Biomedical subjects

F Schroeder

Publications and source records attributed to F Schroeder.

At least 109 records · Page 6Linked to original sources

PubMed1993

Expression of liver fatty acid binding protein alters plasma membrane lipid composition and structure in transfected L-cell fibroblasts.

Liver fatty acid binding protein, L-FABP, is an abundant protein that binds fatty acids in vitro. The effects of L-FABP on plasma membrane lipid composition, distribution, and physical structure were determined in intact L-cell fibroblasts transfected with cDNA encoding L-FABP. L-FABP expression altered plasma membrane phospholipids by decreasing both phosphatidylethanolamine and esterified oleic acid content, and increasing sphingomyelin. L-FABP also binds sterols and stimulates sterol uptake and esterification. The fluorescent sterol dehydroergosterol was used to examine sterol distribution in the transfected cell plasma membrane. Dehydroergosterol codistributed equally with the cholesterol in both the bulk membrane and the individual bilayer leaflets. The sterol/phospholipid ratio was decreased in the inner leaflet due to sterol depletion. Concomitantly, intermembrane sterol transfer from the rapidly exchangeable lateral sterol domains as measured by exchange of dehydroergosterol, was reduced. The fluidity of the plasma membrane was measured with the fluorescent molecule diphenylhexatriene by multifrequency (1-250 MHz) phase and modulation fluorometry. Both the bulk plasma membrane and the plasma membrane outer leaflet lipids were fluidized in transfected cells. These alterations of plasma membrane structure and composition are consistent with a role for L-FABP in regulating intracellular sterol and fatty acid distribution and thereby membrane lipid domain structure.

Animals↗

PubMed1993

Kinetics and size of cholesterol lateral domains in synaptosomal membranes: modification by sphingomyelinase and effects on membrane enzyme activity.

Cholesterol domains and transport have been well-studied in non-neuronal membranes in contrast to neuronal membranes. The purpose of the experiments reported in this paper was to determine: (1) exchangeable and non-exchangeable cholesterol domains or pools were present in brain synaptosomal membranes; (2) effects of hydrolysis of sphingomyelin on cholesterol pools, that has previously been shown to alter membrane cholesterol in non-neuronal membranes and; (3) sphingomyelin hydrolysis and enzyme activity. Cholesterol pools were determined using cholesterol exchange between radiolabeled small unilamellar vesicles and mouse synaptosomes. Activity of Ca(2+)+Mg(2+)-ATPase and Na(+)+K(+)-ATPase were measured in synaptosomal membranes following treatment with sphingomyelinase. The size of the exchangeable pool of synaptosomal membrane cholesterol was approx 50% of total membrane cholesterol when measured at 37 degrees C. The t1/2 of cholesterol exchange at 37 degrees C in synaptosomes was approx 10 h. Lowering the incubation temperature to 25 degrees C, significantly reduced the size of the exchangeable pool and significantly increased the t1/2 of cholesterol exchange. Sphingomyelinase treatment of synaptosomes significantly slowed cholesterol exchange but did not modify the size of the exchangeable pool of cholesterol. Ca(2+)+Mg(2+)-ATPase activity was significantly inhibited by sphingomyelinase treatment as compared to Na(+)+K(+)-ATPase activity. Cholesterol domains were described in neuronal tissue and the size and kinetics of those pools were altered by temperature-induced changes in fluidity and hydrolysis of sphingomyelin. Sphingomyelinase-induced changes in Ca(2+)+Mg(2+)-ATPase activity were not affected by hydrolysis of sphingomyelin but appeared to be associated with a reduction in cytofacial phosphatidylinositol.

Animals↗

PubMed1992

Na pump and plasma membrane structure in L-cell fibroblasts expressing rat liver fatty acid binding protein.

