Biomedical subjects

A M Kleinfeld

Publications and source records attributed to A M Kleinfeld.

At least 19 recordsLinked to original sources

PubMed1996

Kinetics of fatty acid interactions with fatty acid binding proteins from adipocyte, heart, and intestine.

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.

Adipocytes↗

PubMed1995

Continuous measurement of phospholipase A2 activity using the fluorescent probe ADIFAB.

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.

Animals↗

PubMed1995

Activated adhesion of CTL to MHC class I but not to fibronectin is inhibited by cis unsaturated fatty acids and phenylarsine oxide.

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.

Animals↗

PubMed1995

Thermodynamics of fatty acid binding to fatty acid-binding proteins and fatty acid partition between water and membranes measured using the fluorescent probe ADIFAB.

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.

Adipocytes↗

PubMed1995

Unbound free fatty acid levels in human serum.

Total serum free fatty acids (FFAt) levels provide an important measure of the physiologic state. Most of the FFA in serum is bound to albumin; a small portion, however, is unbound. This study presents the first measurements of serum unbound free fatty acid (FFAu) concentrations. These measurements were made possible by the development of the fluorescence probe ADIFAB (acrylodated intestinal fatty acid binding protein). In the present study ADIFAB was shown to provide the correct value of the sum of the [FFAu] of each molecular species of long chain FFA: 1) in aqueous mixtures of FFA and ADIFAB; 2) in mixtures of FFA, human serum albumin, and ADIFAB; and 3) in serum and ADIFAB. Human serum [FFAu] were measured in 283 samples from healthy donors and yielded a mean value of 7.5 nM with a standard deviation of 2.5 nM. This measured value is as much as 1000-fold smaller than [FFAu] values previously estimated. The distribution of [FFAu] values was found to be invariant with donor gender and age. Average [FFAu] values do, as expected, exhibit a small (1.5 nM) but significant (P < 0.001) increase after overnight fasting. FFAu levels were found to be well correlated with total serum FFA or, equivalently, [FFAt]/[HSA]. Given their ease and accuracy, and because [FFAu] values increase exponentially with [FFAt]/[HSA], [FFAu] measurements could provide a sensitive method for assessing the physiologic state.

Age Factors↗

PubMed1994

Equilibrium constants for the binding of fatty acids with fatty acid-binding proteins from adipocyte, intestine, heart, and liver measured with the fluorescent probe ADIFAB.

Affinities of long chain fatty acids (FA) for fatty acid-binding proteins (FABPs) have been measured by monitoring the concentrations of the unbound or free fatty acids (FFA) in equilibrium with the FABPs using the fluorescent probe ADIFAB. This probe allows the measurement of the concentration of FFA in equilibrium with FABPs, without physical separation of any of the reactants. Equilibrium characteristics were measured at 37 degrees C for palmitate, stearate, oleate, linoleate, linolenate, and arachidonate binding to six FABPs from intestine, heart, adipose, and liver from different species. Equilibrium constants for each FA were found to be extremely sensitive to the tissue origin of the FABP but largely independent of species differences. The measured values of the dissociation constants (Kd) ranged from about 2 to 1000 nM, depending upon the tissue origin of the FABP and the FA. Binding constants for some FABPs varied considerably with different FA, as much as 80-fold in the case of the intestinal FABP. In contrast, Kd values for adipocyte FABPs exhibited less than 4-fold variation with FA type and are generally larger (lower affinities) than for the other FABPs. For all FABPs, Kd values for fatty acids with the same chain length were considerably lower for saturated as compared to polyunsaturated FA. This characteristic likely reflects the lower aqueous solubilities of the saturated fatty acids. In contrast to the other FABPs, rat liver FABP was found to have two FA-binding sites/monomer. Each of these two sites had similar high affinities for the saturated FA, while for the unsaturated FA the two sites exhibited affinities that differ by more than 7-fold. This study disagrees with earlier investigations in finding that equilibrium binding of FA to FABPs is a sensitive function of FA type and FABP tissue origin and that FA-FABP dissociation constants are submicromolar. These results provide a framework with which to understand better the biological function of FABPs and the FA-FABP interaction.

Adipocytes↗

PubMed1994

A tyrosine phosphorylation requirement for cytotoxic T lymphocyte degranulation.

