Kinetics of aggregation of UV-irradiated glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscle. Effect of agents possessing chaperone-like activity.

An aggregation test system based on the aggregation of UV-irradiated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle has been proposed. On the basis of the measurements of the enzyme activity and differential scanning calorimetry data a conclusion has been made that UV radiation results in formation of damaged protein molecules with lower thermostability. It was shown that the order of aggregation rate for UV-irradiated GAPDH with respect to the protein was close to 2. This means that such a test system allows detecting the effect of various agents exclusively on the stage of aggregation of unfolded protein molecules. The influence of α-crystallin and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) on aggregation of UV-irradiated GAPDH was studied. Despite the fact that HP-ß-CD accelerates thermal aggregation of non-irradiated GAPDH, in the case of aggregation of UV-irradiated GAPDH HP-ß-CD reveals a purely protective effect.

Effect of 2-hydroxypropyl-beta-cyclodextrin on thermal inactivation, denaturation and aggregation of glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscle.

The effect of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on thermal aggregation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle at 45 degrees C has been studied using dynamic light scattering. In the presence of HP-beta-CD higher values of the rate of aggregation and larger aggregates were registered. The acceleration of GAPDH aggregation was due to destabilization of the enzyme molecule under the action of HP-beta-CD. This is evidenced by the data on thermal inactivation of GAPDH and differential scanning calorimetry.

Comparative analysis of the effects of alpha-crystallin and GroEL on the kinetics of thermal aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

Effects of alpha-crystallin and GroEL on the kinetics of thermal aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using dynamic light scattering and analytical ultracentrifugation. The analysis of the initial parts of the dependences of the hydrodynamic radius of protein aggregates on time shows that in the presence of alpha-crystallin or GroEL the kinetic regime of GAPDH aggregation is changed from the regime of diffusion-limited cluster-cluster aggregation to the regime of reaction-limited cluster-cluster aggregation, wherein the sticking probability for the colliding particles becomes lower the unity. In contrast to alpha-crystallin, GroEL does not interfere with formation of the start aggregates which include denatured GAPDH molecules. On the basis of the analytical ultracentrifugation data the conclusion has been made that the products of dissociation of GAPDH and alpha-crystallin or GroEL play an important role in the interactions of GAPDH and chaperones at elevated temperatures.

Effect of alpha-crystallin on thermal denaturation and aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

The study of thermal denaturation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of alpha-crystallin by differential scanning calorimetry (DSC) showed that the position of the maximum on the DSC profile (T(max)) was shifted toward lower temperatures with increasing alpha-crystallin concentration. The diminishing GAPDH stability in the presence of alpha-crystallin has been explained assuming that heating of GAPDH induces dissociation of the tetrameric form of the enzyme into dimers interacting with alpha-crystallin. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. Suppression of thermal aggregation of GAPDH by alpha-crystallin was studied by dynamic light scattering under the conditions wherein temperature was elevated at a constant rate. The construction of the light scattering intensity versus the hydrodynamic radius (R(h)) plots enabled estimating the hydrodynamic radius of the start aggregates (R(h,0)). When aggregation of GAPDH was studied in the presence of alpha-crystallin, the start aggregates of lesser size were observed.

Mechanism of thermal aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

Thermal denaturation and aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and analytical ultracentrifugation. The maximum of the protein thermal transition (T(m)) increased with increasing the protein concentration, suggesting that the denaturation process involves the stage of reversible dissociation of the enzyme tetramer into the oligomeric forms of lesser size. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. The DLS data support the mechanism of protein aggregation that involves a stage of the formation of the start aggregates followed by their sticking together. The hydrodynamic radius of the start aggregates remained constant in the temperature interval from 37 to 55 degrees C and was independent of the protein concentration (R(h,0) approximately 21 nm; 10 mM sodium phosphate, pH 7.5). A strict correlation between thermal aggregation of GAPDH registered by the increase in the light scattering intensity and protein denaturation characterized by DSC has been proved.

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.

Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme.

We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5'-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants K(d) of 0.4 and 0.9 muM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity.