RESUMO
A highly selective and sensitive method for the quantitative determination of L-arginine (Arg) with a fluorescent detection of the reaction product has been developed. The method is based on the use of human liver arginase I isolated from a recombinant producer strain, yeast Hansenula polymorpha, and 2,3-butanedione monoxime, which is used to detect carbamide--the product of enzymatic reactions. The linear concentration range for determining Arg in the final reaction mixture varies from 0.2 to 250 µM, and the detection limit is 0.16 µM. Tests of the new method using commercial Arg-containing pharmaceutical preparations showed a high correlation (R = 1.0) of the results with the manufacturer's data and the results of other methods for Arg detection.
Assuntos
Arginase/química , Arginina/análise , Bioensaio/métodos , Arginase/genética , Humanos , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sensibilidade e EspecificidadeAssuntos
Aldeído Oxirredutases/metabolismo , Formaldeído/metabolismo , Proteínas Fúngicas/metabolismo , Engenharia Genética/métodos , Pichia/enzimologia , Proteínas Recombinantes/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Aldeído Oxirredutases/genética , Southern Blotting , Proteínas Fúngicas/genética , Dosagem de Genes , Cinética , Metanol/metabolismo , Metilaminas/metabolismo , NAD/metabolismo , Pichia/citologia , Pichia/genética , Proteínas Recombinantes/genéticaAssuntos
Cromatos/metabolismo , Complexos de Coordenação/metabolismo , Proteínas Fúngicas/biossíntese , Pichia/isolamento & purificação , Pichia/metabolismo , Compostos de Potássio/metabolismo , Sulfato de Amônio/metabolismo , Meios de Cultura , Proteínas Fúngicas/metabolismo , Engenharia Genética/métodos , Fenômenos Microbiológicos , Mutação , Oxirredução , Pichia/genética , Pichia/crescimento & desenvolvimento , Seleção Genética , Ureia/metabolismo , Purificação da Água/métodosRESUMO
Purified human arginase I preparations homogeneous in SDS-PAAG test were obtained by the affinity chromatography on the synthesized sorbent L-arginine-macroporous glass. Some physico-chemical characteristics of the isolated arginase preparation have been estimated: thermo- and pH-stability, temperature- and pH-optima of the enzyme. The influence of some bivalent metal ions and other additives on enzymatic activity for stabilization of the enzyme and optimization of its storage conditions was studied.
Assuntos
Arginase/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Animais , Arginase/antagonistas & inibidores , Arginase/biossíntese , Arginase/genética , Arginina/metabolismo , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Vidro , Glicerol/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Cinética , Metais/metabolismo , Metais/farmacologia , Peso Molecular , Pichia/genética , Porosidade , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , TemperaturaRESUMO
Screening of strains producing a stable form of L-lactate cytochrome c oxidoreductase (flavocytochrome b2, FC b2) was carried out among 14 yeast species. Enzyme activity was detected in polyacrylamide gel after the electrophoresis of cell-free extracts. The FC b2 of Hansenula polymorpha, Rhodotorula pilimanae, and Kluyveromyces lactis are characterized by high thermostability; in particular, the FC b2 of H. polymorpha retains its activity and tetrameric structure even after heating at 60 degrees C for 10 min. Constitutive synthesis of FC b2 was observed in H. polymorpha grown on either glucose, ethanol, or glycerol. L-Lactate induces de novo synthesis of FC b2, as proved by the use of cycloheximide, an inhibitor of protein synthesis.
Assuntos
Proteínas Fúngicas/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , L-Lactato Desidrogenase (Citocromo)/biossíntese , Saccharomycetales/enzimologia , Saccharomycetales/crescimento & desenvolvimentoRESUMO
A new method of visualization of the activity of flavocytochrome b2 (FC b2; L-lactate: ferricytochrome c oxidoreductase, EC 1.1.2.3) in electrophoretograms was developed, based on the interaction between ferrocyanide (generated during the enzymatic reaction) and Fe23+, resulting in the formation of intensely colored precipitates of Berlin blue. The main advantages of this method were its high sensitivity (less than 0.005 U FC b2 was detected within a suitable time period) and the stability of the dye formed. The method developed can be used for determining FC b2 activity in cell-free extracts (e.g., in the selection of FC b2 producers) and monitoring chromatographic purification of proteins, as well as in other cases associated with FC b2 assessment.
Assuntos
L-Lactato Desidrogenase/análise , Eletroforese/métodos , Compostos Férricos , Ferrocianetos/metabolismo , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase (Citocromo) , Pichia/enzimologia , Sensibilidade e EspecificidadeRESUMO
A set of Escherichia coli 16S rRNA having unique breaks were prepared using the method of oligodeoxyribonucleotide-directed fragmentation with RNAse H. 16S RNA remained compact or dissociated to separate fragments, depending on the cleavage site location in the RNA structure. 16S rRNAs which have been split at different sites or their isolated fragments were used for a reconstitution of the 30S ribosomal subunits. These reconstituted 30S subunits carrying unique breaks at positions 301, 772, 1047 have the same sedimentation coefficients and electron microscopy images as the native subunit. They were active in the poly(U)-directed cell-free system of synthesis of polyphenylalanine.
