RESUMO
We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-kappaB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-kappaB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.
Assuntos
Benzoquinonas/metabolismo , Propionatos/metabolismo , Receptores de Droga/química , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Humanos , Células Jurkat , Dados de Sequência Molecular , NF-kappa B/antagonistas & inibidores , Receptores de Droga/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
A cDNA for a bovine brain-specific protein p25 which had been originally found as a major protein in a partially purified fraction of tau protein kinases was cloned. The deduced amino-acid sequence consists of 218 amino acids (M(r) 23,472) and has no significant homology with previously reported proteins. p25 is a basic protein and has a consensus sequence for ATP-binding in the C-terminal region.
Assuntos
Química Encefálica , DNA Complementar/genética , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , DNA Complementar/química , Dados de Sequência MolecularAssuntos
Infecções por Citomegalovirus/etiologia , Citomegalovirus/crescimento & desenvolvimento , Síndromes de Imunodeficiência/complicações , Ativação Viral , Citomegalovirus/imunologia , Infecções por Citomegalovirus/microbiologia , Infecções por Citomegalovirus/transmissão , Feminino , Humanos , Síndromes de Imunodeficiência/imunologia , GravidezRESUMO
Structure of the complex of Streptomyces subtilisin inhibitor (SSI) with subtilisin BPN' was studied by examining the thermal denaturation and reducibility of disulfide bonds. The denaturation temperature of the complex was significantly higher than that of the enzyme. Two disulfide bonds localized in the inhibitor side were completely reduced in the complex, whereas only one of them was reduced in the free SSI. Gel filtration of the reduced complex solution showed clearly that the main products of reduction of the complex were two peptide fragments of SSI divided at the active site. The resistive disulfide bond in the complexed inhibitor became accessible as a result of a large conformational change due to splitting of the half-reduced inhibitor.
Assuntos
Proteínas de Bactérias/metabolismo , Subtilisinas/antagonistas & inibidores , Dicroísmo Circular , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Nucleoside diphosphate (NDP) kinase is an enzyme that transfers the gamma-phosphate of nucleoside triphosphates to nucleoside diphosphates. Besides this well-defined role, recent evidence suggests that NDP kinase may be implicated in a wide variety of essential cellular processes. In this paper, we showed that the NDP kinase of Drosophila exhibited protein kinase activity as well as autophosphorylation. Ovalbumin was phosphorylated with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) or ATP. Protein kinase activity was not detected in NDP kinase mutant, abnormal wing discs (awd). These results suggest that this activity could be one of the functions of NDP kinase essential for normal fly development, since awd gene is lethal.