RESUMEN
The effect of prothymosin alpha on transcriptional elongation has been examined. The addition of prothymosin alpha to COS-1 and NIH3T3 cell nuclei engaged in run-on transcription stimulated RNA synthesis approximately two- to threefold in a dose-dependent manner. Polyglutamic acid or a random polypeptide composed of glutamic acid, alanine, and tyrosine, did not substitute for prothymosin alpha. Enhanced transcription occurred in the presence of high and low doses of actinomycin D and in the presence of alpha-amanitin, but not in nuclear extracts. The stimulatory effect was dependent on a limiting concentration of one nucleoside triphosphate and was nearly abrogated by saturating levels of precursors. In the presence of Sarkosyl, which itself increases transcription, prothymosin alpha was almost ineffectual. The data are consistent with a model in which prothymosin alpha does not interact directly with polymerases but, instead, nonspecifically decreases the barriers to diffusion of charged molecules in electrostatically charged environments.
Asunto(s)
Precursores de Proteínas/fisiología , Sarcosina/análogos & derivados , Timosina/análogos & derivados , Timosina/fisiología , Transcripción Genética , Células 3T3 , Animales , Células COS , Citidina Trifosfato/metabolismo , Células HeLa , Humanos , Ratones , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Precursores de Proteínas/genética , ARN/metabolismo , Sarcosina/farmacología , Timosina/genéticaRESUMEN
OBJECTIVE: To assess the potential benefits of the antioxidant activity of certain pharmacological agents that may be beneficial in the treatment of cardiovascular disease, including coronary heart disease and heart failure, by reducing irreversible cell injury due to oxyradical damage. METHODS: The antioxidant activities of representative calcium antagonists were examined and correlated with the molecular membrane interactions of the compounds, as measured by radioligand binding assays and high resolution differential scanning calorimetry. RESULTS: The results of these experiments show a direct relationship between the antioxidant activities of the calcium antagonists and their affinity for the membrane lipid bilayer, as well as their ability to modulate membrane thermodynamic properties (amlodipine > verapamil >> diltiazem). The charged 1,4-dihydropyridine calcium antagonist amlodipine had the highest affinity for the membrane bilayer (Kp10(4)) and produced the largest changes in membrane thermodynamic properties, including a reduction in the thermal phase transition temperature (-11%), enthalpy (-14%) and cooperative unit size (-59%), relative to control phosphatidylcholine liposomes. CONCLUSIONS: These findings indicate that lipophilic calcium antagonists inhibit lipid peroxidation in cellular membranes as a result of modulating physicochemical properties of the membrane lipid bilayer, independently of calcium channel inhibition. The antioxidant activity of highly lipophilic calcium antagonists, such as amlodipine, may contribute to new cytoprotective mechanisms of action in cardiovascular disease.
Asunto(s)
Amlodipino/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Membrana Celular/fisiología , Peroxidación de Lípido , Animales , Antioxidantes/farmacología , Sitios de Unión , Fenómenos Biofísicos , Biofisica , Enfermedades Cardiovasculares/prevención & control , Membrana Celular/efectos de los fármacos , Masculino , Microsomas , Ratas , Ratas Sprague-Dawley , TermodinámicaRESUMEN
Our study examines the effect of apoptosis on prothymosin alpha, an abundant, nuclear protein intimately involved with proliferation of all mammalian cells. When HeLa cells were treated with actinomycin D, with etoposide, or with staurosporine following synchronization with hydroxyurea, they underwent apoptosis based on several specific criteria, including fragmentation of DNA and activation of specific caspases. Similarly treated NIH3T3 cells arrested and displayed no indicators of apoptosis. In HeLa, but not in NIH3T3 cells, prothymosin alpha levels declined precipitously and a truncated version of the protein was formed. The following observations implicate caspase activity: (1) The truncated polypeptide arose only in the treated HeLa cell cultures. (2) The appearance of the truncated polypeptide coincided with the activation of caspase 3 and the cleavage of poly(ADP-ribose) polymerase, a known caspase substrate. (3) Carbobenzoxy-DEVD-fluoromethylketone, a cell-permeable caspase 3 inhibitor, blocked cleavage and degradation of prothymosin alpha. (4) The same inhibitor, when added to mixed extracts of apoptotic and normal cells, prevented cleavage of intact prothymosin alpha. (5) Recombinant caspase 3 and, to a much lesser extent, caspase 7 truncated purified prothymosin alpha. (6) In HeLa cells, cleavage occurred at three overlapping caspase 3-like sites with the consensus sequence D-X-X-D and released 10 to 14 residues from the carboxyl terminus, including the core nuclear localization signal. Two immediate consequences of the cleavage were observed: truncated prothymosin alpha was no longer confined to the nucleus and it was deficient in phosphate. These data suggest that the disabling of prothymosin alpha is a significant event in apoptosis. J. Cell. Physiol. 182:256-268, 2000. Published 2000 Wiley-Liss, Inc.
Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/fisiología , Timosina/análogos & derivados , Células 3T3/metabolismo , Células 3T3/fisiología , Animales , Células HeLa/metabolismo , Células HeLa/fisiología , Humanos , Ratones , Fragmentos de Péptidos/metabolismo , Fosforilación , Timosina/química , Timosina/fisiología , Distribución TisularRESUMEN
The antioxidant activities of representative calcium antagonists, including amlodipine, verapamil, and diltiazem, were measured in hepatic microsomal membranes by the Fe-catalyzed, hydroxyl radical-producing system (dihydroxyfumarate + Fe3+) and assessed by malondialdehyde (MDA) formation. Despite the absence of L-type calcium channels in this membrane preparation, the calcium antagonists showed dose-dependent antioxidant activity. The biophysical mechanism for calcium-antagonist antioxidant activity was evaluated using radioligand binding assays, high-resolution differential scanning calorimetry, and small-angle x-ray diffraction approaches. These analyses demonstrated that calcium-antagonist antioxidant potency correlated directly with the compounds' relative affinity for the membrane lipid bilayer and ability to modulate membrane thermodynamic properties (amlodipine >> verapamil > diltiazem). The charged 1,4-dihydropyridine calcium antagonist, amlodipine, had the highest affinity for the membrane lipid bilayer (Kp>10(4)) and produced the largest changes in membrane thermodynamic properties, including a reduction in thermal phase transition temperature (-11%), enthalpy (-14%), and cooperative unit size (-59%), relative to control phosphatidylcholine liposomes. Electron density profiles generated from x-ray diffraction data demonstrated that amlodipine effected a broad and dose-dependent increase in molecular volume associated with the membrane hydrocarbon core. These data indicate that lipophilic calcium antagonists inhibit lipid peroxidation in cellular membranes as a result of modulating physicochemical properties of the membrane lipid bilayer, independently of calcium channel inhibition. Amlodipine had the most potent antioxidant activity as a result of distinct biophysical interactions with the membrane lipid bilayer. The nonreceptor-mediated antioxidant activity of calcium antagonists may contribute to cytoprotective mechanisms of action in cardiovascular diseases.
Asunto(s)
Antioxidantes/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Peroxidación de Lípido/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Amlodipino/química , Amlodipino/farmacología , Animales , Antioxidantes/química , Bloqueadores de los Canales de Calcio/química , Rastreo Diferencial de Calorimetría , Diltiazem/farmacología , Relación Dosis-Respuesta a Droga , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Masculino , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Verapamilo/farmacología , Difracción de Rayos XRESUMEN
The effect of the highly lipophilic calcium channel antagonist (CCA) amlodipine on membrane oxyradical damage was examined and compared to that of other CCA analogs and a sulfhydryl-containing ACE inhibitor in isolated membrane vesicles enriched with polyunsaturated fatty acids (PUFA). Under physiological-like conditions, the dihydropyridine CCA amlodipine significantly (P<0.001) inhibited lipid peroxide formation (>10(2) microM) at concentrations as low as 10.0 nM. Under identical conditions, inhibition of lipid peroxide formation was not observed with representative CCA analogs (felodipine, verapamil, diltiazem) or the ACE inhibitor, captopril, at concentrations as high as 1.0 microM. The potent antioxidant activity of amlodipine is attributed to distinct membrane physico-chemical interactions. High-resolution differential scanning calorimetry showed that amlodipine effected marked changes in membrane thermodynamic properties as compared to other CCA analogs, including a marked reduction in the thermal phase transition temperature (-2.6 degrees C), enthalpy (-4.8 J/g) and cooperative unit size (-59%), relative to control samples. These findings indicate that the chemical structure of amlodipine contributes to distinct membrane biophysical interactions that lead to potent lipid antioxidant effects, independent of calcium channel modulation. These findings provide insights into potential new mechanisms of action for the charged CCA amlodipine.
