Assuntos
Doenças Ósseas/patologia , Articulação do Quadril/patologia , Artropatias/patologia , Acetábulo/patologia , Adolescente , Adulto , Fatores Etários , Idoso , Doenças Ósseas/diagnóstico , Cartilagem Articular/patologia , Criança , Pré-Escolar , Feminino , Cabeça do Fêmur/patologia , Imunofluorescência , Humanos , Lactente , Recém-Nascido , Artropatias/diagnóstico , Masculino , Pessoa de Meia-Idade , Modelos BiológicosAssuntos
Fosfatase Alcalina/antagonistas & inibidores , Fenilalanina , Placenta/enzimologia , Fosfatase Alcalina/isolamento & purificação , Ligação Competitiva , Cromatografia por Troca Iônica , Ésteres , Feminino , Humanos , Concentração de Íons de Hidrogênio , Cinética , Métodos , Modelos Biológicos , Ácidos Fosfóricos , Isótopos de Fósforo , Relação Estrutura-AtividadeAssuntos
Fosfatase Alcalina/metabolismo , Placenta/enzimologia , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/isolamento & purificação , Sítios de Ligação , Cumarínicos , Estabilidade de Medicamentos , Eletroforese em Gel de Amido , Feminino , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas/metabolismo , Cinética , Nitrofenóis , Oscilometria , Fenilalanina , Ácidos Fosfóricos , EspectrofotometriaRESUMO
1. A pseudomonad capable of utilizing 4-chlorophenoxyacetate (CPA) as sole source of organic carbon was isolated from soil. 2. The organism was grown in liquid culture and the following compounds were isolated and identified in culture extracts: 4-chloro-2-hydroxyphenoxyacetate, 4-chlorocatechol, beta-chloromuconate probably the cis-trans isomer and gamma-carboxymethylene-Delta(alphabeta)-butenolide. 3. Cells grown on 4-chlorophenoxyacetate were able to metabolize 4-chloro-2-hydroxyphenoxyacetate, 4-chlorocatechol and gamma-carboxymethylene-Delta(alphabeta)-butenolide without a lag period. They were not adapted to 4-chlorophenol, or to either culture isolated or synthetic beta-chloromuconate, possibly because of stereospecificity towards the cis-cis isomer. 4. On the basis of isolation and induction evidence, the following metabolic pathway is proposed for the breakdown of 4-chlorophenoxyacetate by this organism: 4-chlorophenoxyacetate --> 4-chloro-2-hydroxyphenoxyacetate --> 4-chlorocatechol --> cis-cis-beta-chloromuconate --> gamma-carboxymethylene-Delta(alphabeta)-butenolide --> maleylacetate and fumarylacetate --> fumarate and acetate.
Assuntos
Bactérias/metabolismo , Glicolatos/metabolismo , Herbicidas/metabolismo , Acetatos/metabolismo , Catecóis , Centrifugação , Cloretos/análise , Cromatografia , Cresóis/metabolismo , Meios de Cultura , Manometria , Fenóis/análise , Fenóis/metabolismo , Microbiologia do Solo , Espectrofotometria , Relação Estrutura-Atividade , Raios UltravioletaRESUMO
1. Two Pseudomonas strains isolated from soil metabolized 2,4-dichlorophenoxyacetate (2,4-D) as sole carbon source in mineral salts liquid medium. 2. 2,4-Dichlorophenoxyacetate cultures of Pseudomonas I (Smith, 1954) contained 2,4-dichlorophenol, 2-chlorophenol, 3,5-dichlorocatechol and alpha-chloromuconate, the last as a major metabolite. 3. Dechlorination at the 4(p)-position of the aromatic ring must therefore take place at some stages before ring fission. 4. Pseudomonas N.C.I.B. 9340 (Gaunt, 1962) cultures metabolizing 2,4-dichlorophenoxyacetate contained 2,4-dichloro-6-hydroxyphenoxyacetate, 2,4-dichlorophenol, 3,5-dichlorocatechol and an unstable compound, probably alphagamma-dichloromuconate. 5. Cell-free extracts of the latter organism grown in 2,4-dichlorophenoxyacetate cultures contained an oxygenase that converted 3,5-dichlorocatechol into alphagamma-dichloromuconate, a chlorolactonase that in the presence of Mn(2+) ions converted the dichloromuconate into gamma-carboxymethylene-alpha-chloro-Delta(alphabeta)-butenolide, and a delactonizing enzyme that gave alpha-chloromaleylacetate from this lactone. 6. Pathways of metabolism of 2,4-dichlorophenoxyacetate are discussed.