Although the intracellular fatty acid binding proteins have been investigated for nearly two decades and purified proteins are now available, little is known regarding the function of these proteins in intact cells. Therefore, L-cell fibroblasts transfected with cDNA encoding for rat liver fatty acid binding protein (L-FABP) were examined as to whether L-FABP expression in intact cells modifies plasma membrane enzyme activities, fluidity, and lipids. Plasma membrane Na/K-ATPase activity was 65.9 +/- 18.7 and 38.6 +/- 22.8 (P less than 0.001) nmol/mg protein x min for control and high-expression transfected cells, respectively. Consistent with this observation, [3H] ouabain binding to whole cells was significantly decreased from 3.7 +/- 0.3 to 2.0 +/- 0.8 pmol ouabain bound/mg cell protein in control and high-expression cells, respectively, whereas the cell's affinity for ouabain was not significantly altered. Unexpectedly, Western blot analysis indicated that transfected cells had higher levels of Na+, K(+)-ATPase protein; in contrast, the activities of 5'-nucleotidase and Mg-ATPase were unaltered. The effects of L-FABP expression on plasma membrane Na/K-ATPase function appeared to be mediated through alterations in plasma membrane lipids and/or structure. The plasma membrane cholesterol/phospholipid ratio decreased and the bulk plasma membrane fluidity increased in the high-expression cells. In conclusion, plasma membrane Na/K-ATPase activity in L cells may be regulated in part through expression of cytosolic L-FABP.

Animals↗

PubMed1992

Sterol domains in phospholipid membranes: dehydroergosterol polarization measures molecular sterol transfer.

The domain structure of cholesterol in membranes and factors affecting it are not well understood. A method, based on kinetics of delta 5,7,9,(11),22-erogostatetraen-3 beta-ol (dehydroergosterol) fluorescence polarization change and not requiring separation of donor and acceptor membranes, was used to examine sterol domains in three-component cholesterol:dehydroergosterol:phospholipid small unilamellar vesicles (SUV). A new mathematical data treatment was developed to provide a direct correlation between molecular sterol exchange and steady-state dehydroergosterol fluorescence polarization measurements. The method identified multiple kinetic pools of sterol in SUV: a small but rapidly exchanging pool, a predominant slowly exchanging pool, and a very slowly exchangeable (nonexchangeable) pool. The relative sizes of the pools and half-times of exchange were highly dependent on the presence of acidic phospholipids and on cytosolic proteins involved in sterol transfer. Thus, the method provides a direct measure of molecular sterol transfer between membranes without separating donor and acceptor membranes.

Biological Transport↗

PubMed1991

Polyene fatty acid interactions with recombinant intestinal and liver fatty acid-binding proteins. Spectroscopic studies.

Binding and proximity relationships of fatty acids with recombinant rat liver fatty acid-binding protein (L-FABP) and intestinal fatty acid-binding protein (I-FABP) were studied with absorption and fluorescence spectroscopy. Protein aromatic amino acids were examined in the absence and presence of bound fatty acid. Second derivative absorbance spectroscopy of the apo- and holoproteins suggested that fatty acid binding altered the conformation of L-FABP, but not of I-FABP. Fatty acid binding also blocked the accessibility of L-FABP tyrosine and I-FABP tryptophan to Stern-Volmer quenching by acrylamide, indicating that these amino acids were present in the fatty acid-binding pocket. Forster energy transfer from I-FABP tryptophan to bound cis-parinaric acid resulted in quenching of tryptophan lifetime and appearance of sensitized lifetime of bound cis-parinaric acid. The calculated donor-acceptor distances were 16.9 +/- 0.6 and 19.2 +/- 0.3 A for I-FABP and L-FABP, respectively. Absorbance spectral shifts and ratios of fluorescence excitation maxima indicated that the parinaric acid microenvironment in the fatty acid-binding site of I-FABP was much less polar than that of L-FABP. Parinaric acids displayed similar rotational correlation time and limiting anisotropy when bound to I-FABP and to L-FABP. These results are consistent with a close proximity of bound fatty acids to the tyrosine and tryptophan residues and with immobilization of the polyene fatty acids in the fatty acid-binding site(s) of L-FABP and I-FABP. The two proteins differ in that only L-FABP has two fatty acid-binding sites and appears to undergo significant conformational change upon fatty acid binding.