Phorbol myristate acetate (PMA) plus ionomycin induces the tyrosine phosphorylation of several cytotoxic T lymphocyte (CTL) substrates, including one with an apparent molecular weight of 100,000 (pp100) in cloned murine CTL. cis-Unsaturated fatty acids and low concentrations of phenylarsine oxide specifically inhibit the tyrosine phosphorylation of pp100. Genistein also inhibits tyrosine phosphorylation of pp100, but not with the same specificity as cis-fatty acids or low concentrations of phenylarsine oxide. Degranulation triggered by PMA plus ionomycin is inhibited by cis-fatty acids, low concentrations of phenylarsine oxide, and genistein, under the same conditions that these agents inhibit tyrosine phosphorylation of pp100. Depleting CTL of protein kinase C (PKC) activity by prolonged exposure to PMA eliminates the increase in tyrosine phosphorylation when challenged by PMA plus ionomycin, but not when these PKC-depleted CTL are activated by cognate target cells, immobilized anti-T cell receptor (TCR) antibodies, or concanavalin A. Tyrosine phosphorylation of pp100 triggered by TCR engagement in PKC-depleted cells is inhibited by cis-fatty acids and phenylarsine oxide, indicating that the inhibitory mechanism of the tyrosine phosphorylation of pp100 is independent of PKC. Furthermore, because all three tyrosine phosphorylation inhibitors are unlikely to inhibit PKC, these results suggest that, in addition to PKC activation and a rise in intracellular Ca2+, CTL degranulation requires the tyrosine phosphorylation of a CTL substrate(s), in addition to phospholipase C, and the present results are consistent with pp100 as that substrate. Taken together with previous studies, these results suggest that tyrosine phosphorylation of pp100 may play a central role in CTL function.

Animals↗

PubMed1993

Tyrosine phosphorylation of a 100-kDa protein is correlated with cytotoxic T-lymphocyte function. Evidence from cis unsaturated fatty acid and phenylarsineoxide inhibition.

Cis unsaturated but not saturated fatty acids (FA) have been shown to specifically inhibit certain early responses in cytotoxic T-lymphocytes (CTL) including the rise in intracellular calcium, degranulation, and lethal hit delivery. They do not, however, affect other early events such as the turnover of phosphatidylinositol, T-cell receptor recognition, and CTL-cognate target cell conjugation. Antigen stimulation of CTL results in an increase in the tyrosine phosphorylation levels of more than 10 substrates. We now find that cis unsaturated but not saturated FA specifically inhibit antigen-stimulated tyrosine phosphorylation of only a single substrate, a 100-kDa protein (pp100). The characteristics of cis FA inhibition of the tyrosine phosphorylation of pp100 are virtually identical to the cis FA inhibition of the CTL functional responses. Inhibition occurs within seconds of cis FA addition, can be reversed by extracting the unesterified FA with albumin, can be initiated after stimulation, and has the same dose and FA molecular species dependence as the inhibition of the CTL responses. In addition, low concentrations of phenylarsine oxide specifically inhibit the tyrosine phosphorylation of pp100 and the CTL responses, but have no effect on other tyrosine phosphorylation events. These striking correlations suggest that pp100 may be central to specific early signaling events including lethal hit delivery and its tyrosine phosphorylation may be regulated by a plasma membrane protein tyrosine phosphatase.

Animals↗

PubMed1993

Interactions of long-chain fatty acids and albumin: determination of free fatty acid levels using the fluorescent probe ADIFAB.

Equilibrium binding of long-chain fatty acids (FA) with albumin from human serum (HSA), bovine serum (BSA), and murine serum (MSA) has been studied by measuring the equilibrium levels of free fatty acids (FFA). FFA levels were measured directly, using a new fluorescent probe composed of acrylodan-derivatized intestinal fatty acid binding protein (ADIFAB). Measurements of [FFA] were done as a function of the ratio of total FA to total albumin (v) for v values between 0 and 6, at pH 7.4 and 37 degrees C. Under conditions observed in normal human physiology (v < or = 2), [FFA] values of the most abundant serum FA (palmitate, stearate, oleate) in equilibrium with human or bovine albumin are less than 15 nM. These values are considerably smaller than the generally quoted values of [FFA] in equilibrium with albumin: more than 20-fold for palmitate and more than 50-fold for oleate. FFA levels were found to increase monotonically with for all three albumins and all FA. In most cases [FFA] increased, for the same chain length, with increasing degree of acyl chain unsaturation, suggesting that FA aqueous solubility may play a significant role in the equilibrium between FA association with albumin and the aqueous phase. [The highest FFA levels (approximately 3000 nM), for example, were observed for linoleate (18:3) at the maximum v value (6).] Although aqueous-phase solubility of the FA may be important in understanding the interaction between FA and albumin, protein structure, as reflected in differences among the three albumins, also significantly affects the equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Transfer of long-chain fluorescent fatty acids between small and large unilamellar vesicles.

