RESUMEN
Cellular injury during tissue hypoxia is due, in part, to reactive intermediates released by activated leukocytes. We found that the inflammatory mediators tumor necrosis factor (TNF)-alpha, IL-6, and IL-1beta are elevated in situ in lung macrophages on day 14 following exposure of rats to intermittent or chronic hypoxia from birth. Because inflammatory mediators can increase lipolysis in adipocytes, we also measured serum unbound free fatty acids (FFAu)--the biologically active compartment of FFA--in rat pups exposed to intermittent or chronic hypoxia. FFAu values were markedly elevated during the first 2 days of life in all rats, displaying an approximately 3-fold decrease from day 2 to day 3. Exposure to chronic hypoxia significantly increased FFAu levels measured on day 13. Since elevated serum FFAu are known to suppress leukocyte activation, we speculate that increased FFAu levels represent a mechanism for attenuating inflammation and tissue injury following sublethal hypoxia in the perinatal period, either physiologically in the immediate newborn period, or as a late response to ongoing hypoxic insult.
Asunto(s)
Citocinas/metabolismo , Ácidos Grasos no Esterificados/sangre , Hipoxia/metabolismo , Pulmón/metabolismo , Envejecimiento , Animales , Femenino , Hipoxia/sangre , Hipoxia/etiología , Inflamación/metabolismo , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Pulmón/patología , Macrófagos/metabolismo , Oxígeno/administración & dosificación , Embarazo , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Fatty acid binding proteins (FABP) form a family of proteins displaying tissue-specific expression. These proteins are involved in fatty acid (FA) transport and metabolism by mechanisms that also appear to be tissue-specific. Cellular retinoid binding proteins are related proteins with unknown roles in FA transport and metabolism. To better understand the origin of these tissue-specific differences we report new measurements, using the acrylodated intestinal fatty acid binding protein (ADIFAB) method, of the binding of fatty acids (FA) to human fatty acid binding proteins (FABP) from brain, heart, intestine, liver, and myelin. We also measured binding of FA to a retinoic acid (CRABP-I) and a retinol (CRBP-II) binding protein and we have extended to 19 different FA our characterization of the FA-ADIFAB and FA-rat intestinal FABP interactions. These studies extend our previous analyses of human FABP from adipocyte and rat FABPs from heart, intestine, and liver. Binding affinities varied according to the order brain approximately myelin approximately heart > liver > intestine > CRABP > CRBP. In contrast to previous studies, no protein revealed a high degree of selectivity for particular FA. The results indicate that FA solubility (hydrophobicity) plays a major role in governing binding affinities; affinities tend to increase with increasing hydrophobicity (decreasing solubility) of the FA. However, our results also reveal that, with the exception of the intestinal protein, FABPs exhibit an additional attractive interaction for unsaturated FA that partially compensates for their trend toward lower affinities due to their higher aqueous solubilities. Thermodynamic potentials were determined for oleate and arachidonate binding to a subset of the FABP and retinoid binding proteins. FA binding to all FABPs was enthalpically driven. The DeltaH degrees values for paralogous FABPs, proteins from the same species but different tissues, reveal an exceptionally wide range of values, from -22 kcal/mol (myelin) to -7 kcal/mol (adipocyte). For orthologous FABPs from the same tissue but different species, DeltaH degrees values were similar. In contrast to the enthalpic dominance of FA binding to FABP, binding of FA to CRABP-I was entropically driven. This is consistent with the notion that FA specificity for FABP is determined by the enthalpy of binding. Proteins from different tissues also revealed considerable heterogeneity in heat capacity changes upon FA binding, DeltaC(p) values ranged between 0 and -1.3 kcal mol(-1) K(-1). The results demonstrate that thermodynamic parameters are quite different for paralogous but are quite similar for orthologous FABP, suggesting tissue-specific differences in FABP function that may be conserved across species.
Asunto(s)
Proteínas Portadoras/metabolismo , Ácidos Grasos/metabolismo , Proteína P2 de Mielina/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Proteínas Supresoras de Tumor , Animales , Cromatografía Líquida de Alta Presión , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Humanos , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Vaina de Mielina/metabolismo , Miocardio/metabolismo , Unión Proteica , Ratas , Receptores de Ácido Retinoico/metabolismo , TermodinámicaRESUMEN
The mechanism by which fatty acids are transported across cell membranes is controversial. The essence of the controversy is whether transport requires membrane protein mediation or whether the membrane's lipid phase provides a pathway so rapid that a protein is not needed. This review focuses on the mechanisms of fatty acid transport across lipid bilayer membranes. These results for lipid membranes are used to help evaluate transport across cell membranes. Within the context of this analysis, a lipid phase mediated process is consistent with results for the transport of fatty acids across erythrocytes but provides a less adequate explanation for fatty acid transport across more complex cells.