Asunto(s)
Amlodipino/farmacología , Amlodipino/uso terapéutico , Antioxidantes/uso terapéutico , Antihipertensivos , Calcio/antagonistas & inhibidores , Rastreo Diferencial de Calorimetría , Diltiazem/farmacología , Dimiristoilfosfatidilcolina/farmacología , Relación Dosis-Respuesta a Droga , Ácidos Grasos Insaturados/metabolismo , Concentración 50 Inhibidora , Peroxidación de Lípido , Fosfatidilcolinas/farmacología , Verapamilo/farmacologíaRESUMEN
Prothymosin alpha gene expression accompanies growth of all mammalian cells. The protein, which is abundant, exceedingly acidic, and localized to the nucleus, is further distinguished by the presence of clustered phosphorylated glutamic acid residues (Trumbore et al., 1997, J Biol Chem 272:26394-26404). These glutamyl phosphates are energy rich and unstable in vivo and in vitro (Wang et al., 1997, J Biol Chem 272:26405-26412). To understand the function of prothymosin alpha in greater detail, the turnover of its phosphates was examined in metabolically manipulated cells. Phosphate half-lives in growing, mock transfected, and vector-transfected COS cells were compared with the half-life in cells transfected with the prothymosin alpha gene to determine the fate of the predominantly ectopic phosphorylated protein. The values obtained--72-75 min in cells with normal levels of the protein, but 118 min in cells with surplus prothymosin alpha--led us to conclude that underutilized phosphates persist whereas functioning phosphates disperse. Cell-cycle-specific differences in the half-lives were observed in NIH3T3 cells: 72 min while cycling, 83 or 89 min during arrest in or progression through S phase, but 174 min during M-phase arrest. In the presence of actinomycin D, the value was about 145 min regardless of whether cells were quiescent or growing. In these experiments, reduced utilization of prothymosin alpha's glutamyl phosphates, signaled by an increase in their half-lives, accompanied the attenuation or abolition of transcription. Our data suggest that prothymosin alpha fuels an energy-requiring step in the production, processing, or export of RNA.
Asunto(s)
Glutamatos/metabolismo , Fosfatos/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Células 3T3 , Animales , Células COS , Dactinomicina/farmacología , Semivida , Ratones , Mitosis , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Unión Proteica , Precursores de Proteínas/genética , ARN/biosíntesis , Fase S , Timosina/genética , Timosina/metabolismo , TransfecciónRESUMEN
According to published accounts, prothymosin alpha exhibits high evolutionary conservation from yeast to man (Makarova, T., Grebenshikov, N., Egorov, C., Vartapetian, A., and Bogdanov, A. FEBS Lett. 257, 247-250, 1989). We report here our failure to find evidence for prothymosin alpha in yeast using three biochemical approaches: hybridization of yeast mRNA and genomic DNA with human prothymosin alpha coding region probes, performance of the polymerase chain reaction with yeast genomic template DNA and three sets of primers recognizing human prothymosin alpha coding region sequences, and isolation of yeast proteins essentially as described in the publication above. A survey of the Saccharomyces cerevisiae complete genome database using the program BLASTp verified our findings: there is no prothymosin alpha-homologue in yeast. Furthermore, DNA representing organisms from bacteria to amphibians also failed to hybridize with the same probes. Therefore, the presence of a prothymosin alpha gene in animals other than mammals is highly unlikely.
