RESUMEN
A beta-rutinosidase, which was specific for releasing the disaccharide rutinose from the flavonoid glycoside rutin, was purified from Penicillium rugulosum IFO 7242. This enzyme had the molecular weight of 245,000, a very low optimum pH of 2.2, and the remarkable specificity that the glycosidase did not hydrolyze any other substrates like 4-nitrophenyl beta-glucoside and cellobiose, but only rutin and isoquercitrin.
Asunto(s)
Glicósido Hidrolasas/aislamiento & purificación , Penicillium/enzimología , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Concentración de Iones de Hidrógeno , Peso Molecular , Quercetina/análogos & derivados , Rutina , Especificidad por SustratoRESUMEN
4-aminobenzoate hydroxylase (4ABH) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 4-aminobenzoate to 4-hydroxyaniline. For use as a clinical reagent, the gene encoding 4ABH from Agaricus bisporus was cloned by the RACE method. Also, the cDNA encoding 4ABH was expressed in Escherichia coli cells as a fusion protein with glutathione S-transferase (GST). The expressed GST-4ABH fusion protein (recombinant 4ABH) in the soluble fraction exhibits decarboxylative hydroxylation and additional NADH oxidation activities.We investigated a new ultraviolet spectrometric method for determining serum gamma-glutamyltransferase (gamma-GT) using recombinant 4ABH as a coupling enzyme. The principle of the method is as follows. Using gamma-glutamyl-3-choloro-4-aminobenzoate (L-gamma-glu-PAClBA) and glycylglycine as the donor and acceptor substrates, 3-choloro-4-aminobenzoate (PAClBA) is formed by the catalysis of serum gamma-GT. PAClBA is stoichiometrically converted to 3-choloro-4-hydroxyaniline (PHClA) and NAD(+) by 4ABH and NADH. However, NADH oxidation results in a high reagent blank, which is considered as a drawback for use as a clinical reagent. Using recombinant 4ABH, we examined the effects of pH and detergents on these two activities, and found that several detergents suppress the additional NADH oxidation activity with little or no effect on hydroxylation activity. The results indicate a promising approach to establishing an ultraviolet spectrophotometric method for determining serum gamma-GT activity using L-gamma-glu-PAClBA as the donor substrate and recombinant 4ABH as a coupling enzyme.
Asunto(s)
Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Espectrofotometría/métodos , Rayos Ultravioleta , gamma-Glutamiltransferasa/sangre , Secuencia de Aminoácidos , Detergentes/farmacología , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Hidroxilación/efectos de los fármacos , Modelos Químicos , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , NAD/metabolismo , NADH NADPH Oxidorreductasas/química , Peroxidasas/química , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
1. Serum cholesterol levels in Japan were unrelated to total fat intake and its quality. 2. A significant positive correlation was found between serum cholesterol level and BMI, and the total energy intake was significantly higher in hypercholesterolemic group than in normocholesterolemic group. 3. Hypercholesterolemia seems to occur in the background of accumulation of body fat due to relative excess of energy intake in Japan.
Asunto(s)
Colesterol/sangre , Obesidad/sangre , Adulto , Anciano , Anciano de 80 o más Años , Estatura/fisiología , Índice de Masa Corporal , Peso Corporal/fisiología , Colesterol/fisiología , Femenino , Humanos , Hipercolesterolemia/epidemiología , Hipercolesterolemia/etiología , Japón/epidemiología , Masculino , Persona de Mediana Edad , Estado Nutricional , Obesidad/etiología , Obesidad/fisiopatologíaRESUMEN
The extracellular poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis T1, which hydrolyzes both hydrophobic poly(3-hydroxybutyrate) and water-soluble oligomers of D(-)-3-hydroxybutyrate, lost its hydrolyzing activity toward the hydrophobic substrate on mile trypsin treatment, but retained its activity toward water-soluble oligomers. The molecular mass of the trypsin-treated enzyme was 44 kDa, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, which was 6 kDa smaller than that of the native enzyme (50 kDa). The trypsin-treated enzyme seemed to be less hydrophobic than the native one, because it was rather weakly adsorbed to a hydrophobic butyl-Toyopearl column compared with the native enzyme, and showed no ability to bind to poly(3-hydroxybutyrate), to which the native enzyme tightly bound. These results suggest that, in addition to a catalytic site, the enzyme has a hydrophobic site, which is not essential for the hydrolysis of water-soluble oligomers, but is necessary for the hydrolysis of hydrophobic substrates, and this hydrophobic site is removed from the enzyme by the action of trypsin.
Asunto(s)
Alcaligenes/enzimología , Hidrolasas de Éster Carboxílico/metabolismo , Cinética , Peso Molecular , Fragmentos de Péptidos/metabolismo , Especificidad por Sustrato , Tripsina/farmacologíaRESUMEN
The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T1 has two disulfide bonds, one of which appears to be necessary for the full enzyme activity. This depolymerase hydrolyzed not only hydrophobic poly(3-hydroxybutyrate) but also water-soluble trimer and larger oligomers of D-(-)-3-hydroxybutyrate, regardless of their solubilities in water. Kinetic analyses with oligomers of various sizes indicated that the substrate cleaving site of the enzyme consisted of four subsites with individual affinities for monomer units of the substrate. Analyses of the hydrolytic products of oligomers, which had labeled D-(-)-3-hydroxybutyrate at the hydroxy terminus, showed that the enzyme cleaved only the second ester linkage from the hydroxy terminus of the trimer and tetramer, and acted as an endo-type hydrolase toward the pentamer and higher oligomers. The enzyme appeared to have a hydrophobic site which interacted with poly(3-hydroxybutyrate) and determined the affinity of the enzyme toward the hydrophobic substrate.