Asunto(s)
Membrana Celular/metabolismo , Ácidos Grasos/metabolismo , Lípidos de la Membrana/metabolismo , Animales , Transporte Biológico , Membrana Eritrocítica/metabolismoRESUMEN
The interactions of long chain fatty acids (FA) with wild type (WT) fatty acid binding proteins (FABP) and engineered FABP mutants have been monitored to determine the equilibrium binding constants as well as the rate constants for binding and dissociation. These measurements have been done using the fluorescent probes, ADIFAB and ADIFAB2, that allow the determination of the free fatty acid (FFA) concentration in the reaction of FA with proteins and membranes. The results of these studies indicate that forWT proteins from adipocyte, heart, intestine, and liver, Kd values are in the nM range and affinities decrease with increasing aqueous solubility of the FA. Binding affinities for heart and liver are generally greater than those for adipocyte and intestine. Moreover, measurements of the rate constants indicate that binding equilibrium at 37 degrees C is achieved within seconds for all FA and FABPs. These results, together with the level of serum (unbound) FFA, suggests a buffering action of FABPs that helps to maintain the intracellular concentration of FFA so that the flux of FFA between serum and cells occurs down a concentration gradient. Measurements of the temperature dependence of binding reveal that the free energy is predominately enthalpic and that the enthalpy of the reaction results from FA-FABP interactions within the binding cavity. The nature of these interactions were investigated by determining the thermodynamics of binding to engineered point mutants of the intestinal FABP. These measurements showed that binding affinities did not report accurately the changes in protein-FA interactions because changes in the binding entropy and enthalpy tend to compensate. For example, an alanine substitution for arginine 106 yields a 30 fold increase in binding affinity, because the loss in enthalpy due to the elimination of the favorable interaction between the FA carboxylate and Arg106, is more than compensated for by an increase in entropy. Thus understanding the effects of amino acid replacements on FA-FABP interactions requires measurements of enthalpy and entropy, in addition to affinity.
Asunto(s)
Proteínas Portadoras/metabolismo , Ácidos Grasos/metabolismo , Proteína P2 de Mielina/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Animales , Proteínas Portadoras/química , Entropía , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Ácidos Grasos/química , Colorantes Fluorescentes/metabolismo , Cinética , Modelos Biológicos , Modelos Moleculares , Proteína P2 de Mielina/química , Ratas , Termodinámica , Distribución TisularRESUMEN
The aqueous phase monomers of fatty acids (FFA) appear in many steps of fat metabolism. Understanding metabolism requires that accurate measurements of FFA levels be determined in enzyme-mediated as well as in membrane and protein binding reactions. Measuring long chain FFA levels with sufficient sensitivity and temporal resolution is now possible using fluorescent probes constructed by ligating fluorescent groups and fatty acid binding proteins. In this paper we provide a practical description of the use of ADIFAB, the acrylodan labeled intestinal fatty acid binding protein. We describe with specific examples how ADIFAB can be used to determine, (1) FFA concentrations in aqueous solutions, (2) binding affinities of fatty acid binding proteins, (3) membrane/water partition coefficients, (4) lipase activities, and (5) serum levels of FFA.
Asunto(s)
Bioquímica/métodos , Proteínas Portadoras/metabolismo , Ácidos Grasos/análisis , Colorantes Fluorescentes/metabolismo , Proteínas Recombinantes , Bioquímica/instrumentación , Química Física/instrumentación , Química Física/métodos , Proteínas de Unión a Ácidos Grasos , Cinética , Fosfolipasas A/análisis , Factores de TiempoRESUMEN
To better understand the mechanism by which fatty acids bind to and dissociate from the binding cavities of fatty acid binding proteins (FABPs), we constructed 31 single amino acid mutants of the intestinal FABP (I-FABP) and determined the rate constants for binding and dissociation, primarily for long-chain fatty acids (FA). FA dissociation from these proteins was measured both by the ADIFAB method and by the change in tryptophan fluorescence of the FABPs. Rate constants for binding (kon) were calculated from the rate constants for dissociation (koff) and the equilibrium binding affinities. Amino acid substitutions were made at locations within the binding cavity, in the region of the gap between the betaD- and betaE-strands, and within the "portal" region of the protein. The koff values for the mutant proteins ranged from about 20-fold slower to 4-fold faster than the wild-type (WT) protein. Values for kon were as much as 20-fold slower than the WT protein, but in no case was kon significantly faster than the WT. Mutants with slower and faster koff values were generally those involving sites within the binding cavity and, relative to the WT protein, revealed higher and lower affinities, respectively. Reduced rates of binding were generally, but not exclusively, associated with sites within the portal region. For example, for F68A which is located closer to the opposite end of the protein from the portal region, the kon is more than 10-fold slower than WT. Even for these distal sites, however, the evidence is consistent with reductions in kon being due to alterations of the portal region. Binding affinities and rate constants measured as a function of ionic strength also suggest that the FA initially binds, through an electrostatic interaction, to Arg-56 on the surface of the protein, before inserting into the binding cavity. Thus, the results of this study are consistent with FA binding to I-FABP involving an initial interaction with Arg-56 followed by insertion of the FA, through the portal region, into the binding cavity and with a reversal of these steps for the dissociation reaction.