Acrylamides↗

PubMed1991

Selective binding of cholesterol by recombinant fatty acid binding proteins.

The sterol binding specificity of rat recombinant liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP) was characterized with [3H]cholesterol and a fluorescent sterol analog dehydroergosterol. Ligand binding analysis, fluorescence spectroscopy, and activation of microsomal acyl-CoA:cholesterol acyltransferase activity showed that L-FABP-bound sterols. 1) Lipidex-1000 assay showed a dissociation constant Kd = 0.78 +/- 0.18 microM and stoichiometry of 0.47 +/- 0.16 mol/mol for [3H]cholesterol binding to L-PABP. 2) With [3H]cholesterol/phosphatidylcholine liposomes, the cholesterol binding parameters for L-FABP were Kd = 1.53 +/- 0.28 microM and stoichiometry 0.83 +/- 0.07 mol/mol. 3) L-FABP interaction with dehydroergosterol altered the fluorescence intensity and polarization of dehydroergosterol. Dehydroergosterol bound to L-FABP with Kd = 0.37 microM and a stoichiometry of 0.83 mol/mol. 4) Cholesterol and dehydroergosterol decreased L-FABP tyrosine lifetime. Dehydroergosterol binding produced sensitized emission of bound dehydroergosterol with longer lifetime.5) L-FABP bound two cis-parinaric acid molecules/molecule of protein. Cholesterol displaced one of these bound cis-parinaric acids. 6) L-FABP enhanced acyl-CoA:cholesterol acyltransferase in a concentration-dependent manner. In contrast, these assays indicated that I-FABP did not bind sterols. Thus, L-FABP appears able to bind 1 mol of cholesterol/mol of L-FABP, the L-FABP sterol binding site is equivalent to one of the two fatty acid binding sites, and L-FABP stimulates acyl-CoA:cholesterol acyltransferase by transfer of cholesterol.

Animals↗

PubMed1991

Transmembrane distribution of sterol in the human erythrocyte.

The transbilayer cholesterol distribution of human erythrocytes was examined by two independent techniques, quenching of dehydroergosterol fluorescence and fluorescence photobleaching of NBD-cholesterol. Dehydroergosterol in conjunction with leaflet selective quenching showed that, at equilibrium, 75% of the sterol was localized to the inner leaflet of resealed erythrocyte ghosts. NBD-cholesterol and fluorescence photobleaching displayed two diffusion values in both resealed ghosts and intact erythrocytes. The fractional contribution of the fast and slow diffusion constants of NBD-labelled cholesterol represent its inner and outer leaflet distribution. At room temperature the plasma membrane inner leaflet of erythrocyte ghosts as well as intact erythrocytes cells contained 78% of the plasma membrane sterol. The erythrocyte membrane transbilayer distribution of sterol was independent of temperature. In conclusion, dehydroergosterol and NBD-cholesterol data are consistent with an enrichment of cholesterol in the inner leaflet of the human erythrocyte.

Cholesterol↗

PubMed1991

Intracellular sterol distribution in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein.