Transfer of 12-(9-anthroyloxy)stearic acid (12AS) was measured between small unilamellar vesicles (SUV) and between large unilamellar vesicles (LUV), over a temperature range of 5-50 degrees C. The results of this study clearly establish the biexponential nature of the time dependence of the transfer in a variety of vesicle types and confirm our previous results using egg phosphatidylcholine (EPC) SUV at 25 degrees C (Storch & Kleinfeld, 1986). In our previous study we developed a kinetic model of the transfer process and concluded that the observed time dependence of the transfer of long-chain 12-(9-anthroyloxy) fatty acids (AOFA) was due to transbilayer flip-flop that was much slower than the rate at which the fatty acids (FA) move from the vesicle and into the surrounding aqueous phase (the off step). In the present study, experimental and theoretical advances have allowed us to examine, in detail, predictions of the kinetic model that critically depend upon the slow rate of flip-flop. The current results verify these predictions and demonstrate that slow AOFA flip-flop is rate limiting in at least three different vesicle systems and at all temperatures studied. Moreover, both flip-flop and the off rate constants were almost an order of magnitude smaller in EPC-LUV than in EPC-SUV. Flip-flop was found to be asymmetric (the rate constant for transfer from the inner to outer hemileaflet of the bilayer is approximately twice that from the outer to inner hemileaflet) in SUV but virtually symmetric in LUV. The temperature dependence of transfer was used to determine the thermodynamic activation potentials for the flip-flop and off rate constants.(ABSTRACT TRUNCATED AT 250 WORDS)

Dimyristoylphosphatidylcholine↗

PubMed1993

Membrane partition of fatty acids and inhibition of T cell function.

Short-term exposure to elevated levels of free fatty acids (FFA) perturbs a variety of cellular functions. It is frequently observed that cis-unsaturated fatty acids (FA) mediate these perturbations while trans-unsaturated or saturated FA are relatively inert. This dichotomy has generally been ascribed to the differential effects of these FA on membrane structure, specifically, that cis but not saturated FA alter lipid acyl chain order. Direct support for this view, however, is lacking because membrane partition of FA has not been determined for the relevant FA and for the conditions of the functional studies. Previous measurements of membrane partition of natural long-chain FA have relied on the determination of the amount bound to the membrane rather than the aqueous-phase concentration of FA. Because [FFA] is low, however, the partition coefficient (Kp) is relatively insensitive to the membrane-bound concentration and, therefore, accurate determinations of Kp require direct [FFA] measurements. In this study FA partition between the aqueous phase and either lipid bilayer vesicles or cytotoxic T lymphocytes (CTL) was measured using a recently developed fluorescent indicator of FFA. This indicator is composed of recombinant intestinal fatty acid binding protein derivatized with acrylodan and is termed ADIFAB. Using ADIFAB, partition coefficients were determined for seven saturated and cis-unsaturated FA under conditions that parallel those in which cis FA have been shown to inhibit CTL signaling. In general, Kp values were approximately an order of magnitude greater than previous determinations and were found to be greater for the noninhibitory saturated FA than the inhibitory cis FA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

A fluorescently labeled intestinal fatty acid binding protein. Interactions with fatty acids and its use in monitoring free fatty acids.

The fatty acid-binding protein from rat intestine (I-FABP) has been covalently modified with the fluorescent compound Acrylodan. Acrylodan was found to label Lys27, one of the few amino acid residues found by x-ray diffraction studies to change orientation upon fatty acid (FA) binding to I-FABP. Binding of FA to this Acrylodan-modified I-FABP (ADIFAB) induces a large shift in fluorescence emission wavelength from 432 to 505 nm. As a consequence, the ratio of emission intensities provides a direct measure of the concentration of FA bound to the protein. Binding of FA is well described by single site equilibrium for FA concentrations below the critical micelle concentration. ADIFAB dissociation constants (Kd) determined at 37 degrees C and at concentrations below the critical micelle concentration for oleate, palmitate, linoleate, arachidonate, and linolenate were, respectively, 0.28, 0.33, 0.97, 1.6, and 2.5 microM. The variation of these Kd values with FA molecular species is highly correlated with the solubility of the FA in water, suggesting that all these FA bind with a similar conformation in the I-FABP binding site. The ADIFAB response together with the measured equilibrium constants allows a direct determination of the concentration of long chain free fatty acid (FFA) in the concentration range, depending upon the FA molecular species, between 1 nM and > 20 microM. As an example of its use as a probe to measure FFA levels, ADIFAB is used here to monitor the time course for FFA release from IgE receptor- and ionomycin-activated rat basophilic leukemia (RBL) cells.