Asunto(s)
Precursores de Proteínas/genética , Saccharomyces cerevisiae/genética , Timosina/análogos & derivados , Secuencia de Aminoácidos , ADN/genética , Bases de Datos Factuales , Genoma Fúngico , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Precursores de Proteínas/química , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Timosina/química , Timosina/genéticaRESUMEN
Human and monkey prothymosin alpha contain activated carbonyl groups on glutamic acid residues. Three lines of evidence indicate the existence of unusual phosphates. 1) Prothymosin alpha continued to be metabolically labeled with [32P]orthophosphoric acid despite a mutation at Ser1, the sole site of phosphate in purified bovine prothymosin alpha (Sburlati, A. R., De La Rosa, A., Batey, D. W., Kurys, G. L., Manrow, R. E., Pannell, L. K., Martin, B. M., Sheeley, D. M., and Berger, S. L. (1993) Biochemistry 32, 4587-4596). 2) Immediately upon cell lysis, the pH stability curves of metabolically labeled native [32P]prothymosin alpha or a [32P]histidine-tagged variant resembled the pH stability curve of acetyl phosphate. 3) After a brief incubation at pH 7, these curves changed from a pattern diagnostic for an acyl phosphate to that characteristic of a serine or threonine phosphate, an observation consistent with transfer of phosphate in vitro. Our data indicate that most of prothymosin alpha's phosphates are subject instantaneously to hydrolysis, based on the observation that greater than 90% of the phosphate initially found at pH 7 disappeared at the extremes of pH. Rapid loss of phosphate was not affected by the presence of phosphatase inhibitors including 50 mM sodium fluoride, 1 mM okadaic acid, and 0.5 mM calyculin A. The amount of phosphate missing could not be ascertained, but the trifling amount recovered on Ser or Thr depended heavily on conditions favoring the transient survival of labile phosphate. Further analysis using COS cells lysed in the presence of sodium borohydride showed that: 1) phosphate recovered on prothymosin alpha decreased 8-fold when lysates were treated with borohydride; 2) the reagent caused 4-8 glutamic acid residues/molecule to vanish; 3) using [3H]NaBH4, label was introduced into proline, a product derived from reductive cleavage of phosphoglutamate; and 4) [3H]proline was localized almost exclusively to a peptide with pronounced homology to the histone binding site of nucleoplasmin, a chromatin remodeling protein found in Xenopus laevis. Our data demonstrate that prothymosin alpha is energy-rich by virtue of stoichiometric amounts of glutamyl phosphate.
Asunto(s)
Ácido Glutámico/química , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Secuencia de Aminoácidos , Animales , Borohidruros/química , Células COS , Bovinos , Clonación Molecular , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Fosforilación , Precursores de Proteínas/química , Precursores de Proteínas/genética , Timosina/química , Timosina/genética , Timosina/metabolismoRESUMEN
Prothymosin alpha is a small, highly acidic, abundant, nuclear, mammalian protein which is essential for cell growth. Our laboratory has recently shown that primate prothymosin alpha contains stoichiometric amounts of phosphate on the glutamyl groups of the protein and that in vitro the phosphate undergoes rapid hydrolysis or transfer to a nearby serine residue. Here an assay for the presence of acyl phosphates in vivo has been developed by measuring stable phosphoserine and phosphothreonine in vitro. The assay was used to determine the half-life of the acyl phosphates on prothymosin alpha in vivo by pulse-labeling HeLa cells with [32P]orthophosphate and chasing using three different techniques: permeabilization with digitonin to allow extracellular ATP to equilibrate with the intracellular pool; electroporation in the presence of ATP to reduce the specific activity of [32P]ATP by expansion of the pool; and incubation with inorganic phosphate. Regardless of the method, the phosphate turned over with a half-life of 75-90 min. The ability of cells to phosphorylate old prothymosin alpha molecules was established by demonstrating equivalent labeling of the protein with [32P]orthophosphate in the presence and absence of cycloheximide. The half-life of the acyl phosphates was also studied in resting and growing NIH3T3 cells, with measured values of 30-35 and 70 min, respectively. Our data suggest that the "activity" of prothymosin alpha involves the turnover of its acyl phosphates and that it participates in a function common to all nucleated mammalian cells regardless of whether they are quiescent or undergoing rapid proliferation. This is the first measurement of the stability of protein-bound acyl phosphates in vivo.
