ABSTRACT
Este trabalho objetivou investigar a dependência entre o equilíbrio de desenovelamento da invertase por uréia, segundo um mecanismo de equilíbrio entre dois estados, com algumas propriedades termodinâmicas do solvente. Os valores das propriedades termodinâmicas do solvente foram calculados com aplicação da teoria de McMillian-Mayer. O equilíbrio de desenovelamento da invertase foi acompanhado por titulação espectrofotométrica em 280nm com uma solução de uréia a 8 mol.L-¹. O bom ajuste a um modelo de primeira ordem apresentado pelos dados de equilíbrio da invertase, bem como da RNase A e da RNase T1, é consistente com a idéia de que o alto conteúdo em carboidratos da invertase não afeta o mecanismo geral de desenovelamento. Entretanto, um aumento na concentração de uréia produziu uma diminuição na energia livre de desenovelamento (DeltaG°U ) para a intertase, e um aumento nos valores de DeltaG°U para Rnase A e Rnase T1. A invertase também exigiu maiores valores de atividade de água (Aw) do que a RNAse A e a RNAse T1 para ser desenovelada. O diferente comportamento da invertase em relação a aquelas enzimas pode ser relevante para fornecer informação adicional sobre os mecanismos detalhados do enovelamento e desenovelamento de proteínas.
Subject(s)
Ribonuclease T1 , Ribonuclease, Pancreatic , Ribonucleases , Solvents , Thermodynamics , Urea , WaterABSTRACT
<p><b>OBJECTIVE</b>To study the inhibition of maxizyme (Mz) directed against the mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG --> AGT) both in cell-free system and in MHCC97 cell lines.</p><p><b>METHODS</b>Maxizyme and control mutant maxizyme (G5 --> A5) were designed by computer and cloned into the eukaryotic expression vector pBSKneoU6 (pU6Mz, pU6asMz). Mz was driven by T7 RNA polymerase promoter in vitro. In the cell lines, U6 promoter was driven by RNA PolIII. The mutant type p53 gene fragment was cloned into the pGEM-T vector under the T7 promoter control. The 32P-labeled mtp53 transcript was the target RNA. Cold maxizyme transcripts were incubated with 32P-labeled target RNA in vitro. pU6Mz was introduced into MHCC97 cells by Lipofectamine2000 and mtp53 expression was analyzed by RT-PCR and Western blot.</p><p><b>RESULTS</b>In vitro cleavage showed that pU6Mz was very active with cleavage efficiency of 42% while pU6asMz was not. The wild type p53 was not cleaved. Partial down-regulation of mtp53 mRNA and mtp53 protein were observed in MHCC97 cells transfected with pU6Mz but not those with pU6asMz. The proliferation of MHCC cells was inhibited by MTT analysis.</p><p><b>CONCLUSION</b>Our findings suggest that the chimeric U6 maxizyme against the mtp53 is a new promising gene therapeutic agent in treating hepatocellular carcinoma.</p>
Subject(s)
Humans , Carcinoma, Hepatocellular , Genetics , Cell Line, Tumor , Genetic Therapy , Methods , Genetic Vectors , Liver Neoplasms , Genetics , Nucleic Acid Conformation , Point Mutation , Protein Conformation , RNA, Catalytic , RNA, Messenger , Metabolism , Recombinant Fusion Proteins , Ribonuclease T1 , Pharmacology , Tumor Suppressor Protein p53 , GeneticsABSTRACT
The mode of binding of the substrate analog 2'-deoxy-2'-fluoroguanylyl- (3',5')-cytidine (GfpC) to RNase T1 was determined by computer modelling studies. The results obtained are in good agreement with the observations of 1H-nmr studies. The modes of binding of the substrate analog GfpC and the substrate GpC to the enzyme RNase T1 have been compared. Though the guanine base favours to occupy the same site of the enzyme in both the complexes, significant differences are observed in the local environment around the 2'-substituent group of guanosine ribose moiety. In the RNase T1-GpC complex, the 2'-OH group is in close proximity to the side chain carboxylic acid of Glu58 which leads to the formation of a hydrogen bond. However, in the RNase T1-GfpC complex, 2'-fluorine is positioned away from Glu58 due to electrostatic repulsion and instead forms a hydrogen bond with His40 imidazolium group. The results obtained rule out the possibility of His40 serving as the base group in catalysis as suggested by 1H-nmr studies and further support the primary role assigned to Glu58 as the general base group by earlier computer modelling and the recent site directed mutagenesis studies. This study also implies that the 2'-deoxy-2'-fluoro substrate analog may not serve as a good model for determining the amino acid residue which serves as the general base group in ribonuclease catalysed reactions.