RESUMO
The 20S proteasome from yeast cells of Candida albicans was purified by successive chromatographic steps to apparent homogeneity, as judged by nondenaturing and denaturing polyacrylamide gel electrophoresis. Its molecular mass was estimated to be 640 kDa by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave at least 10 bands in the range 20-32 kDa. Two-dimensional electrophoresis revealed the presence of at least 14 polypeptides. By electron microscopy after negative staining, the proteasome preparation appeared as typical symmetrical barrel-shaped particles. The enzyme cleaved the peptidyl-arylamide bonds in the model synthetic substrates Cbz-G-G-L-p-nitroanilide, Cbz-G-G-R-beta-naphthylamide, and Cbz-L-L-E-beta-naphthylamide (chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide-hydrolyzing activities). The differential sensitivity of these activities to aldehyde peptides and sodium dodecyl sulfate supported the multicatalytic nature of this enzyme. Three proteasomal subunits were identified as alpha6/Pre5, alpha3/Y13, and alpha5/Pup2 by internal sequencing of tryptic fragments. Their sequences perfectly matched the corresponding deduced amino acid sequences of the C. albicans genes. A fourth subunit was identified as alpha7/Prs1 by immunorecognition with a monoclonal antibody specific for C8, the human proteasome subunit homologue. Treatment of the intact isolated 20S proteasome with acid phosphatase and Western blot analysis of the separated components indicated that the alpha7/Prs1 subunit is obtained as a multiply phosphorylated protein.
Assuntos
Candida albicans/enzimologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/isolamento & purificação , Complexos Multienzimáticos/química , Complexos Multienzimáticos/isolamento & purificação , Fosfatase Ácida/metabolismo , Aldeídos/farmacologia , Sequência de Aminoácidos , Western Blotting , Candida albicans/citologia , Catálise/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/ultraestrutura , Eletroforese em Gel Bidimensional , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Dados de Sequência Molecular , Peso Molecular , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforilação , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma , Dodecilsulfato de Sódio/farmacologia , Especificidade por Substrato , Tripsina/metabolismoRESUMO
A theoretical method was applied to consensus sequences of several members of the snake toxin family as a further approach to examining their conformational homology. Some secondary-structure predictions as well as hydropathy profiles were also examined. A comparison of long neurotoxins themselves reveals a high homology degree. However, their C-terminal fragments show poor homology and the N-terminal fragments appear as the region of maximum variability. Moreover, when the matrix includes the consensus sequence of the genus Laticauda (LNTX1), lacking the disulfide bridge 31-35, the method detects a lower conformational homology in a molecular region centered at position 31. Unlike long neurotoxins, the N-terminal segments of short neurotoxins show a high homology degree, but when comparing short with long neurotoxins, a poor correlation is found in this zone of the molecule. Cytotoxins studied exhibit an excellent conformational homology except when the consensus sequence of cytotoxin homologues CTXE is one of the proteins in the matrix. A comparison between cytotoxins and short neurotoxins reveals homology only in two segments belonging to a beta-sheet structure. A considerable degree of homology is found between the short neurotoxin group and calciseptin and fasciculin as well as between the long neurotoxin group and kappa-neurotoxins.
Assuntos
Citotoxinas/química , Neurotoxinas/química , Conformação Proteica , Venenos de Serpentes/química , Sequência de Aminoácidos , Sequência Consenso , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Alinhamento de SequênciaRESUMO
Monoclonal antibodies (MAb) to human GH (hGH) were used to correlate the antigenic topography of the hormone with its structure. Competition experiments performed in a solid phase RIA system allowed us to measure the reactivity toward the MAb of the following hGH derivatives: hGH 20K (a natural variant lacking residues 32-46), hGH selectively modified in His or Met residues, hGH with the C and/or N-terminal disulfide bond reduced and carbamidomethylated, and hGH cleaved between residues 142-143. Results indicated that fragment 32-46 participates in the structure of epitopes EB1/EB3 and that the C-terminal bridge is located in epitope 10D6, whereas opening of both disulfide bridges alters the entire hGH antigenic surface. His-151 and Met-170 were placed in epitopes NA71 and AC8, respectively, whereas His-18 and Met-14 would be involved in the hGH antigenic domain formed by overlapping epitopes 3C11, 10C1, and HG3. MAb AE5, AE12, and AC3 define a flexible hGH region related to sequence 134-150; the respective epitopes show high conformational mobility induced by modifications in other regions of the molecule. Binding of the different hGH derivatives to lactogenic receptors from female rat liver gave some insights on the localization of the hormone-binding site. Epitopes EB1/EB3 and 10D6 were discarded because there was not a direct correlation between their drastic immunological alterations and the binding properties of the respective hGH derivatives. In the same way, epitopes AE5, AE12, and AC3 were excluded from the hGH-binding domain because a disruption in those sites did not affect the hGH interaction with receptors. We conclude that the hGH structure defined by epitopes 3C11, 10C1, and HG3 is probably related to the binding properties of the hormone.