RESUMO
One component, the i form, of acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) produced by Aspergillus niger was purified from the mycelial extract. The purified enzyme was homogenous on Sephadex G-200 gel filtration, disc electrophoresis and heat inactivation. The purified enzyme was studied and the following results were obtained: 1. The enzyme catalyzed the hydrolysis of a wide variety of phosphomonoesters, but not that of bis(p-nitrophenyl)phosphate, adenosine 3',5'-cyclic monophosphate, fructose 1,6-diphosphate, adenosine 5'-diphosphate or adenosine 5'-triphosphate. 2. Fluoride, orthophosphate, arsenate, borate, molybdate and (+)-tartrate acted as inhibitors. This enzyme was inactivated by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, and was not affected by p-chloromercuribenzoate, N-acetylimidazole, p-diazobenzenesulfonic acid and tetranitromethane. From these results, tryptophan was estimated to play an important role in the enzyme activity. 3. The apparent molecular weight was 310000 by Sephadex G-200 gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggested that the molecular weight of the subunit was approximately 89000. 4. The purified enzyme contained 29% carbohydrate consisting of glucosamine, mannose and galactose. The amino acid composition of this enzyme was not specific compared with other known acid phosphatases.
Assuntos
Fosfatase Ácida/metabolismo , Aspergillus niger/enzimologia , Aspergillus/enzimologia , Fosfatase Ácida/antagonistas & inibidores , Fosfatase Ácida/isolamento & purificação , Aminoácidos/análise , Bromosuccinimida/farmacologia , Carboidratos/análise , Concentração de Íons de Hidrogênio , Peso MolecularRESUMO
Close and distant relationship among 31 strains of Orientia tsutsugamushi (20, two, one and eight strains were isolated in Japan, Korea, China and southeast Asia, respectively) were clarified using phylogenetic analyses based on homologies of 56-kDa type-specific antigen genes. Isolates in Japan, Korea and China were located in eight separate clusters in the phylogenetic tree, and each was designated as JG (Japanese Gilliam type), JP-1 and JP-2 (Japanese Karp 1 and 2 types), Kato, Kawasaki, Kuroki, Shimokoshi and LX-1 types. All isolates originated in southeast Asia, including the prototype Gilliam and Karp strains isolated in Burma and New Guinea, respectively, were distantly located in the phylogenetic tree from those isolates in Japan, Korea and China, indicating that strains of O. tsutsugamushi distributed in northeastern and southeastern Asia are different types.
Assuntos
Antígenos de Bactérias/genética , Orientia tsutsugamushi/classificação , Orientia tsutsugamushi/genética , Filogenia , Tifo por Ácaros/microbiologia , Animais , DNA Bacteriano/genética , Genes Bacterianos , Humanos , Dados de Sequência Molecular , Orientia tsutsugamushi/imunologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Especificidade da EspécieAssuntos
Nucleotídeos de Adenina/farmacologia , Candida/metabolismo , Citratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Candida/crescimento & desenvolvimento , Dinitrofenóis/farmacologia , Relação Dose-Resposta a Droga , Isocitrato Desidrogenase/metabolismo , Isocitratos/metabolismo , Mitocôndrias/metabolismo , Piruvatos/metabolismoRESUMO
The two protein components of Pseudomonas putida tryptophan synthetase have been purified to homogeneity. Although there is general similarity between the Pseudomonas enzyme and that of the enteric bacteria, many differences were found. Components from Escherichia coli and P. putida do not stimulate each other enzymatically, and the enzymes differ in their response to monovalent cations. Serine deamination occurs best with the intact enzyme of P. putida, not with the beta(2) subunit alone as in E. coli. The amino acid compositions of the alpha subunits differ appreciably. These findings extend earlier studies showing differences between enteric organisms and pseudomonads in the regulation and genetic organization of the enzymes of the tryptophan pathway.
