RESUMEN
Pure and mixed monolayers of mono- and dihexoside cerebrosides with cholesterol have been characterized at the air/water interface. Cholesterol oxidase was used as a reporter enzyme for the cholesterol-cerebroside interaction in the mixed monolayers. The cerebrosides either were derived from bovine brain extracts or were synthetic. The dihexoside cerebrosides were synthesized by coupling of the hepta-O-acetyl-alpha-lactosyl- or maltosylphosphoramidates with D-erythro-N-acylceramides in dichloromethane, in the presence of trimethylsilyl triflate and molecular sieves, followed by hydrolysis of the acetate-protecting groups. All of the bovine-brain-derived cerebrosides [galactosyl cerebroside (GalCer, types I and II), glucosyl cerebroside (GlcCer), and lactosyl cerebroside (LacCer)] had very condensed force-area isotherms (compressibility values of 3-5 x 10(-3) m/mN at 20 mN/m), as did the synthetic N-stearoylmaltosylceramide (N-18:0 MaltCer). Shorter-chain synthetic cerebrosides (N-8:0 LacCer and N-8:0 MaltCer) had more expanded isotherms, with compressibility values of 15-17 x 10(-3) m/mN. When cholesterol was included in mixed monolayers of monohexoside cerebroside, it did not induce significant condensation of packing (indicating that cholesterol did not increase the order of the acyl chains). However, with dihexoside cerebrosides, a cholesterol-induced condensing effect was observed, which amounted to a 11-19% reduction in the observed mean molecular area. When cholesterol oxidase was used to titrate the stoichiometry of cholesterol/cerebroside in mixed monolayers, at which pure cholesterol clusters appeared, it was observed that in monohexoside cerebroside monolayers cholesterol clusters were present even below a 1:1 molar stoichiometry.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Cerebrósidos/química , Colesterol/química , Animales , Secuencia de Carbohidratos , Bovinos , Cerebrósidos/síntesis química , Colesterol Oxidasa , Datos de Secuencia Molecular , Oxidación-Reducción , TermodinámicaRESUMEN
In this study we have examined the cholesterol oxidase (Streptomyces cinnamomeus) catalyzed conversion of either 5-cholesten-3 beta-ol or 5-cholesten-3-one into 4-cholesten-3-one in pure sterol or mixed phospholipid-containing monolayers at the air/buffer interface. The mean molecular area requirement of 5-cholesten-3-one in a pure monolayer was slightly smaller than the comparable area required by 5-cholesten-3 beta-ol (although the collapse pressure was markedly lower for 5-cholesten-3-one), and both sterols were about equally capable of condensing the lateral packing density of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine at a lateral surface pressure of 20 mN/m. Both sterols were converted by cholesterol oxidase to 4-cholesten-3-one, the reaction being faster with 5-cholesten-3-one as compared to 5-cholesten-3-beta-ol. When the temperature-dependency of the cholesterol oxidase catalyzed conversion of the sterols to 4-cholesten-3-one was examined, the Arrhenius activation energy was calculated to +30 kJ/mol and +27 kJ/mol for 5-cholesten-3 beta-ol and 5-cholesten-3-one, respectively, when the sterols were presented to the enzyme as pure sterol monolayers at a lateral surface pressure of 20 mN/m. With a mixed monolayer containing 40 mol% sterol and 60 mol% EPC, the corresponding activation energies were +107 kJ/mol and +96 kJ/mol for 5-cholesten-3 beta-ol and 5-cholesten-3-one, respectively. With the monolayer system used, it appeared that the over all rate-limiting step in the enzyme-catalyzed conversion of 5-en-sterols to 4-en-3-one was the desorption of the sterol molecules from the monolayer into the active site of the enzyme at the interface. This appeared to be true both with pure sterol monolayers as well as with mixed monolayers containing phosphatidylcholine.
Asunto(s)
Colestenonas/metabolismo , Colesterol Oxidasa/metabolismo , Colesterol/metabolismo , Isomerismo , Oxidación-Reducción , Fosfolípidos/metabolismo , Esteroles/metabolismoRESUMEN
Addition of increasing amounts of interleukin-4 (IL-4) to large granular lymphocytes (LGL) had a selective downregulative effect on the interleukin-2 beta-chain (p70) receptor expression. A 40% inhibition of the p70 expression compared to untreated cells was already observed after a 24-h incubation with IL-4. This decrease in p70 receptor expression had a marked suppressive effect on their proliferative response to IL-2. In addition, LGL cultured in the presence of both IL-2 and IL-4 substantially decreased the cytotoxic activity against the erythroleukemia cell line K562. Our data therefore indicate an important regulative role for IL-4 on the LAK-generation.