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Ácidos Grasos/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/metabolismo , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Sustitución de Aminoácidos/genética , Animales , Sitios de Unión/genética , Proteínas Portadoras/química , Proteínas de Unión a Ácidos Grasos , Colorantes Fluorescentes/química , Mucosa Intestinal/metabolismo , Cinética , Ácidos Láuricos/metabolismo , Modelos Moleculares , Ácido Mirístico/metabolismo , Concentración Osmolar , Fenilalanina/genética , Unión Proteica/genética , Ratas , Proteínas Recombinantes/químicaRESUMEN
Evidence from a number of laboratories suggests that membrane proteins may meditate the transport of physiologic fatty acids (FA) across cell membranes. However, actual transport of unbound free fatty acids (unbound FFA) from the aqueous phase on one side of a cell membrane to the aqueous phase on the other side has not been measured previously. In this study, we have used the fluorescent probe of unbound FFA, ADIFAB, to monitor the time course of FA movement from the outer to the inner aqueous compartments, and from the lipid membrane to the outer aqueous compartment of red cell ghosts. These two measurements, together with measurements of the lipid/aqueous partition coefficients, allowed the determination of the rate constants for binding (kon), flip-flop (kff), and dissociation (koff) for the transport of long-chain natural FA across red cell ghosts. Measurements done using palmitate, oleate, and linoleate at temperatures between 20 and 37 degreesC revealed that the overall transport times ranged from about 0.5 to more than 10 s, depending upon FA type and temperature. Analysis of these time courses yielded kff values between 0.3 and 3.0 s-1, and these values were consistent with those obtained using ghosts containing pyranine to detect intracellular acidification by the translocating FA. The measured koff values ranged from about 0.3 to 5 s-1, while the rate of binding, for the ghost concentrations used in this study (>50 microM phospholipid), exceed both kff and koff. Thus, long-chain FA transport across red cell ghost membranes is rate-limited by a combination of flip-flop and dissociation rates. Binding of FA to ghost membranes was well described by simple, nonsaturable, aqueous/membrane partition, and that partition appears to be governed by the aqueous solubility of the FA. Transport rates did not reveal any evidence of saturation and were not affected by a variety of protein-specific reagents. These FA binding and transport characteristics are similar to those observed previously for lipid vesicles, although the rate constants are generally about 2-3 fold larger for ghosts as compared to the lipid vesicles. We suggest, therefore, that FA transport across red cell ghosts is reasonably well described by transport across the lipid phase of the membrane.
Asunto(s)
Membrana Eritrocítica/metabolismo , Ácidos Grasos/metabolismo , Proteínas Recombinantes , Arilsulfonatos/metabolismo , Transporte Biológico , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/metabolismo , Membrana Eritrocítica/química , Proteínas de Unión a Ácidos Grasos , Ácidos Grasos/química , Ácidos Grasos no Esterificados/metabolismo , Colorantes Fluorescentes , Humanos , Cinética , Ácido Linoleico/metabolismo , Ácido Oléico/metabolismo , Ácido Palmítico/metabolismoRESUMEN
We constructed 18 single amino acid mutants of the adipocyte fatty acid-binding protein (A-FABP) and 17 of the intestinal fatty acid-binding protein (I-FABP), at locations in the fatty acid (FA) binding sites. For each mutant protein, we measured thermodynamic parameters that characterize FA binding. Binding affinities ranged from about 200-fold smaller to 30-fold larger than the wild type (WT) proteins. Thermodynamic parameters revealed that binding affinities often inaccurately reported changes in protein-FA interactions because changes in the binding entropy and enthalpy were usually compensatory and larger than the binding free energy. FA-FABP interactions were quite different for I-FABP and A-FABP proteins. Binding affinities were larger and decreased to a greater degree with increasing FA solubility for most of the I-FABP as compared with the A-FABP proteins, consistent with a more hydrophobic binding site for the I-FABP proteins. In A-FABP, Ala substitutions for Arg106 and Arg126, which interact with the FA carboxylate, reduce affinities by about 100-fold, but in I-FABP, R106A increases affinities up to 30-fold. Moreover, in A-FABP, the thermodynamic parameters predict that the FA carboxylate location switches from the 126-position in R106A to the 106 position in R126A. Finally, the A-FABP proteins, in contrast to the I-FABP proteins, reveal significant heat capacity changes (DeltaCp) upon FA binding, and substitutions at residues Arg106 and Arg126 reduce the magnitude of DeltaCp.