Assuntos
Hidroliases , Pseudomonas/enzimologia , Aminoácidos/análise , Sulfato de Amônio , Autoanálise , Centrifugação com Gradiente de Concentração , Precipitação Química , Cromatografia em Gel , Cromatografia por Troca Iônica , Meios de Cultura , Desaminação , Eletroforese Descontínua , Escherichia coli/enzimologia , Genética Microbiana , Temperatura Alta , Hidroliases/análise , Hidroliases/isolamento & purificação , Hidroliases/metabolismo , Mutagênicos , Mutação , Nitrosoguanidinas , Fosfatos , Potássio , Protaminas , Pseudomonas/crescimento & desenvolvimento , Piruvatos/biossíntese , Serina/metabolismo , Especificidade da Espécie , Triptofano/biossíntese , UltracentrifugaçãoRESUMO
The killer character of strain isolated from the main mash of sake brewing which produces a killer substance for sake yeast was transmitted to hybrids of the strain and a standard strain of Saccharomyces cerevisiae through a cytoplasmic determinant. The character was eliminated at 41 degrees C by incubation followed by growth at 30 degrees C. The killer strain produced the killer toxin in a growth-associated manner. A preparation of crude killer toxin extract showed first-order inactivation and a linear Arrhenius plot between 25 and 40 degrees C, with an activation of energy of 55.0 kcal/mol. Addition of 1% of synthetic polymer protected the toxin from inactivation by agitation but not by heat. Enhancement of the killer action toward sensitive yeast cells by only the nucleotide adenosine 5'-diphosphate (ADP) was observed after plating on agar medium as well as after incubation in liquid medium. The addition of CaCl2 reversed the enhancing effect of ADP on killing activity. This action of CaCl2 was inhibited by cycloheximide, suggesting that protein synthesis is required for recovery of toxin-induced cells in the presence of CaCl2. Further, CaCl2 overcame the decrease in the intracellular level of adenosine 5'-triphosphate (ATP) enhanced by ADP in killer-treated cells and also inhibited leakage of ATP from the cells with immediate response. The mode of killing action is discussed in terms of a transient state of the cells and the action of ADP and CaCl2.
Assuntos
Difosfato de Adenosina/farmacologia , Cálcio/farmacologia , Micotoxinas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Bebidas Alcoólicas , Cicloeximida/farmacologia , Resistência Microbiana a Medicamentos , Fermentação , Concentração de Íons de Hidrogênio , Micotoxinas/biossíntese , Oryza , Fenótipo , Saccharomyces cerevisiae/metabolismo , TemperaturaRESUMO
The first four enzymatic activities of the tryptophan synthetic pathway in Pseudomonas putida were found on separate molecules. Gel filtration and density gradient centrifugation experiments did not disclose any associations or aggregations among them. These findings contrast with the situation found in the enteric bacteria, where the first two activities are found in an aggregate and the third and fourth are catalyzed by a single enzyme. Tryptophan synthetase, the last enzyme of the pathway, consists of two dissociable components. The affinity of these components is less in P. putida than is the case in Escherichia coli.
Assuntos
Pseudomonas/enzimologia , Triptofano/biossíntese , Centrifugação com Gradiente de Concentração , Cromatografia em Gel , Isomerases/análise , Ligases/análise , Transferases/análiseRESUMO
Three strains of Orientia tsutsugamushi were isolated from patients in Anan City, Tokushima Prefecture. The strains were identified as Karp type by analyses of reactivities with type-specific monoclonal antibodies. One strain, Okazaki, was isolated in L cells cultivated at 31 C, but not in cells at 36 C or in mice. This strain showed better growth at 31 C than 36 C. This is the first report of an O. tsutsugamushi strain which grows preferentially at low temperatures.
Assuntos
Orientia tsutsugamushi/crescimento & desenvolvimento , Orientia tsutsugamushi/isolamento & purificação , Tifo por Ácaros/microbiologia , Idoso , Animais , Humanos , Japão , Células L , Masculino , Camundongos , Pessoa de Meia-Idade , Peritônio/microbiologia , Especificidade da Espécie , Baço/microbiologia , TemperaturaRESUMO
In our attempts to isolate Orientia tsutsugamushi from trombiculid mites, a strain was successfully isolated from Leptotrombidium fuji collected in Aichi Prefecture, Japan. This is the first case of isolation of O. tsutsugamushi from L. fuji. A phylogenetic analysis based on the base-sequence homology of the 56-kDa type-specific antigen-gene indicated that the strain is a new type which is not closely related to any strains analyzed previously. Three strains isolated from Leptotrombidium pallidum harvested at the same area were identified as being closely related to the JP-2 type (subtype-2 of Karp type in Japan) by phylogenetic analysis.