Stereochemistry of reactions of the inhibitor/substrates L- and D-beta-chloroalanine with beta-mercaptoethanol catalysed by L-aspartate aminotransferase and D-amino acid aminotransferase respectively.

Two members of the alpha-family of PLP-dependent enzymes, L-aspartate aminotransferase and D-amino acid aminotransferase, have been shown to catalyse beta-substitution of L- and D-beta-chloroalanine respectively with beta-mercaptoethanol, reactions typical of the beta-family of PLP-dependent enzymes. The reaction catalysed by L-aspartate aminotransferase has been shown to occur with retention of stereochemistry, a typical outcome for reactions catalysed by beta-family enzymes. There are also indications that the reaction catalysed by D-amino acid aminotransferase may involve retention of stereochemistry. Both enzymes have been shown to catalyse exchange at C-3 when the appropriate enantiomer of beta-chloroalanine is the substrate.

Calmodulin-containing substructures of the centrosomal matrix released by microtubule perturbation.

Calmodulin redistribution in MDCK and HeLa cells subjected to microtubule perturbations by antimitotic drugs was followed using a calmodulin-EGFP fusion protein that preserves the Ca(2+) affinity, target binding and activation properties of native calmodulin. CaM-EGFP targeting to spindle structures in normal cell division and upon spindle microtubule disruption allows evaluation of the dynamic redistribution of calmodulin in cell division. Under progressive treatment of stably transfected mammalian cells with nocodazole or vinblastine, the centrosomal matrix at the mitotic poles subdivides into numerous small 'star-like' structures, with the calmodulin concentrated centrally, and partially distinct from the reduced microtubule mass to which kinetochores and chromosomes are attached. Prolonged vinblastine treatment causes the release of localised calmodulin into a uniform cytoplasmic distribution, and tubulin paracrystal formation. By contrast, paclitaxel treatment of metaphase cells apparently causes limited disassembly of the pericentriolar material into a number of multipolar 'ring-like' structures containing calmodulin, each one having multiple attached microtubules terminating in the partially disordered kinetochore/chromosome complex. Thus drugs with opposite effects in either destabilising or stabilising mitotic microtubules cause subdivision of the centrosomal matrix into two distinctive calmodulin-containing structures, namely small punctate 'stars' or larger polar 'rings' respectively. The 'star-like' structures may represent an integral subcomponent for the attachment of kinetochore microtubules to the metaphase centrosome complex. The results imply that microtubules have a role in stabilising the structure of the pericentriolar matrix, involving interaction, either direct or indirect, with one or more proteins that are targets for binding of calmodulin. Possible candidates include the pericentriolar matrix-associated coiled-coil proteins containing calmodulin-binding motifs, such as myosin V, kendrin (PCNT2) and AKAP450.

Stability and conformational properties of doppel, a prion-like protein, and its single-disulphide mutant.

Both prion protein and the structurally homologous protein doppel are associated with neurodegenerative disease by mechanisms which remain elusive. We have prepared murine doppel, and a mutant with one of the two disulphide bonds removed, in the expectation of increasing the similarity of doppel to prion protein in terms of conformation and stability. Unfolding studies of doppel and the mutant have been performed using far-UV CD over a range of solution conditions known to favour the alpha-->beta transformation of recombinant prion protein. Only partial unfolding of doppel or the mutant occurs at elevated temperature, but both exhibit full and reversible unfolding in chemical denaturation with urea. Doppel is significantly less stable than prion protein, and this stability is further reduced by removal of the disulphide bond between residues 95-148. Both doppel and the mutant are observed to unfold by a two-state mechanism, even under the mildly acidic conditions where prion protein forms an equilibrium intermediate with enhanced beta-structure, potentially analogous to the conversion of the cellular form of the prion protein into the infectious form (PrP(C)-->PrP(Sc)). Furthermore, no direct interaction of either doppel protein with prion protein, either in the alpha-form or the beta-rich conformation, was detectable spectroscopically. These studies indicate that, in spite of the similarity in secondary structure between the doppel and prion protein, there are significant differences in their solution properties. The fact that neither doppel nor its mutant exhibited the alpha-->beta transformation of the prion protein suggests that this conversion property may be dependent on unique sequences specific to the prion protein.