Asunto(s)
Organofosfatos/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Células 3T3 , Animales , Células COS , División Celular , Electroporación , Semivida , Células HeLa , Humanos , Ratones , Fosfatos/metabolismo , Radioisótopos de Fósforo , Biosíntesis de Proteínas , ARN/metabolismo , Timosina/metabolismoRESUMEN
We have studied the thermotropic properties of a wide variety of cannabinoids in DPPC bilayers. The molecules under study were divided into four classes: (a) classical cannabinoids possessing a phenolic hydroxyl group; (b) delta9-THC metabolites with an additional hydroxyl group on the C ring; (c) non-classical cannabinoids, and (d) cannabinoids with a protected phenolic hydroxyl group. The results showed that the first three groups have similar effects on the thermotropic properties of DPPC bilayers up to x = 0.05 (molar ratio) and that these effects do not parallel their biological activity. For concentrations less than x = 0.01, cannabinoids affect mainly the pretransition temperature in a progressive manner until its final abolishment. At x = 0.05, they further affect the main phase transition by lowering its phase transition temperature and broadening its half width. At high concentrations the thermograms have multiple components, indicating that membranes are no longer homogeneous but rather consist of different domains. At these concentrations cannabinoids with more hydroxyl groups give simpler thermograms. Low concentrations of cannabinoids in group d affect significantly the pretransition temperature, while high concentrations affect only marginally the main phase transition by slightly lowering its temperature and broadening its half width. These results point out the importance of the phenolic hydroxyl group in inducing membrane perturbations. The d-spacing data from our small angle X-ray diffraction experiments show that delta8-THC produces significant structural changes in the lipid bilayer, including the gel-phase tilting angle, the intermolecular cooperativity and the gauche:trans conformer ratio. Conversely, the inactive analog Me-delta8-THC does not cause drastic changes to the bilayer structure.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química , Rastreo Diferencial de Calorimetría , Cannabinoides/farmacología , Membrana Dobles de Lípidos/química , Difracción de Rayos X , Cannabinoides/química , Estructura Molecular , TermodinámicaRESUMEN
Lipid peroxidation causes cellular damage during aging and various diseases, including atherosclerosis. Chronic administration of highly lipophilic calcium channel blockers (CCB) may reduce lipid peroxidation as a result of concentration in cell membranes and altering physico-chemical properties of the lipid bilayer. In the study, small angle X-ray scattering was used to examine reconstituted cardiac membrane lipid bilayers in the presence of CCB with various antioxidant activities, including nisoldipine, nifedipine, and diltiazem. Analysis of one-dimensional electron density profiles demonstrated that these compounds have different molecular distributions relative to the center of the membrane: diltiazem (+/- 14-22 A), nifedipine (+/- 12-22 A), and nisoldipine (+/- 7-22 A). The overall hydrocarbon core width for control samples was 44 A and was unaffected by the addition of drugs at these concentrations (< 1% by mass). High resolution differential scanning calorimetry indicated that CCB markedly perturbed the thermotropic properties of liposomes, including thermal phase transition temperature and enthalpy, relative to control samples. The effects of these compounds on membrane thermotropic properties correlate with their reported antioxidant activities. These data support the hypothesis that calcium channel blockers have potent physico-chemical interactions with the membrane lipid bilayer, which may underlie their antioxidant activity.