2-Naphthylamine↗

PubMed1991

Free fatty acids are produced in and secreted from target cells very early in cytotoxic T lymphocyte-mediated killing.

Conjugation of CTL with their cognate targets elicits a number of early changes within the target cell that are thought to play an important role in the lytic mechanism. We now report that at times earlier than 5 min after conjugation with allospecific CTL, free fatty acids (FFA) are produced in and then secreted from alloantigen-bearing target cells. Using murine CTL clones with different alloantigen specificities, stimulation of FFA production from target cells was found to be Ag specific. FFA production does not appear to be specific for any particular FFA species. Indeed, a wide spectrum of cis unsaturated as well as saturated FFA are produced. FFA production is well correlated with, and specific for, CTL-mediated target cell lysis. Other means of perturbing or lysing target cells, including freeze/thaw disruption, detergent solubilization, or increasing membrane permeabilization with ionomycin, do not stimulate FFA production. In particular, FFA production is not stimulated by treatment with pore-forming granules under conditions that cause more than 90% target cell lysis. These results suggest that FFA production plays an important role in CTL-mediated lysis because stimulation of FFA release specifically requires an event that is CTL induced, occurs very early after conjugation, and is strongly correlated with CTL-mediated lysis.

Cell Line↗

PubMed1991

Direct determination of free fatty acid transport across the adipocyte plasma membrane using quantitative fluorescence microscopy.

Movement of free fatty acids (FFA) across the plasma membrane has been directly measured for the first time, using fluorescent FFA analogs and quantitative fluorescence microscopy. The rate of short chain FFA (less than or equal to 12 carbons) transport from the extracellular medium into intracellular lipid droplets of 3T3F442A adipocytes was more than 40-fold faster than long chain FFA (16 and 18 carbons). The membrane-impermeable amino reagent 4,4'-diisothiocyanostilbene-2,2'-disulfonate, inhibited greater than or equal to 50% of the long chain FFA transport but had no effect on short chain FFA transport. Oleic acid (2 microM) inhibited 90% of the fluorescent oleate transport but had no effect on the 11-carbon analog. These results indicate that a large fraction of long chain FFA uptake is mediated by a plasma membrane protein (s).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1990

Free fatty acids inhibit cytotoxic T lymphocyte-mediated lysis of allogeneic target cells.

Short term exposure of murine CTL clones to long chain cis unsaturated free fatty acid (FFA) inhibits alloantigen specific lysis of cognate target cells, whereas long-chain saturated FFA have no effect. Inhibition of lysis occurs when cis FFA is added before or within 10 min after CTL-target cell conjugate formation and thus appears to interfere with lethal hit delivery. Our previous studies have shown that similar treatment with cis FFA inhibits, in CTL, the Ag stimulated increase in intracellular calcium and degranulation, suggesting that inhibition of lysis probably results from perturbation of the CTL signaling pathway. However, inhibition of lysis is probably not due to the inhibition of the rise in intracellular calcium or degranulation, because lysis can occur under conditions in which FFA inhibit degranulation and because cis FFA inhibit calcium-independent killing. Inhibition of lysis is detectable at unbound FFA concentrations less than 1 microM and is generally complete at concentrations less than 5 microM. Although these levels of FFA are somewhat higher than reported for normal physiologic conditions, plasma FFA levels can be elevated into this range in states of stress and disease, suggesting that FFA modulation of the immune response has important physiologic consequences.

Calcium↗

PubMed1990

Short term exposure to cis unsaturated free fatty acids inhibits degranulation of cytotoxic T lymphocytes.

