Effect of diet overloaded by fats on the enzyme systems of metabolism in rats

The feeding of rats with high-fat diet (a part of fats was 50% of energy value of ration against 20% in control) during 4 weeks increased the level of cytochrome P-450 in the liver and enhanced aniline- and p-nitrophenol hydroxylase activity of CYP2E1 as well as erythromycin N-demethylase activity of CYP3A; the activity of aldehyde dehydrogenase class 3, UDP-glucuronosyl transferase, glutathione-S-transferase and N-acetyl transferase. At the same time, the moderate decrease of indomethacin-O-demethylase of CYP2C both phenolsulfotransferase activity were fixed. The changes of enzymatic activity correlated with activating of processes of gluconeogenesis, glycogenolysis and, especially, ketogenesis. A high-fat diet enhanced reactions of biotransformation of amidopyrine, acetanilide, toluene, sulfadimezine, were catalyzed with CYP2E1, CYP3A and enzymes of conjugation, simultaneously it increased the hepatotoxicity of paracetamol.     

A simple method for detection of enzyme activities involved in the initial step of phthalate degradation in microorganisms

We have developed a simple method for the detection of phthalate 4,5-dioxygenase and 4,5-dihydro-4,5-dihydroxyphthalate dehydrogenase activities in the initial step of phthalate degradation in bacteria. It was found that cells of a Pseudomonas putida strain adapted for phthalate could convert quinolinic acid to a hydroxylated product having λ_max at 315 nm. The occurrence of this compound was visualized by reaction with diazotized p-nitroaniline with which a red compound having λ_max at 512 nm was produced. In practice, if cells in colonies developed on an agar plate containing mineral salt medium supplemented with 0.4% of disodium phthalate and 0.1% of quinolinic acid are active with respect to the 4,5-dihydroxyphthalate pathway, then the colonies would be colored red immediately upon spraying with the diazotized p-nitroaniline reagent. The method was used to identify the phthalate degradative pathway for 27 phthalate-utilizing strains of the genera Pseudomonas (18 strains), Agrobacterium (3 strains), Alcaligenes (5 strains), and Micrococcus (1 strain). It was found that 24 of the 26 Gram-negative bacteria have the 4,5-dihydroxyphthalate pathway and that the remaining two strains of Pseudomonas sp. may metabolize via an unidentified pathway other than the dihydroxyphthalate pathways, and the Gram-positive strain of Micrococcus sp. metabolizes phthalate via the 3,4-dihydroxyphthalate pathway.     

Studies on prephenoloxidase-activating enzyme from cuticle of the silkworm Bombyx mori. I. Activation reaction by the enzyme

Prephenoloxidase-activating enzyme from larval cuticle of the silkworm catalyzes the activation reaction of hemolymphal prephenoloxidase to the active enzyme. The activation reaction of prephenoloxidase by the enzyme has been analyzed with respect to effect of salts, dependency on pH and substrate concentration, and susceptibility to inhibitors. It has been demonstrated that the reaction is highly sensitive to specific inhibitors for “serine enzyme.”Difference in substrate specificity of phenoloxidase preparations, produced by two enzyme fractions which can be separated by chromatography on DEAE-cellulose, is also described.     

Purification of a novel enzyme involved in catechin degradation by Calvatia gigantea

Summary A novel enzyme, involved in the degradation of catechin by Calvatia gigantea, was purified 114-fold over the crude extract yielding 24% purified enzyme with a specific activity 16.1 U/mg protein. Two isozymic forms (I and II) were isolated, both exhibiting the same kinetic characteristics with maximum activity at pH 8 and 35°C. SDS electrophoresis of I and II revealed the presence of two identical components in each form with molecular weights 50 500 and 49 500.     

Studies on the enzyme systems involved in electron and energy transfer in isolated chloroplasts. I. Effect of endogenous phosphate on the photophosphorylation coupled with noncyclic electron transport in intact chloroplasts

1. It was found that the P/2e ratio was independent of the degree of electron transport stimulation in the presence of ADP and P_i and exceeded 1.0 in the preparations with slight (30 %) as well as with high (80 %) stimulation.

2. Chloroplast preparations having a low content of endogenous P_i showed higher stimulation than those with higher contents.

3. Illumination of the chloroplasts in the presence of ADP and electron acceptor led to a decrease of endogenous P_i content that resulted in an increase of electron transport stimulation in the presence of exogenous P_i.

4. Electron transport in the absence of exogenous P_i was inhibited by both exogenous ADP and ATP.

5. It appears that the electron transport in the absence of exogenous P_i is coupled to phosphorylation, which occurs because isolated chloroplasts contain endogenous P_i. Stimulation of the electron transport by the addition of ADP and P_i seems to be caused by acceleration of the existing electron transport pathway, and not from the initiation of a new one.     

Novel heme-containing enzyme possibly involved in lignin degradation by the white-rot fungus Junghuhnia separabilima

Abstract The white-rot fungus Junghuhnia separabilima (Pouz.)Ryv, showed high levels of laccase production in cultures supplemented with veratric acid. Laccase, lignin peroxidase and an unknown peroxidase were separated from the extracellular culture fluid using anion-exchange FPLC. Three laccase species, three lignin peroxidases and a novel heme-containing protein were characterized by gel electrophoresis and isoelectric focusing. The new hemoprotein has a molecular mass of 44 kDa, isoelectric point of 3,4 and pH optimum of 5.5 for oxidation of o -dianisidine in the presence of H₂O₂. However it oxidised diaminobenzidine and guaiacol in the absence of H₂O₂. Veratryl alcohol and phenol red were not substratesfor this enzyme with or without addition of H₂O₂ and Mn(II). In addition the enzyme did not produce H₂O₂.     

Effect of fungal pellet morphology on enzyme activities involved in phthalate degradation

Pellet size of white rot fungus, Pleurotus ostreatus may affect the secretion of its degradative enzymes and accompanying biodegrading capability, but could be controlled by several physical culture conditions in liquid culture. The pellet size of P. ostreatus was affected by the volume of inoculum, flask, and medium, but the agitation speed was the most important control factor. At the lower agitation speed of 100 rpm, the large pellets were formed and the laccase activity was higher than that of small pelleted culture at 150 rpm, which might be due to loose intrapellet structure. However, the biodegradation rates of benzylbutylphthalate and dimethylphthalate were higher in the small pelleted culture, which indicated the involvement of other degradative enzyme rather than laccase. The activity of esterase which catalyzes the nonphenolic compounds before the reaction of ligninolytic enzymes was higher in the small pelleted culture, and coincided with the degradation pattern of phthalates. This study suggests the optimization of pellet morphology and subsequent secretion of degradative enzymes is necessary for the efficient removal of recalcitrants by white rot fungi.