Asunto(s)
Antioxidantes/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Membrana Dobles de Lípidos/química , Rastreo Diferencial de Calorimetría , Peroxidación de Lípido/efectos de los fármacos , Difracción de Rayos XRESUMEN
Alzheimer's disease is characterized by changes in phospholipid metabolism leading to a perturbation in the levels of phosphomonoesters, including L-Phosphoserine (L-PS). These early changes in lipid metabolism may result in a defect in membrane bilayer structure, leading to increased rates of beta-amyloid formation. To investigate the effect of L-PS on membrane lipid bilayers, small angle x-ray diffraction and high resolution differential scanning calorimetry (DSC) approaches were used with liposomes composed of lecithin and cholesterol. A one-dimensional electron density profile of a control dimyristoyl phosphatidylcholine (DMPC)/cholesterol lipid bilayer with a unit cell dimension of 52 A at 37 degrees C was generated from the x-ray diffraction data. Following incubation with 2.0 mM L-PS, a broad decrease in electron density +/- 4.12A from the lipid bilayer center was observed concomitant with an increase in the width of the phospholipid headgroup electron density and a 3A reduction in lipid bilayer width. The interactions of L-PS with DMPC lipid bilayers were concentration-dependent, highly affected by cholesterol content and reproduced in egg phosphatidylcholine/cholesterol liposomes. DSC analysis showed that millimolar (1.0-5.0 mM) L-PS levels decreased the phase transition cooperative unit size of DMPC liposomes in a highly concentration-dependent manner which was significantly greater in preparations containing 10 mol% cholesterol. These data provide direct evidence that phosphomonoester levels modulate the biophysical properties of the membrane lipid bilayer which may, in turn, lead to altered structure/function relationships in AD.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Membrana Dobles de Lípidos , Fosfoserina/farmacología , Rastreo Diferencial de Calorimetría , Colesterol/análisis , Dimiristoilfosfatidilcolina , Microanálisis por Sonda Electrónica , Humanos , Membrana Dobles de Lípidos/química , Termodinámica , Factores de TiempoRESUMEN
Phosphoserine (L-PS) is among several phosphomonoesters found to be elevated in autopsied Alzheimer's disease (AD) brain tissue. To investigate the molecular interactions of L-PS with membrane lipid bilayers, small angle X-ray diffraction and high resolution differential scanning calorimetry (DSC) approaches were used with liposomes composed of lecithin and cholesterol. A one-dimensional electron density profile of a control dimyristoyl phosphatidylcholine (DMPC)/cholesterol lipid bilayer with a unit cell dimension of 52 A at 37 degrees C was generated from the X-ray diffraction data. Following incubation with 2.0 mM L-PS, a broad decrease in electron density +/- 4-12 A from the lipid bilayer center was observed concomitant with an increase in the width of the phospholipid headgroup electron density and a 3 A reduction in lipid bilayer width. The interactions of L-PS with DMPC lipid bilayers were concentration-dependent, highly affected by cholesterol content and reproduced in egg phosphatidylcholine/cholesterol liposomes. DSC analysis showed that millimolar (1.0-5.0 mM) L-PS levels decrease the phase transition cooperative unit size of DMPC liposomes in a highly concentration-dependent manner which was significantly greater in preparations containing cholesterol. The endotherm width at half-maximum doubled at 5.0 mM and 1.25 mM L-PS, respectively, for DMPC and DMPC/cholesterol liposomes. These data provide direct evidence that elevated phosphomonoester levels modulate the biophysical properties of the membrane lipid bilayer which may, in turn, lead to altered structure/function relationships in membranes during AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fosfoserina/metabolismo , Fenómenos Biofísicos , Biofisica , Rastreo Diferencial de Calorimetría , Colesterol/metabolismo , Dimiristoilfosfatidilcolina/metabolismo , Humanos , Liposomas/metabolismo , Difracción de Rayos XRESUMEN
MK-801, a noncompetitive antagonist of the NMDA (N-methyl-D-aspartate) receptor, has protective effects against excitotoxicity and ethanol withdrawal seizures. We have determined membrane/buffer partition coefficients (Kp[mem]) of MK-801 and its rates of association with and dissociation from membranes. Kp[mem] (+/- SD) = 1137 (+/- 320) in DOPC membranes and 485 (+/- 99) in synaptoneurosomal (SNM) lipid membranes from rat cerebral cortex (unilamellar vesicles). In multilamellar vesicles, Kp[mem] was higher: 3374 (+/- 253) in DOPC and 6879 (+/- 947) in SNM. In cholesterol/DOPC membranes, Kp[mem] decreased as the cholesterol content increased. MK-801 associated with and dissociated from membranes rapidly. Addition of ethanol to SNM did not affect Kp[mem]. MK-801 decreased the cooperative unit size of DMPC membranes. The decrease was smaller than that caused by 1,4-dihydropyridine drugs, indicating a weaker interaction with the hydrocarbon core. Small angle x-ray diffraction, with multilayer autocorrelation difference function modeling, indicated that MK-801 in a cholesterol/DOPC membrane (mole ratio = 0.6) causes a perturbation at approximately 16.0 A from the bilayer center. In bilayers of cholesterol/DOPC = 0.15 (mole ratio) or pure DOPC, the perturbation caused by MK-801 was more complex. The physical chemical interactions of MK-801 with membranes in vitro are consistent with a fast onset and short duration of action in vivo.
