RESUMEN
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).
Asunto(s)
Animales/química , Animales/efectos de los fármacos , Animales/enzimología , Animales/metabolismo , Animales/farmacología , Antineoplásicos/química , Antineoplásicos/efectos de los fármacos , Antineoplásicos/enzimología , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Biocatálisis/química , Biocatálisis/efectos de los fármacos , Biocatálisis/enzimología , Biocatálisis/metabolismo , Biocatálisis/farmacología , Proliferación Celular/química , Proliferación Celular/efectos de los fármacos , Proliferación Celular/enzimología , Proliferación Celular/metabolismo , Proliferación Celular/farmacología , Estabilidad de Enzimas/química , Estabilidad de Enzimas/efectos de los fármacos , Estabilidad de Enzimas/enzimología , Estabilidad de Enzimas/metabolismo , Estabilidad de Enzimas/farmacología , Glutaminasa/química , Glutaminasa/efectos de los fármacos , Glutaminasa/enzimología , Glutaminasa/metabolismo , Glutaminasa/farmacología , Glutamina/química , Glutamina/efectos de los fármacos , Glutamina/enzimología , Glutamina/metabolismo , Glutamina/farmacología , Células HeLa/química , Células HeLa/efectos de los fármacos , Células HeLa/enzimología , Células HeLa/metabolismo , Células HeLa/farmacología , /química , /efectos de los fármacos , /enzimología , /metabolismo , /farmacología , Humanos/química , Humanos/efectos de los fármacos , Humanos/enzimología , Humanos/metabolismo , Humanos/farmacología , Cinética/química , Cinética/efectos de los fármacos , Cinética/enzimología , Cinética/metabolismo , Cinética/farmacología , Streptomyces/química , Streptomyces/efectos de los fármacos , Streptomyces/enzimología , Streptomyces/metabolismo , Streptomyces/farmacología , Especificidad por Sustrato/química , Especificidad por Sustrato/efectos de los fármacos , Especificidad por Sustrato/enzimología , Especificidad por Sustrato/metabolismo , Especificidad por Sustrato/farmacologíaRESUMEN
The effect of 2-naphthylamine, p-nitroaniline, o-phenanthroline, sodium deoxycholate and hydrocortisone succinate on the activity of human urine aminopeptidase, rat kidney methionyl and arginyl aminopeptidase, soybean and Enterolobium contortisiliquum seed aminopeptidase was studied using aminoacyl-2-naphthylamide and L-Leu-p-nitroanilide as substrates. Ki values ranged from 10 microM to 2.7 mM. On the basis of Ki and Km values, and catalytic efficiency for each enzyme, it is clear that the aminopeptidases from human urine and from soybean seed should be assayed with both substrates, whereas L-Leu-p-nitroaniline is a more appropriate substrate for the rat kidney aminopeptidases. Sodium deoxycholate is a better inhibitor than hydrocortisone succinate. Non-competitive inhibition was observed in all cases except for E. contortisiliquum seed aminopeptidase