Degranulation of CTL stimulated by alloantigen-bearing target cells is shown to be inhibited by short term exposure to low concentrations of long chain cis unsaturated free fatty acids (FFA), whereas saturated FFA have no effect. The Ag-specific (TCR mediated) stimulation of cloned murine CTL was monitored by changes in intracellular calcium concentrations [( Ca2+]i) using the fluorescence indicator acetoxymethylester of fura-2 and by degranulation as measured by the release of BLT-esterase. Treatment of the CTL cells with any of the physiologically important FFA; oleic (18:1), linoleic (18:2), linolenic (18:3), or arachidonic (20:4) acid, at concentrations between 1 and 10 microM inhibits the target cell-mediated rise in [Ca2+]i which occurs within seconds of stimulation and the release of BLT-esterase, which occurs over a period of 1 to 3 h. These inhibitory effects are observed within seconds to minutes of FFA addition. Inhibition can be reversed by treating cells with fatty acid free BSA and, in agreement with our previous studies, indicates that the effects of FFA are due to physical perturbations of cellular components. To determine the locus of this perturbation, the effect of FFA on the lipid order of CTL plasma membrane was determined using fluorescence polarization of the membrane impermeable probe trimethylammoniumdiphenylhexatriene. Cis unsaturated FFA were found to disorder the lipid acyl chains and the degree of disorder was found to increase with the degree of cis unsaturation. These results, together with the previous studies, suggest that inhibition results from a physical perturbation of plasma membrane lipid order. Moreover, because degranulation requires elevated levels of [Ca2+]i, it is likely that inhibition of degranulation results from a FFA-induced decrease in Ca2+ permeability through the membrane.

Calcium↗

PubMed1989

Free fatty acid perturbation of transmembrane signaling in cytotoxic T lymphocytes.

Transmembrane signaling in CTL is found to be extremely sensitive to short term exposure to long chain free fatty acids (FFA). Both alloantigen specific target cells and the lectin Con A were used to stimulate cloned murine CTL. This stimulation was monitored by changes in intracellular calcium concentrations ([Ca2+]i) using the fluorescence indicator fura-2. Treatment of the CTL cells with oleic acid (18:1) at concentrations corresponding to less than 10% (mol/mol) bound to the cell, completely inhibits target cell or Con A-mediated rise in [Ca2+]i. The inhibitory effect of oleic acid is observed within seconds of addition and the inhibition is completely reversed by treating cells with fatty acid free BSA. In addition, using the fluorescence indicator 2',7'-bis(carboxyethyl)carboxyfluorescein to monitor intracellular pH, it was found that oleic acid itself acidifies the cytosol by about 0.3 to 0.4 pH units. Acidification is probably necessary, but is not sufficient to inhibit the calcium rise. Stearic acid (18:0), even at concentrations that correspond to a factor of two to three more bound to the cell than for oleic acid, had no effect on either the [Ca2+]i or intracellular pH responses. Oleic acid was found to bind to cells with single site kinetics and with a number of sites and affinity corresponding to membrane lipid binding sites. Esterification of added oleic acid was negligible in the time (seconds to minutes) required to induce inhibition of the [Ca2+]i response. Inasmuch as added FFA primarily binds to membrane lipid, is not appreciably esterified, and the inhibition is reversed by treatment with fatty acid free BSA, it is likely that the oleic acid effects are due to a physical perturbation of membrane lipid. Furthermore, oleic acid does not affect Con A binding or the production of inositol phosphate metabolites, suggesting that the inhibition of the response is distal to surface recognition events or receptor-phospholipase C coupling. Given the relatively low levels of FFA at which these effects occur it is possible, under conditions in which FFA levels are elevated, that FFA perturbation may modulate CTL activity.

Animals↗

PubMed1989

Plasma membrane lipid order and composition during adipocyte differentiation of 3T3F442A cells. Studies in intact cells with 1-[4-(trimethylamino)phenyl]-6-phenylhexatriene.

The plasma membrane lipid order of 3T3F442A cells was examined during the course of adipocyte differentiation by measuring the fluorescence polarization of 1-[4-(trimethylamino)phenyl]-6-phenylhexatriene. This cationic fluorophore labels the plasma membrane but does not rapidly redistribute to intracellular organellar membranes and can, therefore, be used to specifically probe the plasma membrane of intact cells. Studies with whole cells demonstrated that the plasma membrane of 3T3F442A cells becomes less ordered during the course of adipocyte conversion and that this alteration begins relatively early during the differentiation process. In addition, the lipid order of plasma membranes isolated from adipocyte-stage cells was found to be lower than the lipid order of the early, fibroblast-stage cells. Analysis of membrane lipid composition suggests that the molecular bases for the decrease in adipocyte plasma membrane lipid order are a large increase in the level of monounsaturated phospholipid acyl chains and a decrease in the molar ratio of cholesterol to phospholipid. The alteration in plasma membrane lipid composition may be specifically required for integral membrane protein function, since the differentiation-dependent fatty acid desaturase activity is known to be maintained even in the absence of triacylglycerol accumulation.

Adipose Tissue↗