RESUMO
In rat, serine dehydratase (SDH) is abundant in the liver and known to be a gluconeogenic enzyme, while there is little information about the biochemical property of human liver serine dehydratase because of its low content and difficulty in obtaining fresh materials. To circumvent these problems, we purified recombinant enzyme from Escherichia coli, and compared some properties between human and rat liver serine dehydratases. Edman degradation showed that the N-terminal sequence of about 75% of human serine dehydratase starts from MetSTART-Met2-Ser3- and the rest from Ser3-, whereas the N-terminus of rat enzyme begins from the second codon of MetSTART-Ala2-. The heterogeneity of the purified preparation was totally confirmed by mass spectrometry. Accordingly, this observation in part fails to follow the general rule that the first Met is not removed when the side chain of the penultimate amino acid is bulky such as Met, Arg, Lys, etc. There existed the obvious differences in the local structures between the two enzymes as revealed by limited-proteolysis experiments using trypsin and Staphylococcus aureus V8 protease. The most prominent difference was found histochemically: expression of rat liver serine dehydratase is confined to the periportal region in which many enzymes involved in gluconeogenesis and urea cycle are known to coexist, whereas human liver serine dehydratase resides predominantly in the perivenous region. These findings provide an additional support to the previous notion suggested by physiological experiments that contribution of serine dehydratase to gluconeogenesis is negligible or little in human liver.
Assuntos
Imuno-Histoquímica , L-Serina Desidratase/química , L-Serina Desidratase/metabolismo , Fígado/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Humanos , Cinética , L-Serina Desidratase/análise , L-Serina Desidratase/efeitos dos fármacos , L-Serina Desidratase/genética , L-Serina Desidratase/isolamento & purificação , Masculino , Dados de Sequência Molecular , Peptídeo Hidrolases/farmacologia , Proteínas/análise , Ratos , Ratos Wistar , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria , Tripsina/farmacologiaRESUMO
It is reported that ethanol enhances DNA synthesis in E. coli cells [Basu, T and Poddar, R. K. (1994), Folia. Microbiol. 39, 3-6]. This communication reports that during growth of E. coli in the presence of 5% v/v ethanol, the derepressed expression of the cytoplasmic enzymes beta-galactosidase and D-serine deaminase per cell increased approximately three fold, while that of the periplasmic enzyme alkaline phosphatase decreased approximately 40% compared to control cell levels. However, in cells transformed with the plasmid pSM 456, bearing phoA-lacZ fusion, the level of induced synthesis of the hybrid protein PhoA-LacZ, controlled by the phoA promoter, was elevated by 25% in the presence of 5% v/v ethanol. This result suggests that the induction of the alkaline phosphatase precursor has also been enhanced by the ethanol treatment, but the inhibition in the export of the precursor across the cytoplasmic membrane, by the influence of ethanol, may represent the reason for the deficient expression of active alkaline phosphatase. It is proposed that there is an ethanol-mediated increase in DNA synthesis, resulting in gene amplification, which may enhance the synthesis of inducible proteins in ethanol-treated cells.
