RESUMO
Rhizobium etli glutaminase A was purified to homogeneity by conventional procedures that included ammonium sulfate differential precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration, and dye-ligand chromatography. Alternatively, the structural glsA gene that codifies for glutaminase A was amplified by PCR and cloned in the expression vector pTrcHis. The recombinant protein was purified to homogeneity by affinity chromatography. This protein showed the same kinetic properties as native glutaminase A (K(m) for glutamine of 1.5 mM and V(max) of 80 micromol ammonium min(-1) mg protein(-1)). Physicochemical and biochemical properties of native and recombinant glutaminase were identical. The molecular mass of recombinant glutaminase A (M(r) 106.8 kDa) and the molecular mass of the subunits (M(r) 26.9 kDa) were estimated by mass spectrometry. These results suggest that R. etli glutaminase A is composed of four identical subunits. The high-level production of recombinant glutaminase A elevates the possibilities for determination of its three-dimensional structure through X-ray crystallography.
Assuntos
Glutaminase/genética , Glutaminase/metabolismo , Rhizobium/enzimologia , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Glutaminase/química , Glutaminase/isolamento & purificação , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Cinética , Espectrometria de Massas , Peso Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Rhizobium/genética , Análise de Sequência de Proteína , TemperaturaRESUMO
We have recently purified a protease from the marine sponge Spheciospongia vesparia. It consists of a single nonglycosylated polypeptide chain with a molecular weight of 29 600 and has one free thiol group. Metal analysis revealed the presence of zinc at 2.02 +/- 0.05 g-atoms per mole of protein, as measured by atomic absorption spectroscopy. The circular dichroism spectrum in the far UV region (183-259 nm) indicates that the sponge protease contains appreciable amounts of beta sheet. This enzyme resembles very much an aminopeptidase from Aeromonas proteolytica concerning activity and some physiochemical characteristics.
Assuntos
Metaloendopeptidases/química , Poríferos/enzimologia , Conformação Proteica , Estrutura Secundária de Proteína , Zinco/análise , Aeromonas/enzimologia , Aminoácidos/análise , Aminopeptidases/química , Animais , Dicroísmo Circular , Metaloendopeptidases/isolamento & purificação , Peso Molecular , Espectrofotometria Atômica , Espectrofotometria Ultravioleta , Compostos de Sulfidrila/análiseRESUMO
A protein that showed activity against proteic (casein and hide powder azure) and synthetic (BAEE and HLPA) substrates was isolated from the marine sponge Spheciospongia vesparia. The protease was purified from an aqueous extract by ammonium sulfate precipitation, gel filtration, hydrophobic and HPLC-anion exchange chromatographies. The purified protease showed a single band in SDS-PAGE minigels and had a molecular weight of 29,600, but when submitted to isoelectric focusing it showed 2 bands with isoelectric points of 4.56 and 4.43. Its catalytic action was inhibited by EDTA and 1,10-phenanthroline, so it seemed to be a metalloprotease.
Assuntos
Endopeptidases/isolamento & purificação , Poríferos/enzimologia , Animais , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Endopeptidases/química , Endopeptidases/metabolismo , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Peso Molecular , Especificidade por SubstratoRESUMO
Hevein, a small protein from the latex of Hevea brasiliensis, has been crystallized by the vapor diffusion method using 2-methyl-2,4-pentanediol and CaCl2 as the precipitant agents. The crystals are orthorhombic space group P21212 with a = 21.88, b = 31.90, and c = 51.24 A and one molecule in the asymmetric unit. The crystals are quite stable to x-rays and suitable for a high resolution three-dimensional structure determination.