Asunto(s)
Adipocitos/metabolismo , Proteínas Portadoras/metabolismo , Ácidos Grasos/metabolismo , Mucosa Intestinal/metabolismo , Proteína P2 de Mielina/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Sustitución de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Cinética , Modelos Moleculares , Proteína P2 de Mielina/genética , Ácido Oléico/metabolismo , Ingeniería de Proteínas , Ratas , Relación Estructura-Actividad , TermodinámicaRESUMEN
Evidence from a number of laboratories suggests that membrane proteins may meditate the transport of physiologic fatty acids (FA) across cell membranes. However, studies using lipid membranes indicate that FA are capable of spontaneous flip-flip, raising the possibility that rapid transport through the lipid phase obviates the need for a transport protein. Determining the rate-limiting steps for transport of FA across lipid membranes, therefore, is central to understanding FA transport across cell membranes. The transport of long-chain FA across lipid membranes, from the aqueous compartment on one side of the lipid bilayer to the aqueous phase on the other side, has not been measured previously. In this study, we have used the fluorescent probe ADIFAB to monitor the time course of FA movement from the outer to the inner aqueous compartments and from the lipid membrane to the outer aqueous compartment of lipid vesicles. These two measurements, together with measurements of the lipid:aqueous partition coefficients, allowed the determination of the rate constants for binding (kon), flip-flop (kff), and dissociation (koff) for the transport of long-chain natural FA across lipid vesicles. These rates were determined using large unilamellar vesicles (LUV) of approximately 1000 A diameter, prepared by extrusion and giant unilamellar vesicles (GUV), prepared by detergent dialysis, that are >/=2000 A diameter. The results of these studies for vesicles composed of egg phosphatidylcholine (EPC) and cholesterol reveal kff values that range from 3 to 15 s-1 for LUV and from 0.1 to 1.0 s-1 for GUV, depending upon temperature and FA type. For these same vesicles, dissociation rate constants range from 4 to 40 s-1 for LUV and from 0.3 to 2.5 s-1 for GUV. In all instances, the rate constant for flip-flop is smaller than koff, and because the rate of binding is greater than the rate of transport, we conclude that flip-flop is the rate-limiting step for transport. These results demonstrate that (1) kff and koff are smaller for GUV than for LUV, (2) the rate constants increase with FA type according to oleate (18:1) < palmitate (16:0) < linoleate (18:2), and (3) the barrier for flip-flop has a significant enthalpic component. Comparison of the flip-flop rates determined for GUV with values estimated from previously reported metabolic rates for cardiac myocytes, raises the possibility that flip-flop across the lipid phase alone may not be able to support metabolic requirements.
Asunto(s)
Colesterol , Ácidos Grasos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fosfatidilcolinas , Transporte Biológico , Cinética , Ácido Linoleico/metabolismo , Modelos Químicos , Ácido Oléico/metabolismo , Palmitatos/metabolismo , Tamaño de la PartículaRESUMEN
Site-specific variants of rat intestinal fatty acid-binding protein were constructed to identify the molecular interactions that are important for binding to fatty acids (FAs). Several variants displayed affinities that appeared incompatible with the crystal structure of the protein-FA complex. Thermodynamic measurements provided an explanation for these apparent inconsistencies and revealed that binding affinities often inaccurately reported changes in protein-FA interactions because changes in the binding entropy and enthalpy were usually compensatory. These results demonstrate that understanding the effects of amino acid replacements on ligand binding requires measurements of enthalpy and entropy, in addition to affinity.
