RESUMO
The plasmid-encoded lactose genes of the Lactococcus lactis phosphotransferase system encoding Enzyme IIIlac (lacF) and Enzyme IIlac (lacE) have been identified and cloned in Escherichia coli and L. lactis. Nucleotide sequence and transcription analysis showed that these genes are organized into a lactose-inducible operon with the gene order lacF-lacE-lacG-lacX, the latter two genes encoding phospho-beta-galactosidase and a 34-kDa protein with an unknown function, respectively. The lac-operon is immediately followed by an IS element that is homologous to ISS1. Enzyme IIIlac was purified from L. lactis and determination of its NH2-terminal sequence demonstrated that the lacF gene starts with a TTG codon and encodes a 105 amino acid protein (Mr = 11416). Cross-linking studies with the purified enzyme showed that Enzyme IIIlac is active as a trimer. A mutant lacF gene was identified in strain YP2-5 and appeared to encode Enzyme IIIlac containing the missense mutation G18E. The lacF gene could be expressed under control of vector-located promoter sequences resulting in overproduction of Enzyme IIIlac in E. coli and complementation of the L. lactis lacF mutant YP2-5. The deduced amino acid sequence of Enzyme IIlac consists of 586 amino acids (Mr = 61562) and shows the characteristics of a hydrophobic, integral membrane protein. The deduced primary structures of the L. lactis Enzyme IIIlac and Enzyme IIlac are homologous to those of Staphylococcus aureus (72 and 71% identity, respectively) and Lactobacillus casei (48 and 47% identity, respectively). In contrast, the organization of the lactose genes differs significantly between those Gram-positive bacteria. Heterogramic homology in specific domains was observed between the derived amino acid sequences of the lactose-specific enzymes and that of E. coli Enzyme IIIcel and Enzyme IIcel, which suggest a common function in the transport and phosphorylation of these structurally related beta-glucosides.
Assuntos
Genes Bacterianos , Glicosídeo Hidrolases , Lactococcus lactis/genética , Lactose/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , beta-Galactosidase/genética , Sequência de Aminoácidos , Sequência de Bases , Escherichia coli/genética , Lacticaseibacillus casei/genética , Lactococcus lactis/enzimologia , Dados de Sequência Molecular , Óperon , Plasmídeos , Conformação Proteica , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Staphylococcus aureus/genética , Transcrição GênicaRESUMO
Monomeric human growth hormore (hGH) and big hGH obtained by a new purification procedure from human pituitary glands were examined for a possible conversion into different molecular weight forms under in vitro and in vivo conditions. Sephadex G-150 gel filtration was used for separation. Big or monomeric hGH was incubated in hormone-free serum or injected into normal individuals in order to induce a peak of immunoreactive hGH. Specimens from early and late stages of the surge were subjected to gel filtration. It could be shown that not only the big hGH is converted to the smaller form but that monomeric hGH is also recovered in two higher molecular weight regions by gel filtration in vitro and in vivo experiments. A significantly higher conversion of monomeric to big hGH was found in vitro. This suggests an inhibition of big hGH formation from monomeric hGH in normal individuals. There was no apparent time relation of big hGH formation from monomeric hGH in vivo. Regarding the conversion of big hGH, more of this material remained in its original form in vitro. The immunoreactivity recovered in the region of the very large form was found to be higher in vivo. The formation of the monomer from big hGH was similar under in vitro and in vivo conditions.