Assuntos
Acetatos/metabolismo , Herbicidas/metabolismo , Fenóis/metabolismo , Pseudomonas/metabolismo , Ácido 2,4-Diclorofenoxiacético/metabolismo , Catecóis/metabolismo , Cloretos/metabolismo , Cromatografia , Distribuição Contracorrente , Isomerases , Manganês , Oxigenases , Análise Espectral , Raios UltravioletaRESUMO
1. The transient-state and steady-state phases of the reaction between Escherichia coli alkaline phosphatase and 4-methylumbelliferyl phosphate were investigated by using a fluorimetric stopped-flow technique. 2. At low substrate concentration (5mum) in the pH range 3.8-6.3 there was an initial rapid liberation of up to 1mole of 4-methylumbelliferone/mole of enzyme. 3. At very low substrate concentration (0.1mum) in the pH range 4.9-5.9 an initial lag in 4-methylumbelliferone production was observed, from which values for k(+1) and k(-1) could be obtained. 4. The pH profiles for the rates of phosphorylation and dephosphorylation are quite different, and it is postulated that an ionizing group which determines the conformation during the phosphorylation step is not involved in the dephosphorylation step. 5. The binding constants for substrate and P(i) are similar throughout the pH range 4-8. The ionization of substrate or P(i) appeared to have no marked effect on the binding.
Assuntos
Fosfatase Alcalina/metabolismo , Escherichia coli/enzimologia , Sítios de Ligação , Cumarínicos/biossíntese , Concentração de Íons de Hidrogênio , CinéticaRESUMO
1. A purified preparation of alkaline phosphatase from calf-intestinal mucosa was phosphorylated by (32)P-labelled PP(i) at a serine residue on the enzyme. Under the conditions employed, up to 0.15mum-labelled sites were obtained from 1mum-[(32)P]PP(i). 2. The phosphorylated enzyme was labile, the rate of dephosphorylation being similar to the overall rate of substrate hydrolysis. 3. A stopped-flow technique was used to determine the number of phosphomonoesterase active sites, which agreed with the number of (32)P-labelled sites. 4. It is concluded that calf-intestinal alkaline phosphatase is both a phosphomonoesterase and a pyrophosphatase.
Assuntos
Fosfatase Alcalina , Difosfatos , Mucosa Intestinal/enzimologia , Animais , Sítios de Ligação , Bovinos , Esterases , Fluorescência , Cinética , Isótopos de Fósforo , PirofosfatasesRESUMO
1. The kinetics of inhibition of calf-intestinal alkaline phosphatase by inorganic phosphate, fluorophosphate, inorganic pyrophosphate, beta-glycerophosphate and adenosine 5'-triphosphate in the range pH8-10 were investigated. The reference substrate was 4-methylumbelliferyl phosphate. 2. The inhibitions were ;mixed' in that both K(m) and V were affected, but the competitive element was by far the stronger. 3. In each case the pH profile for the competitive K(i) was similar to the pH profile for K(m). Since the K(m) and K(i) values both change 100-fold over the pH range 8-10, it is concluded that the inhibitors compete with the substrate for the same active site. 4. It was also found that the enzyme preparation hydrolysed fluorophosphate, pyrophosphate and adenosine 5'-triphosphate as readily as it hydrolysed 4-methylumbelliferyl phosphate and beta-glycerophosphate. Each pH-activity curve, however, had a different shape, but with the exception of pyrophosphate the activity approached the same maximum value at high pH. 5. Attempts to separate the phosphomonoesterase and pyrophosphatase activities by column chromatography were not successful, and the results of other experiments listed suggest that the two activities are a property of the same enzyme. 6. The effect of Mg(2+) ions is briefly mentioned: the phosphomonoesterase activity is enhanced whereas the pyrophosphatase and adenosine triphosphatase activities are strongly inhibited in the presence of excess of Mg(2+) ions.