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We review some theoretical and experimental aspects of the application of the scattering of neutrons and of Mössbauer radiation to the study of the structure and molecular dynamics of polysaccharide gels. The results demonstrate clearly that one can obtain detailed information on the polysaccharide network over distances up to about 1000 Å, and on the molecular dynamics of the polysaccharide backbone and of the associated hydration water.

The transition of prion protein from a mainly α-structured isoform (PrPC) to a β sheet-containing protein (PrPSc) represents a major pathogenetic mechanism in prion diseases. To study the role of PrP structural conformation in prion-dependent neurodegeneration, we analysed the neurotoxicity of PrP in α and β conformations, using a recombinant protein encompassing amino acids 90-231 of the human PrP (hPrP90-231). Using controlled thermal denaturation (S3°C, 1h) we converted hPrP90-231 in a structural isoform displaying PrPSc-related characteristics: high β sheet content, increased aggregability and a slight increase in the resistance to protease K. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing a caspase-3 and p38- dependent apoptosis. Conversely, in the native α-helix-rich conformation, hPrP90-231 did not show significant cell toxicity. The relationship between the structural state of hPrP90-231 and its neurotoxicity was demonstrated, inducing the thermal denaturation of the peptide in the presence of Congo red that prevented both the transition of hPrP90-231 into a β-rich isoform and the acquisition of toxic properties. In conclusion, we report that the toxicity of hPrP90-231 is dependent on its three-dimensional structure, as is supposed to occur for the pathogen PrP during TSE.

1. Tyrosine aminotransferase from guinea-pig liver is inactivated at neutral pH by a factor localized in the microsomal fraction. The inactivation, independent of exogenous L-cysteine, is rapidly reversed by addition of dithiothreitol. 2. The effects of physiological reducing agents on the enzyme inactivation were investigated. L-Cysteine and L-cysteamine enhance the inactivation rate of the enzyme in the presence of microsomal membranes, and also they are able to bring about the loss in enzyme activity independently of microsomal action. Reduced glutathione, at physiological concentration, and NADPH decrease the inactivation rate. Other physiological reducing compounds, as well as oxidized glutathione and NADP+, are without effect. 3. Neither reduced glutathione nor NADPH, unlike dithiothreitol and mercaptoethanol, is able to restore the activity of partially inactivated tyrosine aminotransferase. 4. It is proposed that the intracellular concentration of reduced glutathione might modulate the rate of inactivation of the enzyme in vivo.

Summary Holo and apoenzyme of aspartate aminotransferase from beef kidney are 80% inactivated by photoxidation in the presence of 2 × 10-6m tetraiodofluroescein with the modification of two histidine residues per enzyme protomer. At a higher concentration (1 × 10-5m) a tyrosine residue is also modified. The keto substrates, ketoglutarate and oxalacetate, protect the enzyme from photoxidation.

Summary Cytoplasmic aspartate aminotransferase from beef kindney loses 25% of its activity on nitration with tetranitromethane while the apoenzyme about 95%. In the holoenzyme 0.5 tyrosine residue and 1.0 tyrosine residue in the apoenzyme are nitrated per enzyme protomer. In addition 1 cysteine residue per protomer is oxidized in both. The presence of substrates,a-ketoglutarate and glutamate, both at ten times their Km values, does not change these results. Mercaptoethanol does not affect the residual activity of either the nitrated holo or apoenzyme. Dithionite abolishes the activity of the nitrated holoenzyme by reducing the coenzyme moiety. It has no effect on the native holoenzyme or on either the native or nitroapoenzyme.