RESUMEN
Conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) have been estimated during the autoperoxidation of chromatographically pure phosphatidylcholine from hen eggs (EYPC) in multilamellar liposomes by UV-VIS spectrophotometry. During the propagation phase of autoperoxidation reaction, n-decane (C10) and n-octadecane (C18) gradually inhibit EYPC peroxidation up to 0:2:1 and 1:1 alkane:EYPC molar ratios, respectively. At higher molar ratios, the yield of estimated autoperoxidation products increases. At the highest molar ratio studied (alkane:EYPC = 2:1), the CD and TBARS concentrations exceed their levels in control sample without alkane added. The changes in the lipid bilayer thickness estimated from the small-angle neutron scattering (SANS) curves of unilamellar dioleoylphosphatidylcholine (DOPC) liposomes have indicated that C10 is located in the bilayer hydrophobic region parallel to the DOPC acyl chains at low molar ratios (C10:DOPC < or = 0.4). At higher molar ratios (0.6 < or = C10:DOPC < or = 1.0), the alkane changes its location into the center of the bilayer between the apposing monolayers. The alkane location parallel to polyunsaturated lipid fatty acyl chains RH separates these chains resulting in a decreased frequency of their encounters, in decreased yields of ROO. + RH-->ROOH + R., 2 ROOH-->RO. + ROO. + H2O and RO. + RH-->R. + ROH free radical reactions, and consequently, in decreased autoperoxidation. Autoperoxidation returns to the control values due to alkane redistribution into the bilayer center. 1H decoupled 31P NMR spectra of EYPC + C10 aqueous dispersions have shown that the lipid bilayer transforms into nonbilayer phases at C10:EYPC > 1:1 molar ratios. The inverted hexagonal HII phase with C10 (or C18) preferentially located in the interstitial regions of the HII unit cell displays higher autoperoxidation than the control bilayer sample due to greater motional freedom of RH chains in the HII phase.
Asunto(s)
Alcanos , Yema de Huevo/química , Peroxidación de Lípido , Liposomas , Fosfatidilcolinas/química , Animales , Pollos , Membrana Dobles de Lípidos , Fosfatidilcolinas/análisisRESUMEN
Lipid peroxidation of aerated multilamellar liposomes composed of egg yolk phosphatidylcholine (EYPC) and cholesterol (CHOL) at molar ratios CHOL:EYPC = 0, 0.1, 0.2, 0.3, 0.4, 0.6 and 1.0 was studied during autooxidation and during Fe2+/H2O2-induced peroxidation by following the formation of conjugated diene and thiobarbituric acid reactive substances. The presence of cholesterol in the fluid lipid bilayers has an inhibiting effect on the EYPC peroxidation in the propagation phase of both the autooxidation and Fe(2+)-induced peroxidation free radical chain reactions. This inhibiting effect increases with the increase in CHOL:EYPC molar ratio. The inhibition of EYPC peroxidation by cholesterol probably originates a) from the increased lateral separation of polyunsaturated EYPC acyl chains caused by insertion of cholesterol between EYPC molecules, b) from the increased molecular packing of both the bilayer polar and hydrophobic regions due to the reduced bilayer hydration, and c) from the antioxidant properties of cholesterol.
Asunto(s)
Colesterol/química , Yema de Huevo/química , Fosfatidilcolinas/química , Portadores de Fármacos , Excipientes , Hierro/química , Peroxidación de Lípido , Liposomas , Oxidación-Reducción , Espectrofotometría Ultravioleta , Sustancias Reactivas al Ácido TiobarbitúricoRESUMEN
Amphiphilic N,N-dimethylalkylamine N-oxides (abbreviation CnNO, n is the number of carbon atoms in the alkyl substituent) modulate the activity of the purified sarcoplasmic reticulum (Ca-Mg)ATPase. The phase of insensivity or slight stimulation of the activity at lower homologue concentrations is followed by the inhibition phase at higher concentrations. The potency to inhibit at high concentrations is maximal for the homologue with the alkyl chain length n = 16. The inhibition could be caused by the binding of homologues to binding sites at the protein/lipid bilayer interface.
