Effect of alpha-crystallin on thermostability of mitochondrial aspartate aminotransferase.

Effect of alpha-crystallin on thermal inactivation, denaturation and aggregation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been in the focus of this study. Acceleration of heat-induced inactivation of mAAT was demonstrated in the presence of alpha-crystallin. According to the data of differential scanning calorimetry, alpha-crystallin induces destabilization of the mAAT molecule. The size of protein aggregates formed at heating of mAAT at a constant rate (1 degree C/min) has been defined by dynamic light scattering. The obtained data show that aggregation of mAAT in the presence of alpha-crystallin proceeds in the regime of reaction-limited cluster-cluster aggregation.

Study of kinetics of thermal aggregation of mitochondrial aspartate aminotransferase by dynamic light scattering: protective effect of alpha-crystallin.

Thermal aggregation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been studied at various temperatures and various protein concentrations by dynamic light scattering. The character of the dependence of protein aggregate size on time indicates that aggregation of mAAT proceeds in the regime of diffusion-limited cluster-cluster aggregation. Suppression of mAAT aggregation by alpha-crystallin is due to transition of the aggregation process into the regime of reaction-limited cluster-cluster aggregation. Realization of this regime of aggregation means that the sticking probability for the colliding particles is less than unity.

Thermal inactivation, denaturation and aggregation of mitochondrial aspartate aminotransferase.

A comparative study of thermal denaturation and inactivation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been carried out (10 mM Na phosphate buffer, pH 7.5). Analysis of the data on differential scanning calorimetry shows that thermal denaturation of mAAT follows the kinetics of irreversible reaction of the first order. The kinetics of thermal inactivation of mAAT follows the exponential law. It has been shown that the inactivation rate constant (k(in)) is higher than the denaturation rate constant (k(den)). The k(in)/k(den) ratio decreases from 28.8+/-0.1 to 1.30+/-0.09 as the temperature increases from 57.5 to 77 degrees C. The kinetic model explaining the discrepancy between the inactivation and denaturation rates has been proposed. The size of the protein aggregates formed at heating of mAAT at a constant rate (1 degrees C min(- 1)) has been characterized by dynamic light scattering.

Hepatoprotective action of prostaglandin A(2) analogs under CCl(4)-induced liver injury in vitro.

AIM: The cytoprotective effects of six novel synthetic prostaglandin A(2) analogs against carbon tetrachloride (CCl(4)) as a toxic agent were studied with isolated rat liver hepatocytes in vitro. RESULTS: It was found that hepatocytes treatment with CCl(4) induced: (i) a significant increase of lactic dehydrogenase (LDH) release from cytoplasm; (ii) leakage of glutamate dehydrogenase (GDH) and acid phosphatase from mitochondria and lysosomes, respectively; (iii) 10-fold increase of trien conjugates formation; and (iv) a reduction of free SH-groups by 50%. Prostanoids U-26, U-9 and U-34 decreased cytotoxic index of CCl(4) on average by 1.5-2.0 times and were more effective than PGI(2), the well-known hepatoprotector of prostanoids type. The protective action of the prostanoids was not a cAMP- or Ca(2+)-dependent process. However, prostanoids U-26, U-9 and U-34 normalized intracellular content of SH-groups, reduced trien conjugates formation by 60-80% and strongly prevented enzyme leakage through cellular membranes. They were also able to inhibit CCl(4) effects via decreasing cytochrome P(450)2E1 activity. CONCLUSION: The results obtained demonstrate that prostanoids provide cytoprotective effects on liver hepatocytes through the prevention of lipid peroxidation of the plasma and the cellular membranes and maintenance of their barrier function.

Interaction of polyanions with basic proteins, 2(a) : influence of complexing polyanions on the thermo-aggregation of oligomeric enzymes.

The ability of synthetic polyanions to suppress thermo-aggregation of the oligomeric enzymes (glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and aspartate aminotransferase) has been established. The ability of the polyanions to reduce the thermo-aggregation increased in the order poly(methacrylic acid) < poly(acrylic acid) < sodium poly(styrene sulphonate), which agreed well with the increase, in the same order, of the charge density of the chains. The lengthening of the chains, as well as the rise in their relative content, resulted in an increase of the ability to reduce thermo-aggregation, mentioned above. Complete prevention of the enzyme aggregation was achieved when highly charged polyanions of a relatively high degree of polymerization were used in a concentration sufficient to solubilize the protein. Complexing with the polyanions prevented thermo-aggregation of the enzymes, but not their thermo-denaturation. The adverse effect of the complexing polyanions on the catalytic activity was reduced by the addition of a synthetic polycation, which resulted in a significant reactivation (up to 40%) of the enzyme. The possibility of preventing the thermo-aggregation of enzyme molecules and then partly restoring the enzyme activity, appears to be of particular interest when studying the aggregation mechanism of proteins that are prone to form the amyloid structures responsible for the development of neurodegenerative diseases like Alzheimer's disease, bovine spongiform encephalopathy and Huntington disease. This finding can also be considered as an important step in the creation of artificial chaperones.