RESUMO
AIMS: To evaluate the ability of the haloarchaeon Haloferax volcanii to produce Ag and Au nanoparticles (NPs) and to characterize the obtained material in order to find relevant properties for future potential applications. METHODS AND RESULTS: Nanoparticles were produced by incubating H. volcanii cells with the corresponding metal salt. In the presence of precursor salts, cultures evidenced a colour change associated to the formation of metallic nanostructures with plasmonic bands located in the visible range of the spectrum. X-ray fluorescence analysis confirmed the presence of Ag and Au in the NPs which were spherical, with average sizes of 25 nmol l-1 (Ag) and 10 nmol l-1 (Au), as determined by electronic microscopy. Fourier transformed infrared spectroscopy indicated that both types of NPs showed a stable protein capping. Ag NPs evidenced antibacterial activity and Au NPs improved the specificity of polymerase chain reaction reactions. Au and Ag NPs were able to reduce 4-nitrophenol when incubated with NaBH4 . CONCLUSIONS: Haloferax volcanii is able to synthesize metallic NPs with interesting properties for technological applications. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data demonstrate the ability of H. volcanii to synthesize metal NPs and constitutes a solid starting point to deepen the study and explore novel applications.
Assuntos
Ouro/metabolismo , Haloferax volcanii/metabolismo , Nanopartículas Metálicas/microbiologia , Prata/metabolismo , Antibacterianos/biossíntese , Antibacterianos/química , Antibacterianos/farmacologia , Boroidretos/metabolismo , Ouro/química , Ouro/farmacologia , Nanopartículas Metálicas/química , Nitrofenóis/metabolismo , Tamanho da Partícula , Prata/química , Prata/farmacologiaRESUMO
Maternal physiological hypercholesterolemia occurs during pregnancy, ensuring normal fetal development. In some cases, the maternal plasma cholesterol level increases to above this physiological range, leading to maternal supraphysiological hypercholesterolemia (MSPH). This condition results in endothelial dysfunction and atherosclerosis in the fetal and placental vasculature. The fetal and placental endothelial dysfunction is related to alterations in the L-arginine/nitric oxide (NO) pathway and the arginase/urea pathway and results in reduced NO production. The level of tetrahydrobiopterin (BH4), a cofactor for endothelial NO synthase (eNOS), is reduced in nonpregnant women who have hypercholesterolemia, which favors the generation of the superoxide anion rather than NO (from eNOS), causing endothelial dysfunction. However, it is unknown whether MSPH is associated with changes in the level or metabolism of BH4; as a result, eNOS function is not well understood. This review summarizes the available information on the potential link between MSPH and BH4 in causing human fetoplacental vascular endothelial dysfunction, which may be crucial for understanding the deleterious effects of MSPH on fetal growth and development.
Assuntos
Biopterinas/análogos & derivados , Endotélio Vascular/metabolismo , Hipercolesterolemia/patologia , Animais , Arginina/metabolismo , Biopterinas/metabolismo , Boroidretos/metabolismo , Colesterol/sangue , Feminino , Humanos , Hipercolesterolemia/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , GravidezRESUMO
The objective of this work was to study the immobilization of penicillin G acylase from Escherichia coli on to chitosan-glutaraldehyde beads by multipoint covalent binding. This process was optimized using a 2(3) experimental design. The parameters selected for the present study were the concentrations of glutaraldehyde, phenylacetic acid and sodium borohydride. Three responses were chosen, namely immobilization yield and stabilization factors of enzyme derivatives at high temperature and at alkaline pH. All the runs at the maximum (+1) and minimum (-1) levels were performed at random. Three experiments were performed at the centre point, coded as zero, for experimental-error estimation. With respect to immobilization yield, the main effectors were the concentrations of glutaraldehyde and phenylacetic acid. For stabilization factors at 50 degrees C and at alkaline pH, the main effectors were the concentrations of glutaraldehyde and sodium borohydride and the interaction between them.
Assuntos
Quitosana/química , Enzimas Imobilizadas/metabolismo , Glutaral/química , Penicilina Amidase/metabolismo , Sítios de Ligação , Boroidretos/química , Boroidretos/metabolismo , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Fenilacetatos/química , Fenilacetatos/metabolismo , Ligação ProteicaRESUMO
Glucosamine-6-phosphate deaminase (EC 3.5.99.6) from Escherichia coli is an allosteric enzyme of the K-type, activated by N-acetylglucosamine 6-phosphate. It is a homohexamer and has six allosteric sites located in clefts between the subunits. The amino acid side-chains in the allosteric site involved in phosphate binding are Arg158, Lys160 and Ser151 from one subunit and the N-terminal amino group from the facing polypeptide chain. To study the functional role of the terminal amino group, we utilized a specific non-enzymic transamination reaction, and we further reduced the product with borohydride, to obtain the corresponding enzyme with a terminal hydroxy group. Several experimental controls were performed to assess the procedure, including reconditioning of the enzyme samples by refolding chromatography. Allosteric activation by N-acetylglucosamine 6-phosphate became of the K-V mixed type in the transaminated protein. Its kinetic study suggests that the allosteric equilibrium for this modified enzyme is displaced to the R state, with the consequent loss of co-operativity. The deaminase with a terminal hydroxy acid, obtained by reducing the transaminated enzyme, showed significant recovery of the catalytic activity and its allosteric activation pattern became similar to that found for the unmodified enzyme. It had lost, however, the pH-dependence of homotropic co-operativity shown by the unmodified deaminase in the pH range 6-8. These results show that the terminal amino group plays a part in the co-operativity of the enzyme and, more importantly, indicate that the loss of this co- operativity at low pH is due to the hydronation of this amino group.
Assuntos
Acetilglucosamina/análogos & derivados , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Escherichia coli/enzimologia , Acetilglucosamina/metabolismo , Acetilglucosamina/farmacologia , Regulação Alostérica , Sítio Alostérico , Aminação , Boroidretos/metabolismo , Catálise/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Metionina/metabolismo , Modelos Moleculares , Ligação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Substâncias Redutoras/metabolismo , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The cleavage of disulfide bonds is the major modification of chloroplast fructose-1,6-bisphosphatase when the light-mediated ferredoxin-thioredoxin system enhances the activity of the enzyme. In vitro, only thiol-bearing compounds are functional in the stimulation of fructose 1,6-bisphosphate hydrolysis. This investigation was undertaken to determine the effectivity of other reductants for enhancing the catalytic capacity. In the presence of 1 mM fructose 1,6-bisphosphate and 0.1 mM Ca2+, the five-fold activation triggered by 3.5 mM tributylphosphine is further potentiated by 15% (v/v) 2-propanol. When the enzyme is incubated in the presence of 0.15 M sodium trichloroacetate in place of the cosolvent, NaH4B initially stimulates the activity but subsequently causes the inactivation of the enzyme. A model developed to analyze this dual effect suggests that the concerted action of fructose 1,6-bisphosphate, Ca2+ and trichloroacetate yields an enzyme form that is slightly activable by reduction (t0.5 = 28 min.). However, chloroplast fructose-1,6-bisphosphatase becomes highly sensitive to trichloroacetate inactivation (t0.5 = 5 min.) when NaH4B reduces fructose 1,6-bisphosphate. Hence, the thiol/disulfide exchange constitutes a particular case of reductive mechanisms that stimulate the activity of chloroplast fructose-1,6-bisphosphatase.