Maria Brzyska

Electrochemical and Conformational Consequences of Copper (CuI and CuII) Binding to β-Amyloid(1–40)

Copper‐induced structural rearrangements of Aβ40 structure and its redox properties are described in this study. Electrochemical and fluorescent methods are used to characterise the behaviour of Aβ–Cu species. The data suggest that time‐dependent folding of Aβ–Cu species may cause changes in the redox potentials.

The influence of metal ions on the soluble and immobilized cytoplasmic cabbage peroxidase activity and its kinetics

Abstract A crude preparation of peroxidase, containing most of the intracellular cabbage proteins, was immobilized and examined as a good model of natural cell membranes. The proteins were covalently bound by free NH 2 or COOH groups to matrices which could modify the stereospecificity of the action of this peroxidase preparation. The immobilization of the peroxidase preparation by NH 2 groups caused an increase in the Michaelis constant, determined at varying H 2 O 2 concentrations. As a result of the immobilization of the same protein preparation by COOH groups, there followed a lowering of the K m value of peroxidase determined against the same substrate, or the preservation of this value on a level similar to that of the soluble enzyme. Our investigations concerned changes in the kinetics of the peroxidase reaction after cabbage protein immobilization in the presence of eight metal ions (Cu 2+ , Ca 2+ , Mg 2+ , Mn 2+ , Zn 2+ , Cd 2+ , Hg 2+ , Pb 2+ ) at a concentration of 0.6 mM. The ions of Mg, Mn, Cu, Ca and Zn, as well as Cd, stimulate the activity of the immobilized peroxidase more effectively than the preparation of soluble proteins. Although the Hg and Pb ions caused some decrease in peroxidase activity, immobilization of the proteins partially protected them from the harmful effect of these ions. It seems that this type of investigation and its results may provide a simple model for the behaviour of plant proteins in natural environment which is so highly exposed today to ecological stresses.

Changes in activity of soluble and immobilized cabbage peroxidase after treatment with seven different proteases

Abstract The following proteases were used in the experiments: cabbage serine protease, papain, pepsin, trypsin, proteinase K, carboxypeptidase Y and pronase. The incubation of cabbage peroxidase, soluble as well as immobilized, with 7 different proteases for 1 to 2 h caused an increase in the enzyme activity of up to 200%. A longer incubation time of peroxidase with proteases caused a decrease in this enzyme activity, but the immobilization stabilized its activity despite the action of proteases. The pH optimum of the immobilized peroxidase in the presence of cabbage serine protease and pronase shifted from 6.0 to 9.0, and a second peak in the activity was found at pH 4.0. The influence of papain on immobilized cabbage peroxidase caused an increase in the peroxidase activity after 1 h of incubation accompanied by a shift in the pH optimum from 9.0 to 3.0. The observed changes in the peroxidase activities after treatment with proteases may be similar to those occurring in nature.

The Effect of the Simultaneous Operation of Two Metal Ions on Soluble and Immobilized Peroxidase

Changes in the activity of peroxidase (EC 1.11.1.7) were compared under the influence of two simultaneously present metal ions, one of which deactivates most enzymes (mercury or cadmium) while the other possesses stimulating properties (magnesium, manganese or calcium). A possibility of a considerable increase in peroxidase activity (50–200%) was found in the presence of the ions of manganese, magnesium or calcium, in spite of the presence of ions known to possess toxic properties (mercury or cadmium). An enzyme immobilization method was employed in order to determine the kind of protein functional groups (COOH or NH2) involved in the processes of peroxidase activity changes due to the presence of metal ions. It was found that by immobilizing peroxidase by free amino groups of the enzyme, one achieves a considerable decrease in the toxic effect of mercury or cadmium, and a higher increase in peroxidase activity in the presence of manganese, magnesium or calcium.

Changes in activity of soluble and immobilized peroxidases after treatment by various proteases and some metal ions

Abstract The proteases used were cabbage serine proteinase, trypsin, proteinase K, papain, pepsin, pronase and carboxypeptidase Y. The metal ions used were Mg, Mn, Cu, Hg, Pb and Ag. The incubation of cabbage peroxidase, soluble or immobilized, with seven different proteases and six different metal ions during 1 to 2 hours caused an increase in enzyme activity up to 250%. Longer incubation times of the peroxidase with the proteases caused a decrease of the enzyme activity but the immobilization stabilized its activity, despite the action of proteases. Especially evident was activation (from 50% to 200%) by serine proteinases, including the cabbage serine proteinase, in the presence of Pb, Hg, Cu and Mg ions after one to two hours of incubation. The observed changes in the peroxidase activities after protease and metal ion treatment may resemble those in nature or in enzymatic bioreactors.

Discrete conformational changes as regulators of the hydrolytic properties of beta-amyloid (1–40)

Beta‐amyloid (1–40) (Abeta), the main component of senile plaques seen in the brains of Alzheimers disease patients, was found to be toxic both as fibrils and smaller soluble globular aggregates. The hydrolytic properties of Abeta, a new biochemical activity described previously [Brzyska M, Bacia A & Elbaum D (2001) Eur J Biochem268, 3443–3454], may contribute to its overall toxicity. In this study, the hydrolysis of fluorescein ester series was studied under predetermined conditions affecting Abeta hydrophobicity and conformation. Reaction products of the most effectively decomposed ester (dibutyrate) were characterized using HPLC and ESI‐MS. Hydrophobicity of Abeta, as measured by bis‐8‐anilinonaphthalene fluorescence, correlated with its hydrolytic abilities. FTIR and CD data analysis showed a relationship between enhanced hydrolytic abilities and Abeta structure. Seriously limited hydrolysis caused by higher peptide concentrations is consistent with monomeric/dimeric Abeta species participation in the process, confirmed by thioflavine T binding. Inhibition of hydrolysis was caused by β‐sheet breaker peptide (LPFFD), indicating that the Abeta central hydrophobic cluster (amino acids 17–21) participates in the process. The reported Abeta properties suggest that small conformational alterations of the peptide structure may have a pronounced effect on its functions and biological activity.

Influence of reduced glutathione (GSH) and metal ions on soluble and immobilized cabbage peroxidase

Abstract Preparations of soluble and immobilized cabbage peroxidase were subjected to the action of six metal ions: Ag, Zn, Cu, Cd, Pb and Hg. Their influence on peroxidase activity in the presence of an excess of reduced glutathione (GSH) was measured. A great increase in the immobilized peroxidase activity was observed, especially in the presence of Cd ions and GSH.