These enzymes are either monomeric or multimeric, compris ing one, two, four or six selleck chem SB203580 subunits. Although members of the M17 family have been mainly described as multi meric, some of them behave as monomeric. For exam ple, recombinant LAPs of Leishmania spp. and P. falciparum exhibit a homohexameric structure, while those of Haemaphysalis longicornis, Schistosoma mon soni and Schistosoma japonicum seem to be monomeric enzymes. In contrast, LAPTc displays an elec trophoretic migration pattern corresponding to a homo tetramer. However, it must be taken into account that some proteins display abnormal migration both in SDS PAGE and size exclusion chromatography, and assembly of recombinant proteins might differ from that of their native forms.

In addition, LAPTc three dimen sional structure may contribute to its fast migration since it was not heated before PAGE. Oligopeptidase B of T. cruzi also displays abnormal electrophoretic migra tion under the same experimental conditions. Nevertheless, other enzymes such as T. cruzi cathepsin B and the hexameric leucyl aminopeptidase of Borrelia burgdorferi show the expected migration. The hexameric nature of LAPTc was thus con firmed by analytical ultracentrifugation and MALLS assays, which are accurate techniques to determine molecular masses of macromolecules in the absence of any interaction with matrices or surfaces. As it has been observed for members of the M17 and M29 families, such as leucyl aminopeptidase of bovine lens, aminopep tidase A of E.

coli, and TAPBb, the oligomeric assembly of LAPTc does not require the presence of interchain disulfide bonds because monomerization occurs in the absence of a reducing agent. The oligo meric structures of these enzymes may be maintained through hydrogen bridges, Van der Waals and hydro phobic interactions as is observed for bovine lens ami nopeptidase. The advantage of multimeric over monomeric structures is still unclear, but it is possible that a quaternary structure allows not only hydrophobic regions to be hidden within the protein assembly but also the reduction of the macromolecule surface in con tact with the medium, thus restraining the amount of water required to stabilize these proteins. The asso ciation between enzymatic activity and multimeric struc ture of leucyl aminopeptidases suggests that either the active sites are formed at the subunit junctions or the three dimensional assembly stabilizes the active site of each monomer. The latter hypothesis is supported by the fact that the activity of bovine lens leucyl Carfilzomib aminopep tidase depends on the stabilization of each monomer active site by the structure of the oligomer. LAPTc comprises several distinctive characteristics of M17 leucyl aminopeptidases.