperature and pH on the phosphohydrolase activity of carp hepatic glucose-6-phosphatase (EC 3.1.3.9) have been investigated. The enzyme activity was maximum at about 308 K and in the pH range 5-6.5. The apparent Michaelis constant (KM) and Vmax of the reaction with glucose-6-phosphate were found to be 14.8 mM and 2.27 nmol/min/mg protein. The enzyme activity was partly inhibited by EDTA, while in the presence of sufficient PCMB virtually total inhibition was observed.

A novel polyacrylamide-type support prepared by p-benzoquinone activation.

A new and simple method for the activation of polyacrylamide gels using p-benzoquinone is described. The optimal conditions of activation have been elaborated. The activated support could be successfully applied to the immobilization of ligands having nucleophilic groups active over a broad pH range.

Morphological and biochemical studies on liver, kidney and gill of fishes affected by pesticides.

Effect of a herbicide, paraquat (1,1'-dimethyl-4,4'-bipyridilium-dichloride), the fungicide copper sulphate, and zinc chloride was studied on the histological structure of liver, kidney and gill of three fish species with different feeding habits, viz.: a herbivorous, silver carp (Hypophthalmichthys molitrix); an omnivorous, common carp (Cyprinus carpio L.) and a carnivorous, sheatfish (Silurus glanis L.). The organs were studied electron microscopically after fixation according to Karnovsky. The toxic effect manifested itself characteristically on the respective species, regardless of the type of the chemical applied and the species specificity. Upon the effect of the treatments applied the cytoplasm of the respiratory cells of the gill became electron transparent and the cytoplasmic organelles disappeared almost totally. In the chloride cells showing focal necrosis, residuals of nuclear, mitochondrial and endoplasmic origin were seen. Pillar cells and the pericytes remained intact. In the nucleus of the liver cells, electron dense heterochromatin was not present. The degree of the damage in the liver cells was indicated by swollen mitochondria with electron transparent matrix and by dilatation and vacuolation of the endoplasmic reticulum system. Epithelial cells decreased in electron density, the endoplasmic reticulum was vesiculated, mitochondria were swollen. Leucocytes increased in number, and empty vacuoles and vacuoles filled with dense granules appeared in them during toxicosis. Copper sulphate or paraquat increased serum transaminase enzyme activities (glutamic acid-oxalacetic acid transaminase, glutamic acid-pyruvic acid transaminase) in all the three fish species. These damages can cause serious disturbances in energy uptake and secretion processes of fish.

Studies on the effect of paraquat on glycogen mobilization in liver of common carp (Cyprinus carpio L.).

1. A herbicide, paraquat (1,1'dimethyl-4,4'-bipyridilium-dichloride) was administered to carp in 0.5-10.0 ppm concentrations, respectively, and blood sugar level, glucose-6-phosphatase and glycogen phosphorylase activities of liver were determined. 2. Paraquat treatment caused an increase of blood sugar level and enhanced phosphorylase and glucose-6-phosphatase activities. 3. Paraquat can induce alterations in endoplasmic reticulum that might contribute to the changes in glucose-6-phosphatase activity, resulting in an increase of blood glucose level and/or all the effects can be attributed to a high level of circulating epinephrine produced by paraquat toxicosis.

Characterization of enzyme-enzyme interaction using an affinity batch system.

NAD-Sepharose 4B gel was used to study the complexation between glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) EC 1.2.1.12) and aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). An affinity sorbent specific for glyceraldehyde-3-phosphate dehydrogenase was utilised in a batch system. The dissociation constant of the enzyme complex was calculated. The method elaborated in our laboratory was used to investigate the effects of temperature and pH on the complex formation.

Comparative studies on the sensitivity of different fish species to metal pollution.

The effects of 10 ppm CuSO4, ZnCl2 on the blood glucose level and serum LDH, GOT, GPT activities of three fish species (carp, silver carp, European wels) were measured. CuSO4 increased the blood glucose level, LDH, GOT and GPT activities in the three species in the following order: silver carp greater than carp greater than European wels. ZnCl2 did not alter the same levels. Our results showed that CuSO4, and presumably several anthropogenic agents, damaged the different fish species specifically and caused different stress effects depending on the fish species.

Investigation of the active center and catalytic mechanism of porcine kidney aminoacylase: a model of the active center.

Procine kidney aminoacylase (E.C. 3.5.1.14) is inhibited neither by phenylmethylsulfonylfluoride nor by alkylating agents (iodoacetate or iodoacetamide). Therefore reaction mechanisms including the formation of acylenzyme through seryl or cysteinyl side chains are ruled out. The enzyme is a metalloprotein that can be inactivated by ECTA and in which Co2+ is an equivalent substitute for the Zn2+ ion. The two SH groups/subunit of aminoacylase exhibit different reactivites to p-hydroxymercuribenzoate. Modification of the less reactive SH group reversibly inactivates the enzyme. We suggest that this cysteinyl side chain is situated in the active center or in its immediate vicinity. On the basis of our results we suppose a close similarity between aminoacylase and carboxypeptidase A with respect to their active center and catalytic mechanism.