Asunto(s)
Proteínas Portadoras/genética , Ácidos Grasos/metabolismo , Mutación , Proteína P2 de Mielina/genética , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Animales , Sitios de Unión , Proteínas Portadoras/metabolismo , Entropía , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Cinética , Ligandos , Modelos Moleculares , Proteína P2 de Mielina/metabolismo , Unión Proteica/genética , Ratas , TermodinámicaRESUMEN
An issue that is central to understanding cellular fatty acid (FA) metabolism is whether physiologic transport of FA across cell membranes requires protein mediation or can be satisfied by the rate of spontaneous movement through the lipid phase. For this reason, considerable effort has been devoted to determining the rate-limiting steps for transport of FA across pure lipid bilayer membranes. Previously, we found that transbilayer flip-flop was the rate-limiting step for transport of long chain anthroyloxy FA (AOFA) across lipid bilayers and that the times for long chain AOFA flip-flop were > or = 100 s, yielding rate constants for flip-flop (k(ff)) that were < or = 0.01 s(-1) [Storch, J., & Kleinfeld, A. M. (1986) Biochemistry 25, 1717-1726; Kleinfeld, A. M., & Storch, J. (1993) Biochemistry 32, 2053-2061]. In those studies, k(ff) values were inferred from the time course of AOFA transfer between lipid vesicles. Recently, Kamp et al. [Kamp, F., Zakim, D., Zhang, F., Noy, N., & Hamilton, J. A. (1995) Biochemistry 34, 11928-11937], using pyranine trapped within lipid vesicles to detect flip-flop more directly, have reported that flip-flop rates of long chain AOFA are extremely rapid (k(ff) > 10 s(-1)) and are not rate limiting for transbilayer transport. Because no defect was apparent in our previous measurements, we have extended, for AOFA, the pyranine method of Kamp et al. (1995) by using stopped-flow fluorometry to resolve flip-flop rates of both short and long chain AOFA in vesicles. In addition, we have monitored the time course of transbilayer AOFA flip-flop using carboxyfluorescein (CF) trapped within the lipid vesicles as a resonance energy transfer (RET) acceptor of AO fluorescence. The differential quenching of AOFA fluorescence in the outer and inner leaflets of the bilayer allows flip-flop to be separated from the time course of AOFA binding to the vesicles. Results obtained from both the pyranine and CF methods indicate, in agreement with our previous results, that flip-flop of the long chain AOFA is slow relative to either the binding or the rate of dissociation from the vesicle. In particular, we find that the time constant (tau) for pyranine quenching by 2-AO-palmitate (2-AOPA) was > 40 s and that k(ff) obtained from RET in CF vesicles was about 0.003 s(-1). Also, in contrast to Kamp et al. (1995) who reported that k(ff) values were independent of FA chain length or structure for the C-12 to C-18 native and the C-18 AOFA within a factor of 2, we find that the rate of pyranine quenching for the shorter chain 11-AO-undecanoic acid is more than 50-fold faster than for the longer chain AOFA. We conclude, therefore, that transbilayer transport of the AOFA is limited by the rate of flip-flop and that this rate is a sensitive function of the AOFA structure.
Asunto(s)
Ácidos Grasos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Arilsulfonatos , Transporte Biológico , Etanol , Ácidos Grasos Volátiles/metabolismo , Fluoresceínas/metabolismo , Colorantes Fluorescentes , Cinética , Vehículos Farmacéuticos , Fosfatidilcolinas/metabolismo , Espectrometría de FluorescenciaRESUMEN
BACKGROUND: Free fatty acids (FFA) play essential roles in maintaining physiologic homeostasis in the newborn infant. Most of the FFA in serum is carried in complex with albumin, but a small fraction remains unbound in the aqueous phase. OBJECTIVE: This study's goal is to report the values of serum levels of unbound free fatty acids (FFAu) in pregnant women and their newborn infants at term gestation. METHODS: The measurements were made possible by the availability of the fluorescent probe for unbound FFA, acrylodated intestinal fatty acid binding protein (ADIFAB). Twenty-two mother-infant pairs were enrolled in the study. Maternal levels were obtained immediately before delivery, cord levels at the time of delivery, and infant levels after 24 hours of age. RESULTS: The level of FFAu measured in maternal samples was 11.8 +/- 4 nM, in cord samples 9.2 +/- 4 nM, and in infants 13.9 +/- 3 nM. These population averages are considerably greater than those observed in healthy adults (7.5 +/- 2.5 nM). No correlation was found between cord levels and birthweight, gestational age, labor duration, mode of deliver, and infant or maternal temperature. CONCLUSIONS: This investigation is the first to measure FFAu in a group of mothers and their infants and provides the technique for future investigations of the biologic activity of free fatty acids.
Asunto(s)
Ácidos Grasos no Esterificados/sangre , Sangre Fetal/química , Recién Nacido/sangre , Embarazo/sangre , Adulto , Femenino , Humanos , Masculino , Intercambio Materno-FetalRESUMEN
BACKGROUND: Fatty acids (FFA) are key nutrients in maintaining physiologic homeostasis and in the form of Intralipid administration, they are important sources of nutrition in the premature newborn infant. Complexed with albumin, fatty acids have a small but important fraction that remains unbound in the aqueous phase. OBJECTIVE: The goal of this study was to examine the levels of serum levels of unbound free fatty acids (FFAu) in premature newborns following Intralipid administration. METHOD: A fluorescent probe acrylodated intestinal fatty acid binding protein (ADIFAB) was used to measure (FFAu) before Intralipid and during increasing rates of infusion. RESULTS: There were significant differences between (FFAu) values obtained before Intralipid and levels after the infusion of 1.0, 2.0, and 3.0 g/kg/day (p < 0.05). Regression analysis of Intralipid dose and FFAu yielded an r = 0.438 and the following relationship: [FFAu] = 26.39 + 3.60 * IL (g/kg/day). CONCLUSIONS: Intralipid administration results in significant elevation of FFAu in the very low birth weight infant.