Asunto(s)
Maleato de Dizocilpina/farmacología , Lípidos de la Membrana/química , Membranas Artificiales , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Fenómenos Biofísicos , Biofisica , Rastreo Diferencial de Calorimetría , Corteza Cerebral/química , Colesterol/química , Maleato de Dizocilpina/química , Maleato de Dizocilpina/farmacocinética , Técnicas In Vitro , Cinética , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Liposomas/química , Lípidos de la Membrana/metabolismo , Fosfatidilcolinas/química , Ratas , Sinaptosomas/química , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Agua/química , Difracción de Rayos XRESUMEN
Nimodipine is a 1,4-dihydropyridine (DHP) calcium channel blocker which is used in the treatment of neurological deficits associated with subarachnoid hemorrhage. Small angle x-ray diffraction, differential scanning calorimetry, and equilibrium and kinetic binding techniques were used to study the interaction of nimodipine with bovine brain phosphatidylcholine (BBPC) membranes of varying cholesterol content. At concentrations (5 x 10(-10) M) near its Kd, the membrane partition coefficient of nimodipine was inversely related to the cholesterol to phospholipid (C:P) mole ratio in both model and native (rat synaptoneurosome) membranes. The nonspecific dissociation rate of nimodipine from BBPC was significantly slower at low C:P mole ratio (0.1:1) than at high C:P mole ratio (0.6:1). Calorimetric analysis showed that nimodipine decreased both the main phase transition temperature and cooperative unit size of melt for dimyristoyl phosphatidylcholine, dependent on membrane cholesterol content. Small angle x-ray diffraction analysis showed that nimodipine occupies a position in BBPC approx +/- 15 A from the center of the hydrocarbon core, near the hydrocarbon core/water interface. These data indicate that nimodipine is an amphiphilic molecule which rapidly washes out of and transports across membrane bilayers, facilitating its interactions with membranes and possibly its transport across the blood-brain barrier.
Asunto(s)
Encéfalo/efectos de los fármacos , Lípidos de la Membrana/química , Nimodipina/química , Nimodipina/farmacología , Animales , Rastreo Diferencial de Calorimetría , Bovinos , Membrana Celular/efectos de los fármacos , Técnicas In Vitro , Cinética , Modelos Estructurales , Ratas , Ratas Sprague-DawleyRESUMEN
Amiodarone is a drug used in the treatment of cardiac arrhythmias and is believed to have a persistent interaction with cellular membranes. This study sought to examine the structure and location of amiodarone in a membrane bilayer. Amiodarone has a high membrane partition coefficient on the order of 10(6). Small angle x-ray diffraction was used to determine the position of the iodine atoms of amiodarone in dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under conditions of low temperature and hydration where the DPPC bilayer is in the gel state. The time-averaged position of the iodine atoms was determined to be approximately 6 A from the center (terminal methyl region) of the lipid bilayer. A dielectric constant of kappa = 2, which approximates that of the bilayer hydrocarbon core region, was used in calculating a minimum energy structure for membrane-bound amiodarone. This calculated structure when compared with the crystal structure of amiodarone demonstrated that amiodarone could assume a conformation in the bilayer significantly different from that in the crystal. The results reported here are an attempt to correlate the position of a membrane-active drug in a lipid bilayer with its time-averaged conformation. This type of analysis promises to be of great use in the design of drugs with greater potency and higher specificity.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina , Amiodarona , Membrana Dobles de Lípidos , Amiodarona/análisis , Animales , Humedad , Modelos Moleculares , Conformación Molecular , Músculos/análisis , Conejos , Retículo Sarcoplasmático/análisis , Espectrofotometría Ultravioleta , Difracción de Rayos XRESUMEN
