RESUMO
The SUMO-1 has been identified as a protein that is highly similar to ubiquitin and shown to conjugate to RanGAP1, PML, Sp200 and I kappa B alpha. The conjugation steps are thought to be similar to those of ubiquitination; and human Ubc9, which is homologous to the E2 enzyme for the ubiquitin conjugation step, was identified and shown to be necessary for the conjugation of SUMO-1 to its target protein. Other essential enzymes involved in this modification, however, remain to be clarified. Here we cloned human Sua1 (SUMO-1 activating enzyme) and hUba2, which are human homologs of yeast Saccharomyces cerevisiae Aos1 and Uba2, respectively. The recombinant proteins, Sua1p and hUba2p, formed a complex. In this complex, hUba2 bound SUMO-1 and this complex had the activity of the SUMO-1 activating enzyme. Furthermore, in an in vitro system, RanGAP1 was modified by SUMO-1 in the presence of Sua1p/Uba2p and hUbc9p, showing that the modification of SUMO-1 could be catalyzed by two enzyme steps, although ubiquitination usually requires three enzyme steps.
Assuntos
Proteínas Ativadoras de GTPase , Enzimas Ativadoras de Ubiquitina , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Clonagem Molecular , Células HeLa , Humanos , Ligases/química , Ligases/genética , Ligases/metabolismo , Dados de Sequência Molecular , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína SUMO-1 , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína LigasesRESUMO
The retention behaviour of beta-lactam antibiotics in micellar electrokinetic chromatography (EKC) was investigated. Sodium dodecyl sulphate (SDS) and sodium N-lauroyl-N-methyltaurate were used an anionic surfactants at concentrations of 0.05-0.3 M. It was found that the retention of ionic substances in micellar EKC is determined by the following three factors: the electrophoretic migration of the ionic substances, the interaction between the ionic substances and ionic surfactants and solubilization of the solute by the micellar phase. A difference in the retention behaviours of cationic substances was observed between the two anionic surfactants, which have different groups neighbouring the charge-bearing groups. The effect of an ion-pairing reagent was also investigated to make the effect of the micelle clearer. All test solutes were successfully separated by micellar EKC at SDS concentrations above 0.1 M, with theoretical plate numbers ranging from 70,000 to 260,000.
Assuntos
Antibacterianos/isolamento & purificação , Cromatografia Líquida/métodos , Fenômenos Químicos , Química , Detergentes , Eletroquímica , Micelas , beta-LactamasRESUMO
The retention behaviour of eleven water-soluble vitamins in micellar electrokinetic chromatography (micellar EKC) was investigated in comparison with capillary zone electrophoresis. Sodium dodecyl sulphate (SDS) and sodium lauroylmethyl taurate were used as the anionic surfactants at concentrations of 0.05-0.2 M in micellar EKC. The retention times of cationic substances increased more rapidly with increasing concentration of the anionic surfactant than those of other substances. This result suggests that ion-pair formation between cationic substances and anionic surfactants contributes to the retention of the former. The difference in the structures of the two surfactants affects the retention behaviour of solutes, especially cationic substances. To clarify the effect of the micelle, an ion-pairing agent that does not form the micelle structure was employed. All solutes were successfully separated within 15 min by using a 650 mm x 0.05 mm I.D. fused-silica tube with a 0.05 M SDS solution (pH 9.0) to give theoretical plates ranging from 100,000 to 350,000.