Asunto(s)
Emulsiones Grasas Intravenosas/farmacología , Ácidos Grasos no Esterificados/sangre , Recien Nacido Prematuro/sangre , Nutrición Parenteral , Emulsiones Grasas Intravenosas/administración & dosificación , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Concentración OsmolarRESUMEN
Serum unbound free fatty acid levels (FFAu) were measured in patients undergoing percutaneous transluminal coronary angioplasty (PTCA) using the fluorescent probe acrylodan intestinal fatty acid binding protein (ADIFAB). These are the first measurements of FFAu, under nonphysiologic conditions. In these studies, FFAu, levels were determined in 22 patients 5 minutes before and 30 minutes after the procedure. Post-PTCA FFAu, levels were higher than pre-PTCA levels in all patients. The average post-PTCA level for all patients was 103 nM, about 14-fold higher than the 7.5 nM value observed in healthy subjects. Although all patients exhibited elevated FFAu levels after PTCA, ischemic ST-segment changes were observed in only 11 of these patients. The average post-PTCA FFAu levels for patients with significant ST-segment changes (123 nM) were significantly higher than those in patients who did not exhibit such changes (47 nM). Average FFAu (22 nM) levels before the procedure were elevated in the patient population relative to healthy subjects and these values correlated positively with post-PTCA levels. These results suggest that increased serum FFAu levels reflect angioplasty-induced ischemia and that FFAu levels may provide a more sensitive measure of ischemia than electrocardiographic measurements. Moreover, because 30% of these patients had post-PTCA FFAu concentrations exceeding those found to alter in vitro cell function, the increased serum FFAu levels that accompany ischemia may be deleterious for myocardial function.
Asunto(s)
Angioplastia Coronaria con Balón , Enfermedad Coronaria/sangre , Ácidos Grasos no Esterificados/sangre , Proteínas Recombinantes , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Portadoras , Enfermedad Coronaria/terapia , Electrocardiografía , Proteínas de Unión a Ácidos Grasos , Femenino , Colorantes Fluorescentes , Humanos , Masculino , Persona de Mediana Edad , Albúmina Sérica/análisisRESUMEN
Fatty acid-binding protein from rat liver (L-FABP) binds 2 fatty acids (FA) per protein, in contrast to FABPs from adipocyte, heart, and intestine, for which binding and structural studies are consistent with a single FA binding site. To understand better the unique characteristics of L-FABP, we have carried out equilibrium binding and kinetic measurements of long chain FA using the fluorescent probes of free fatty acids (FFA), ADIFAB and ADIFAB2, to monitor the concentration of FFA in the reaction of FA with L-FABP. We found that the dissociation constants (Kd) ranged from about 1 nM to 4 microM, being largest for myristate at 45 degrees C and smallest for oleate at 10 degrees C, and that 2 FA were bound per L-FABP for all temperatures and FA. The binding measurements also revealed that at temperatures below 37 degrees C, affinities for the two binding sites differ by between 5- and 20-fold but as the temperature was increased, the affinities converge toward equal values. Off-rate constants (koff) were similar for all FA and for temperatures between 10 and 45 degrees C, ranged from about 0.1 s-1 to 50 s-1. Moreover, for all FA, koff values for dissociation from both the high and low affinity sites were similar, indicating that binding affinity differences at the lower temperatures reflect lower on-rates for binding to the low affinity site. The temperature at which the affinities of the two sites become equivalent depends upon the FA; higher temperatures (45-50 degrees C) are required for the unsaturated FA and myristate than for the longer chain saturated FA (<37 degrees C). This transition from different to equivalent affinity binding sites at specific temperatures reflects a nonlinear van't Hoff behavior of the high affinity site, which in turn is a reflection of large heat capacity changes (between -0.6 and -1.2 kcal K-1 mol-1) that accompany FA binding to the high affinity site. These heat capacity changes, which are unique to L-FABP, do not appear to be correlated with a significant conformational change upon ligand binding. The differences between long chain saturated and unsaturated FA suggest that the conformation of FA bound to L-FABP may differ with both FA type and temperature, and that, in comparison to other FABPs, L-FABP may have distinctly different effects on saturated and unsaturated FA metabolism.
