RESUMO
The lectin, wheat germ agglutinin (WGA), has been shown to have a significant larvicidal effect on the European corn borer, a major insect pest of corn. In order to characterize this toxic effect, we have undertaken structure-function studies on WGA. To this extent, the effect of cyanogen bromide (CNBr) on the conformation, subunit interactions, and biological activity of WGA has been investigated. The CNBr-modified lectin exhibits no toxicity to the ECB, cannot dimerize, does not bind to N-acetylglucosamine or its polymers, has no or vastly reduced hemagglutinating activity against red blood cells of different animals, and shows loss of an antigenic determinant by immunodiffusion. The CD spectrum of CNBr-WGA is not significantly different from that of native WGA, although the intrinsic fluorescence shows about 30% quenching. Our results suggest that the integrity of the N-terminal domain of WGA is essential for dimer formation. Furthermore, toxicity of WGA to ECB may be intrinsically related to its ability to dimerize and bind to sugar residues.
Assuntos
Hemaglutinação , Lepidópteros/efeitos dos fármacos , Aglutininas do Germe de Trigo/farmacologia , Sequência de Aminoácidos , Animais , Carboidratos , Cromatografia em Gel , Dicroísmo Circular , Brometo de Cianogênio , Larva/efeitos dos fármacos , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Conformação Proteica , Aglutininas do Germe de Trigo/química , Aglutininas do Germe de Trigo/toxicidadeRESUMO
Twelve herpesviral deoxythymidine kinases were examined for regions of sequence similarity by multiple alignment. Six highly conserved sites were observed. Site 1 corresponded to a glycine-rich loop that forms part of the ATP-binding pocket in porcine adenylate kinase (PAK), and site 5 corresponded to a region in PAK, located on one lobe of the cleft, that contains arginine residues that bind substrate phosphoryl groups. Site 3, consisting of the motif -DRH-, is thought to be involved in thymine/deoxythymidine recognition; site 4, which is nearby, probably participates in this function as well. The functions of sites 2 and 6 have not been identified. Secondary structure predictions were made by the Garnier method and averaged for each position in the multiple alignment. The structure predicted for all six sites was typically a short flexible region (turn or coil) at or adjacent to the site, flanked by rigid structures (helix or sheet) on either side.
Assuntos
Adenilato Quinase/genética , Arginina , Herpesviridae/enzimologia , Timidina Quinase/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Herpesviridae/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Fosforilação , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , SuínosRESUMO
The 2'-fluoropyrimidine nucleoside analogs 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC). 1(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU), and 1(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) showed higher in vitro activity against herpes simplex virus type 1 (HSV-1), than equine herpesvirus 1 (EHV-1) or pseudorabies virus (PRV). Comparison of the 50% plaque inhibitory doses for HSV-1 and its mutant MMdUr-20 in cell cultures with inhibition constants (Ki's) for the viral deoxythymidine kinases (dTKs) suggests that in the infected cell FMAU is phosphorylated by host enzymes. As compared to HSV-1, EHV-1 and PRV were more resistant to E-5-(2-bromovinyl-2'-deoxyuridine (BVdU) and to the 2'-fluoropyrimidine analogs, as are HSV-2 and the HSV-1 mutants MMdUr-20 and S1. Because the dTKs of the latter lack deoxythymidylate kinase (dTMPK) activity, there appears to be a correlation between resistance to these analogs and BVdU on the one hand, and lack of dTMPK activity on the other. We predict that EHV-1 and PRV dTKs will be shown to lack significant dTMPK activity.