The potential role of liver fatty acid binding protein (L-FABP) in modulating cellular sterol distribution was examined in mouse L-cell fibroblasts transfected with cDNA encoding L-FABP. L-cells were chosen because they contain only a small amount of endogenous FABP which does not bind [3H]cholesterol, does not enhance intermembrane sterol transfer, and whose content is unaltered by the expression of L-FABP. Transfected L-cells expressed 0.34% of cytosolic protein as L-FABP. Transfection alone with low expression of L-FABP (0.008% of cytosolic protein) had no effect on any of the parameters tested. Three aspects of cellular sterol transfer were examined. First, cellular sterol uptake, monitored by [3H]cholesterol and the fluorescent sterol, delta-5,7,9(11),22-ergostatetraen-3 beta-ol, was increased 21.5 +/- 2.6% (p less than 0.001) in L-cells expressing L-FABP. This increase was not accounted for by increased sterol esterification in the cells expressing L-FABP. Inhibition of both cholesterol transfer and esterification with 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide from Sandoz abolished the L-FABP related enhancement of both [3H]cholesterol uptake and esterification. Second, plasma membrane transbilayer distribution of sterol, determined by fluorescence methods indicated that the majority of sterol was in the inner leaflet of the plasma membrane. In transfected cells expressing L-FABP, twice as much sterol (28 +/- 4%) was present in the exofacial leaflet of the plasma membrane as compared to that of control cells (15 +/- 2%). Third, expression of L-FABP enhanced sterol transfer from the plasma membrane to microsomes in intact cells. Treatment of [3H]cholesterol or [3H]oleate-loaded cells with sphingomyelinase resulted in increased formation of radiolabeled cholesterol ester, consistent with enhanced microsomal esterification of plasma membrane derived cholesterol. Concomitantly, plasma membrane [3H]cholesterol became less accessible to oxidation by cholesterol oxidase. Sphingomyelinase-stimulated cholesterol esterification was 21 +/- 3% greater in transfected cells. Concomitantly, accessibility of plasma membrane [3H]cholesterol to cholesterol oxidase was decreased 18 +/- 3% in cells expressing L-FABP. These differences are consistent with the ability of L-FABP to influence sterol transport and plasma membrane transbilayer sterol distribution in intact cells.

Animals↗

PubMed1991

Interaction of fatty acids with recombinant rat intestinal and liver fatty acid-binding proteins.

Intestinal enterocytes contain two homologous fatty acid-binding proteins, intestinal fatty acid-binding protein (I-FABP)2 and liver fatty acid-binding protein (L-FABP). Since the functional basis for this multiplicity is not known, the fatty acid-binding specificity of recombinant forms of both rat I-FABP and rat L-FABP was examined. A systematic comparative analysis of the 18 carbon chain length fatty acid binding parameters, using both radiolabeled (stearic, oleic, and linoleic) and fluorescent (trans-parinaric and cis-parinaric) fatty acids, was undertaken. Results obtained with a classical Lipidex-1000 binding assay, which requires separation of bound from free fatty acid, were confirmed with a fluorescent fatty acid-binding assay not requiring separation of bound and unbound ligand. Depending on the nature of the fatty acid ligand, I-FABP bound fatty acid had dissociation constants between 0.2 and 3.1 microM and a consistent 1:1 molar ratio. The dissociation constants for L-FABP bound fatty acids ranged between 0.9 and 2.6 microM and the protein bound up to 2 mol fatty acid per mole of protein. Both fatty acid-binding proteins exhibited relatively higher affinity for unsaturated fatty acids as compared to saturated fatty acids of the same chain length. cis-Parinaric acid or trans-parinaric acid (each containing four double bonds) bound to L-FABP and I-FABP were displaced in a competitive manner by non-fluorescent fatty acid. Hill plots of the binding of cis- and trans- parinaric acid to L-FABP showed that the binding affinities of the two sites were very similar and did not exhibit cooperativity. The lack of fluorescence self-quenching upon binding 2 mol of either trans- or cis-parinaric acid/mol L-FABP is consistent with the presence of two binding sites with dissimilar orientation in the L-FABP. Thus, the difference in binding capacity between I-FABP and L-FABP predicts a structurally different binding site or sites.

Carrier Proteins↗

PubMed1991

Florid cutaneous papillomatosis, malignant acanthosis nigricans, and pulmonary squamous cell carcinoma.

