RESUMO
Abstract L-glutaminase was produced by Streptomyces canarius FR (KC460654) with an apparent molecular mass of 44 kDa. It has 17.9 purification fold with a final specific activity 132.2 U/mg proteins and 28% yield recovery. The purified L-glutaminase showed a maximal activity against L-glutamine when incubated at pH 8.0 at 40 °C for 30 min. It maintained its stability at wide range of pH from 5.0 11.0 and thermal stable up to 60 °C with Tm value 57.5 °C. It has high affinity and catalytic activity for L-glutamine (Km 0.129 mM, Vmax 2.02 U/mg/min), followed by L-asparagine and L-aspartic acid. In vivo, L-glutaminase showed no observed changes in liver; kidney functions; hematological parameters and slight effect on RBCs and level of platelets after 10 days of rabbit's injection. The anticancer activity of L-glutaminase was also tested against five types of human cancer cell lines using MTT assay in vitro. L-glutaminase has a significant efficiency against Hep-G2 cell (IC50, 6.8 μg/mL) and HeLa cells (IC50, 8.3 μg/mL), while the growth of MCF-7 cells was not affected. L-glutaminase has a moderate cytotoxic effect against HCT-116 cell (IC50, 64.7 μg/mL) and RAW 264.7 cell (IC50, 59.3 μg/mL).
Assuntos
Animais/química , Animais/efeitos dos fármacos , Animais/enzimologia , Animais/metabolismo , Animais/farmacologia , Antineoplásicos/química , Antineoplásicos/efeitos dos fármacos , Antineoplásicos/enzimologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Biocatálise/química , Biocatálise/efeitos dos fármacos , Biocatálise/enzimologia , Biocatálise/metabolismo , Biocatálise/farmacologia , Proliferação de Células/química , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/enzimologia , Proliferação de Células/metabolismo , Proliferação de Células/farmacologia , Estabilidade Enzimática/química , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/enzimologia , Estabilidade Enzimática/metabolismo , Estabilidade Enzimática/farmacologia , Glutaminase/química , Glutaminase/efeitos dos fármacos , Glutaminase/enzimologia , Glutaminase/metabolismo , Glutaminase/farmacologia , Glutamina/química , Glutamina/efeitos dos fármacos , Glutamina/enzimologia , Glutamina/metabolismo , Glutamina/farmacologia , Células HeLa/química , Células HeLa/efeitos dos fármacos , Células HeLa/enzimologia , Células HeLa/metabolismo , Células HeLa/farmacologia , /química , /efeitos dos fármacos , /enzimologia , /metabolismo , /farmacologia , Humanos/química , Humanos/efeitos dos fármacos , Humanos/enzimologia , Humanos/metabolismo , Humanos/farmacologia , Cinética/química , Cinética/efeitos dos fármacos , Cinética/enzimologia , Cinética/metabolismo , Cinética/farmacologia , Streptomyces/química , Streptomyces/efeitos dos fármacos , Streptomyces/enzimologia , Streptomyces/metabolismo , Streptomyces/farmacologia , Especificidade por Substrato/química , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/enzimologia , Especificidade por Substrato/metabolismo , Especificidade por Substrato/farmacologiaRESUMO
We examined the requirement of Tropanosoma cruzi protein tyrosine phosphorylation for parasite entry into mammalian cells and analyzed the profile of phosphorylated proteins in infective trypomastigotes. Treatment of metacyclic or tissue culture trypomastigotes with genistein, an inhibitor of protein tyrosine kinase activity, significantly inhibited invasion of cultured HeLa cells. A soluble factor, contained in HeLa cell extract and absent in the extract ot T. cruzi-resistant K562 cells, greatly enhanced phosphorylation levels of a 175-kDa protein (p175) in trypomastigotes. Genistein inhibited p175 tyrosine phosphorylation. P175 was undetectable in noninvasive epimastigotes. The phosphorylation-inducing activity of HeLa cell extract was abrogated by adsorption with metacyclic trypomastigotes but not with epimastigotes or when it was mixed with recombinant protein J18, which contains the entire peptide sequence of gp82, a metacyclic stage-specific surface glycoprotein implicated in target cell invasion. These data suggest that, in metacyclic trypomastigotes, gp82 is the signaling receptor that mediates protein tyrosine phosphorylation necessary for host cell invasion.
Assuntos
Células HeLa/parasitologia , Proteínas Tirosina Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/patogenicidade , Tirosina/metabolismo , Animais , Extratos Celulares/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Células HeLa/química , Humanos , Camundongos , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas de Protozoários/química , Ratos , Transdução de Sinais , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo , Células Tumorais CultivadasRESUMO
HeLa cells were studied with the interference microscope 1 h after heat shock at temperatures of 40 degrees C and 43 degrees C and also under conditions of recovery from the shock. The aim was to investigate changes in patterns of cellular dry mass distribution with the heat shock, based on variation of interference colours in interphase cells. A change in concentration and distribution of a partly high-salt-resistant material in the nuclear and perinuclear regions of the cells was found to be induced by the heat shock at 43 degrees C, a situation which reverted to control under recovery conditions. A similar interference image response was obtained for the heat shock assay at 40 degrees C, but it was detected only during the 4 h recovery period, suggesting that it could have been elicited later. The material induced by the heat shock and visualized by the analysis of interference images is assumed to be a part of the nuclear matrix-intermediate filament cell fraction.