Asunto(s)
Proteínas Portadoras/metabolismo , Ácidos Grasos/metabolismo , Hígado/metabolismo , Proteína P2 de Mielina/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Proteínas Recombinantes , Animales , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Colorantes Fluorescentes/metabolismo , Cinética , Ratas , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
Tyrosine phosphorylation of proteins plays a central role in T cell activation. Mitogens or anti-receptor antibodies have been employed to study these signaling events, but the extent to which these mimic receptor interactions with native ligands is unclear. Cytotoxic T lymphocytes can be activated for functional responses using purified, native class I ligands presented on a surface. Previous work showed that stimulation with fluid-phase anti-T cell receptor (TCR) monoclonal antibody (mAb) activates CD8 to mediate adhesion to class I proteins and that activated CD8 generates a co-stimulatory signal upon binding to class I. Changes in tyrosine phosphorylation of substrates and activity of the p56lck kinase have now been examined in this two-step process. The observed changes are small in comparison to those found using more potent nonphysiological stimuli, but may more accurately reflect the events required for activation of functional responses. Fluid-phase anti-TCR mAb caused increased tyrosine phosphorylation of a discrete subset of cellular substrates. Increased phosphorylation of additional substrates occurred upon CD8 binding to class I, resulting in a phosphorylation pattern comparable to that found in cells stimulated with class I alloantigen. Anti-TCR mAb alone caused increased tyrosine phosphorylation of p56lck. When CD8 bound to class I, phosphorylation of p56lck decreased to below the basal level found in unstimulated cells, accompanied by a substantial increase in kinase activity. These results are consistent with the two-step model for TCR activation of CD8/class I interactions and directly demonstrate that CD8 binding to class I leads to up-regulation of p56lck activity.
Asunto(s)
Antígenos CD8/fisiología , Antígenos de Histocompatibilidad Clase I/fisiología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T Citotóxicos/metabolismo , Familia-src Quinasas/metabolismo , Animales , Activación de Linfocitos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Ratones , Fosfotirosina/metabolismo , Transducción de SeñalRESUMEN
Rate constants for the interaction of fatty acids (FA) with fatty acid binding proteins (FABP) from adipocyte (A-FABP), heart (H-FABP), and intestine (I-FABP) were determined by using stopped-flow fluorometry and ADIFAB, the fluorescent probe of free fatty acids (FFA), or a new FFA probe, ADIFAB2, constructed by derivatizing with acrylodan the Leu72 --> Ala mutant of I-FABP. ADIFAB2, because its binding affinities are about 10-fold greater than ADIFAB, was found to be more accurate for monitoring the kinetics of the higher affinity reactions. On- (kappa on) and off- (kappa off) rate constants were determined as a function of temperature. Our results reveal that in all cases the FA-FABP equilibrium is achieved within 2 s at 37 degrees C and within 20 s at 10 degrees C. Off-rate constants varied by about 10-fold among the different underivatized FABPs; kappa off values were smallest for H-FABP and largest for A-FABP, while kappa on values for these proteins generally varied by less than 2-fold. The results show that the previously reported larger affinities of I- and H-FABPs as compared to A-FABP are primarily a reflection of larger kappa on values for I-FABP and smaller kappa off values for H-FABP. Eyring transition state theory was used to evaluate the activation thermodynamic parameters for both on- and off-reactions and the results show that in virtually all cases the rate-limiting steps are predominantly enthalpic. Activation free energies for binding to ADIFAB are generally composed of about 8 kcal/mol unfavorable enthalpy and about a 1 kcal/mol favorable entropic contribution. For the underivatized FABPs the activation free energies are all about 7 +/- 0.3 kcal/mol, suggesting that the transition state for entering or leaving the binding site involves a common protein structural change. We suggest that entering or leaving the FABP binding cavity involves similar mechanisms for all 3 FABPs and may involve amino acid residues located within the portal regions of these proteins.
Asunto(s)
Adipocitos/metabolismo , Proteínas Portadoras/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Ácidos Grasos/metabolismo , Mucosa Intestinal/metabolismo , Proteína P2 de Mielina/metabolismo , Miocardio/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Proteínas Supresoras de Tumor , Animales , Proteínas Portadoras/química , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Colorantes Fluorescentes , Humanos , Cinética , Leucina , Matemática , Ratones , Modelos Teóricos , Proteína P2 de Mielina/química , Ácido Oléico , Ácidos Oléicos/metabolismo , Mutación Puntual , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
A new method is described for the continuous quantitation of phospholipase A2 (PLA2) activity with greatly improved sensitivity compared to existing techniques. The method utilizes a fluorescent probe to detect the release of fatty acid monomers (free fatty acids) into the aqueous phase. The fluorescent probe ADIFAB, which is the acrylodan derivative of rat intestinal fatty acid binding protein, exhibits a change in the ratio of its fluorescence upon binding medium- to long-chain native fatty acids. ADIFAB was used to measure the hydrolysis of artificial and natural membranes using PLA2s from porcine pancreas, Naja mocambique mocambique, Crotalus durissus terrificus, and bee venom. Total phospholipid hydrolysis was determined from the free fatty acid concentration using membrane/water partition coefficients, also measured using ADIFAB. The results indicate that continuous monitoring of natural substrates can be determined with a sensitivity limit of less than 1 pmol/min, a more than 10(4)-fold increase in sensitivity over the most commonly used pH stat method.