The interactions of propranolol, nimodipine, and amiodarone with membrane lipids were examined in an effort to explain their different pharmacokinetic and pharmacodynamic properties. Propranolol and nimodipine, which bind with high affinity to plasmalemmal beta-adrenergic and calcium channel receptors, respectively, have membrane partition coefficients of approximately 1200 and 5000 and are readily washed out of membranes with which they had been equilibrated. X-ray and neutron diffraction studies showed that after partitioning into lipid membranes, both propranolol and nimodipine are located approximately 6 A from the phosphate headgroup region of the membrane bilayer, near the hydrocarbon core/water interface. Amiodarone, which blocks Na and K channels with less site specificity than propranolol and nimodipine, has a much higher partition coefficient of approximately 1,000,000, resists washout from membrane bilayers, and is located deeper in the membrane, approximately 12 A from the phosphate headgroup region of the bilayer, nearer to the terminal methyl groups of the fatty acyl chains. The shorter durations of clinical action of propranolol and nimodipine may be related to the reversibility of their interactions with the region of the bilayer exposed to the aqueous media near the hydrocarbon core/water interface, whereas the much longer duration of clinical action of amiodarone may reflect a location more deeply within the fatty acyl region of the bilayer where this hydrophobic drug interacts avidly with the hydrocarbon core of the membrane.
Asunto(s)
Amiodarona/farmacocinética , Lípidos de la Membrana/metabolismo , Nimodipina/farmacocinética , Propranolol/farmacocinética , Sarcolema/metabolismo , Retículo Sarcoplasmático/metabolismo , Amiodarona/farmacología , Animales , Cinética , Membrana Dobles de Lípidos , Conformación Molecular , Neutrones , Nimodipina/farmacología , Propranolol/farmacología , Análisis Espectral , Difracción de Rayos XRESUMEN
A rapid and sensitive method was developed for the preparative separation of laminin subunits. Laminin was extracted and purified from mouse EHS sarcoma. On SDS-PAGE, the reduced and carboxymethylated molecule separated into two components corresponding to molecular weights of about 400 KDa (subunit A) and 200 KDa (subunit B). These two subunits were preparatively separated using heparin-agarose affinity chromatography. The larger subunit quantitatively adhered to the affinity column while the smaller one did not adhere. Amino acid analyses of the separated subunits showed distinct differences. Subunit B was further resolved into two distinct polypeptides of 200 KDa, B1 and B2, by means of reverse-phase HPLC. Although the amino acid compositions of B1 and B2 were very similar, the peptide maps generated by digestion of the B1 and B2 chains with Staphylococcus aureus V8 protease or by cyanogen bromide showed B1 and B2 to differ from each other. Thus, at least three different polypeptide subunits are present in this laminin and probably arise from separate gene origins. These studies provide a basis for the subsequent localization and analysis of the specialized structural and functional domains of laminin.
Asunto(s)
Laminina/análisis , Proteínas de Neoplasias/análisis , Sarcoma Experimental/análisis , Animales , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Densitometría , Ratones , Peso Molecular , Mapeo PeptídicoRESUMEN
The purification procedure for isolating sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from Saccharomyces cerevisiae was improved by the introduction of an ion-exchange step. Enzyme yields were doubled and the specific activity was increased as compared to the original procedure. A new value of 42,000 was obtained for the molecular weight by several denaturing methods. By native gel chromatography the molecular weight appears to be 31,000 as reported earlier. Michaelis constants were found to be 0.37 mM with dihydroxyacetone phosphate as the variable substrate and 0.018 mM for NADH as the variable substrate.