Asunto(s)
Aminas/síntesis química , ATPasa de Ca(2+) y Mg(2+)/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Óxidos/síntesis química , Retículo Sarcoplasmático/enzimología , Aminas/farmacología , Animales , Inhibidores Enzimáticos/farmacología , Femenino , Técnicas In Vitro , Membrana Dobles de Lípidos/química , Óxidos/farmacología , Conejos , Retículo Sarcoplasmático/efectos de los fármacos , Difracción de Rayos XRESUMEN
To produce two xylanases with Trichoderma reesei grown on glucose, recombinant strains which carry either the xyn1 or the xyn2 (xylanase I and II [XYN I and XYN II]-encoding) structural genes under the expression signals of the homologous pki1 (pyruvate kinase-encoding) gene were constructed. The two types of transformants secreted XYN I or II, respectively, during growth on glucose, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostaining. The corresponding specific xylanase activities of the best transformants on glucose were 76 and 145 U/mg of protein for XYN I and XYN II, respectively, as opposed to that obtained by the parent strain (26 U/mg of protein). When related to the amount of biomass formed, however, they produced only about 4 to 5 U/g, in contrast to much higher activities (10 to 12 U/g) during growth on xylan. The ultrastructural location of XYN II in the transformant strain producing the highest constitutive XYN II formation (ATX2-12) was investigated by immunoelectron microscopy and compared with that in the wild-type strain growing on xylan. Cell extracts from both types of transformants grown on glucose exhibited a higher intracellular xylanase activity than did the parent strain grown on xylan. By using electron microscopy and immunogold labelling, XYN II was detected in the endoplasmic reticulum, Golgi-like vesicles, secretory vesicles, vacuoles, and cell walls. The immunolabel in the vacuoles was detected preferentially in subapical cells. When a recombinant strain which expressed xyn2 from the pki1 promoter was compared with the parent strain during growth on xylan, the former exhibited a less proliferated endoplasmic reticulum and a smaller number of secretory vesicles; however, a higher density of labelling was observed. The relationship of these findings to the efficacy of protein secretion during growth on glucose is discussed.
Asunto(s)
Glucosa/farmacología , Trichoderma/enzimología , Xilosidasas/metabolismo , Secuencia de Bases , Genes Bacterianos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Xilano Endo-1,3-beta-Xilosidasa , Xilosidasas/análisis , Xilosidasas/genéticaRESUMEN
Local anesthetic [2-(alkyloxy)phenyl]-2-(1-piperidinyl)ethyl esters of carbamic acid modulate the activity of the purified sarcoplasmic reticulum (Ca-Mg)ATPase. The phase of insensitivity or slight stimulation of the activity at lower anesthetics concentrations is followed by the inhibition phase at higher concentrations. The potency to inhibit the activity at high concentrations is maximal for the homologue with the hexyloxy substituent and decreases for shorter and longer substituents. The inhibition of activity can be partially reversed by addition of n-decane. The inhibition could be caused by the binding of anesthetics to binding sites at the protein--lipid bilayer interface. The changed thickness of the hydrophobic part of this interface might be responsible for the n-decane reversible inhibition and its dependence on the alkyloxy substituent chain length, while the changed structure of the polar part of this interface could be the cause of the n-decane irreversible inhibiton.