Asunto(s)
Proteínas Portadoras , Fosfolipasas A/análisis , Fosfolipasas A/metabolismo , Proteínas Recombinantes , Animales , Venenos de Abeja/análisis , Membrana Celular/enzimología , Colesterol/metabolismo , Venenos de Crotálidos/análisis , Venenos Elapídicos/análisis , Proteínas de Unión a Ácidos Grasos , Colorantes Fluorescentes , Hidrólisis , Cinética , Membranas Artificiales , Ratones , Páncreas/enzimología , Fosfatidilcolinas/metabolismo , Fosfolipasas A2 , Ratas , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/métodos , Porcinos , Termodinámica , Células Tumorales CultivadasRESUMEN
Binding of CTL to MHC class I or fibronectin is activated through TCR signaling. Once activated, CTL adhesion to MHC class I results in tyrosine phosphorylation of CTL substrates, phosphatidylinositol (PI) turnover, and degranulation. Although activated adhesion to fibronectin does not itself initiate PI hydrolysis or degranulation, these responses are amplified once they become activated. In the present study we have examined the effect of cis unsaturated fatty acids (FA) and phenylarsine oxide (PAO) on CD8-mediated adhesion of CTL to immobilized class I protein and on biochemical and functional events that are triggered by this adhesion. Previous studies have shown that FA and low concentrations of PAO inhibit specific tyrosine phosphorylation events and degranulation but have no effect on PI turnover or CTL-target cell conjugates. The present results show that pretreating CTL with cis unsaturated, but not saturated, FA and low concentrations of PAO (< 0.5 microM) inhibit soluble anti-TCR-triggered binding of CD8 to immobilized MHC class I, tyrosine phosphorylation of CTL substrates, PI turnover, and degranulation. Addition of cis unsaturated FA or PAO after CTL have been allowed to bind to immobilized class I protein did not affect the level of adhesion. In contrast, neither cis unsaturated FA nor PAO affected the TCR-activated binding of CTL to fibronectin. These results suggest that activation of adhesion to the class I and fibronectin ligands involves divergent different pathways that can be distinguished by the FA and PAO agents.
Asunto(s)
Arsenicales/farmacología , Ácidos Grasos Insaturados/farmacología , Fibronectinas/metabolismo , Antígenos H-2/inmunología , Activación de Linfocitos/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/efectos de los fármacos , Linfocitos T Citotóxicos/efectos de los fármacos , Animales , Anticuerpos Monoclonales/farmacología , Adhesión Celular , Interleucina-2/farmacología , Activación de Linfocitos/fisiología , Ratones , Fosfatidilinositoles/metabolismo , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/fisiología , Proteínas Recombinantes/farmacología , Transducción de Señal/fisiología , Linfocitos T Citotóxicos/fisiologíaRESUMEN
Using the fluorescent probe ADIFAB (acrylodan-derivatized intestinal fatty acid-binding protein) to determine the equilibrium concentration of the free (unbound) fatty acid (FFA), dissociation constants were measured between 10 and 50 degrees C for the interaction of five different long chain fatty acids (FA) with fatty acid-binding proteins (FABP) from adipocyte, intestine, and heart. Gibbs free energies (delta G) determined from the dissociation constants were between about -9 and -11 kcal/mol at 25 degrees C. Thermodynamic parameters for binding were determined using van't Hoff plots of the dissociation constants, which range, over the entire temperature region, between 2 and 3000 nM. For all the unlabeled FABPs, free energies of binding were dominated by large negative enthalpies that ranged from -7 to -12 kcal/mol, and the enthalpies tended to decrease with increasing FA unsaturation. The entropic contributions (-T detla S) at 25 degrees C between -4 and +2 kcal/mol and tended to increase with increasing FA unsaturation. To assess the role of FA aqueous solubility in FABP binding, measurements of the partition of FA between unilamellar lipid vesicles and water were also done using ADIFAB; the lipid/water partition coefficients (Kp) determined from these measurements were found to be independent of temperature. The binding of FA to FABP is governed by the sum of contributions of various interactions between FA, water, and FABP. An analysis of the individual contributions suggests that the net free energy of binding results from the canceling in part of a number of separate quite large contributions. The entropic contributions sum almost to zero for most FA and FABPs as a result of the canceling of a large increase in bulk solvent entropy by decreases in configurational entropy upon FA binding to FABP. The net, approximately -10 kcal/mol enthalpy of binding, probably results from an increase in FA configurational enthalpy upon binding to FABP plus a large negative enthalpy from the interaction between the FA and the FABP. This large enthalpy of the FA-FABP interaction suggests that in addition to previously identified specific interactions between the carboxylate portion of the FA and charged amino acids within the binding cavity, other significantly larger enthalpic interactions, presumably involving the hydrocarbon portion of the FA, must contribute to the binding energy.