Asunto(s)
Anestésicos Locales/síntesis química , ATPasa de Ca(2+) y Mg(2+)/antagonistas & inhibidores , Carbamatos/síntesis química , Piperidinas/síntesis química , Retículo Sarcoplasmático/enzimología , Anestésicos Locales/farmacología , Animales , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Carbamatos/farmacología , Dibucaína/farmacología , Femenino , Técnicas In Vitro , Piperidinas/farmacología , Conejos , Retículo Sarcoplasmático/efectos de los fármacos , Relación Estructura-Actividad , Tetracaína/farmacologíaRESUMEN
The activity of (Ca(2+)-Mg2+)ATPase reconstituted into egg yolk phosphatidylcholine liposomes is reduced when the lipid has been oxidized before the reconstitution procedure. It is suggested that the reversible decrease in activity is caused by a decrease of the lipid bilayer thickness due to the shortening of lipid acyl chains during autoperoxidation; the irreversible decrease in the activity is caused by chemical reactions of the enzyme SH groups with the lipid autoperoxidation products.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+)/metabolismo , Peroxidación de Lípido , Retículo Sarcoplasmático/enzimología , Animales , Pollos , Femenino , Técnicas In Vitro , Cinética , Membrana Dobles de Lípidos/metabolismo , Liposomas , Óvulo , Fosfatidilcolinas/metabolismo , ConejosRESUMEN
The purpose of the present work was localization of penicillin-binding proteins (PBPs) in the cells of Streptomyces sp. R61 using immunological and autoradiographic methods and electron microscopy. The cells were treated with H3-penicillin G. PBPs of protoplasts were marked with peroxidase labelled IgG to DD-carboxypeptidase of the strain. The results indicate areas privileged in PBPs content at the cytoplasmic membrane and in vesicles located in the periplasmic space. PBPs were visualized in bulges of the protoplasts membrane. In the cells PBPs are present at hyphal tips and in centers at the periphery of cells, which are places of cross wall biosynthesis or branching of hyphae and overproduction of cell wall material.
Asunto(s)
Penicilina G/metabolismo , Receptores de Superficie Celular/análisis , Receptores de Droga/análisis , Streptomyces/química , Sitios de UniónRESUMEN
N-alkyl-N,N,N-trimethylamonium bromides (cnTMA, n = number of carbons in alkyl) stimulate and inhibit the autoperoxidation of egg yolk phosphatidylcholine (EYPC) in liposomes at n less than 12 and n greater than 12, respectively, with maximum stimulation for n = 8. CnTMA intercalate between EYPC molecules (decreasing the yield of ROO. + RH----ROOH+R. reaction, where RH is an unsaturated EYPC acyl chain, R. - EYPC acyl radical, and ROO. - peroxy radical of the EYPC acyl chain) and disorder the hydrophobic region of the bilayer (increasing the oxygen solubility there and thus yield of R. + O2----ROO. reaction). The final level of oxidation is affected by a summation of the EYPC lateral separation and disordering effects.
Asunto(s)
Peroxidación de Lípido/efectos de los fármacos , Liposomas/metabolismo , Fosfatidilcolinas/metabolismo , Compuestos de Amonio Cuaternario/farmacología , Cromatografía en Capa Delgada , Yema de Huevo/química , Cinética , Peroxidación de Lípido/fisiología , Compuestos de Amonio Cuaternario/químicaRESUMEN
During 5 days of autoxidation of egg lecithin liposomes in nonbuffered saline pH dropped from an initial value of 7.4 to 4.5. A linear relationship between oxidation index and pH was obtained. Lipid peroxidation, monitored as conjugated diene and TBA-reactive products, was inhibited significantly by keeping the samples under pH-controlled conditions (7.4 +/- 0.5), compared to controls. Obtained results indicate that the buffering capacity of Tris and Hepes buffers may play a role in their recently reported (D. Fiorentini et al. (1989) Free Radical Res. Commun., 6, 243) inhibitory action against lipid peroxidation of lecithin liposomes.
Asunto(s)
Peróxidos Lipídicos/química , Fosfatidilcolinas/química , Tampones (Química) , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Liposomas/químicaRESUMEN
6-Oxopiperidine-2-carboxylic acid (OCA; cyclic alpha-aminoadipic acid) reverses the L-lysine inhibition of penicillin G production by Penicillium chrysogenum PQ-96. The reaction probably depends on the recovery of L-alpha-aminoadipic acid for penicillin G production from OCA.