A 72-year-old man had florid cutaneous papillomatosis (FCP), which is an obligatory paraneoplastic syndrome always associated with an internal malignancy. The cancer, which is usually intraabdominal and most often gastric in origin, evolves parallel to the FCP. This patient is the first case of FCP occurring in association with a lung malignancy. An association of FCP with other signs of internal cancer is common, with malignant acanthosis nigricans usually appearing many times with the sign of Leser-Trélat. FCP, malignant acanthosis nigricans, and the sign of Leser-Trélat are part of a continuum, developing by a common or similar pathogenic pathway due to an underlying malignancy producing a factor possibly similar to human epidermal growth factor.

Acanthosis Nigricans↗

PubMed1991

Membrane cholesterol dynamics: cholesterol domains and kinetic pools.

Nonreceptor mediated cholesterol uptake and reverse cholesterol transport in cells occur through cellular membranes. Thus, elucidation of cholesterol dynamics in membranes is essential to understanding cellular cholesterol accumulation and loss. To this end, it has become increasingly evident that cholesterol is not randomly distributed in either model or biologic membranes. Instead, membrane cholesterol appears to be organized into structural and kinetic domains or pools. Cholesterol-rich and poor domains can even be observed histochemically and physically isolated from epithelial cell surface membranes. The physiologic importance of these domains is 2-fold: (i) Select membrane proteins (receptors, transporters, etc.) are localized in either cholesterol-rich or cholesterol-poor domains. Consequently, the structure and properties of the domains rather than of the bulk lipid may selectively affect the function of proteins residing therein. (ii) Kinetic evidence suggests that cholesterol transport through and between membranes may occur through specific domains or pools. Regulation of the size and properties of such domains may be controlling factors of cholesterol transport or accumulation in cells. Recent technologic advances in the use of fluorescent sterols have allowed examination of cholesterol domain structure in model and biologic membranes. These techniques have been applied to examine the role of high-density lipoprotein, cholesterol lowering drugs, and intracellular lipid transfer proteins in membrane sterol domain structure and sterol movement between membranes.

Animals↗

PubMed1991

Significance of ethanol-induced changes in membrane lipid domains.

Ethanol both in vitro and in vivo has been shown to affect membrane bulk lipids and it had been previously proposed that such effects may explain sensitivity and tolerance to ethanol. There has been however, criticism of the membrane bulk lipid hypothesis and interest in the role of lipids as a site of the effects of ethanol has declined. This change in how lipids are viewed may have been premature. Several groups including work of our laboratory have shown in the past few years that ethanol has a very pronounced effect on membrane lipids but that these effects are restricted to specific domains of the membrane. This paper will discuss how ethanol affects membrane domains and mechanisms that may be involved in the regulation of lipid domains with emphasis on cholesterol domains. Discussion will also cover how lipid domains interact with protein organization and function.

Alcohol Drinking↗

PubMed1990

Altered membrane structure in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein.

Mouse L cell fibroblasts were transfected with cloned cDNA encoding rat liver fatty acid binding protein (L-FABP) also known as sterol carrier protein. Stable transfectant cell lines were selected and expression of L-FABP determined using Western blot analysis. The nontransfected controls and low expression cells did not differ significantly in any of the properties examined. All cell lines showed similar doubling times but cells expressing high levels of L-FABP attained 2-fold higher cell saturation density and differed significantly in their lipid metabolism as indicated by 1) higher cholesterol ester and phospholipid content, and 2) decreased sterol/phospholipid ratio. The observed changes in the lipid composition predicted a lower degree of membrane-lipid order (higher fluidity) in the plasma membranes of cells expressing high levels of L-FABP. Therefore, fluorescent molecule, 1,6-diphenyl-1,3,5-hexatriene, and multifrequency (1-250 MHz) phase and modulation fluorometry were used to probe the effect of L-FABP expression on membrane structure. Steady-state polarization and limiting anisotropy of diphenylhexatriene were significantly lower in the isolated plasma membrane vesicles from the high expression clones. The observed changes in L-cells as a result of de novo expression of L-FABP are consistent with the ability of this protein to bind sterols and fatty acids, stimulate sterol esterification, and stimulate phospholipid biosynthesis. This evidence is supportive of a physiologic role for L-FABP in modulating cellular lipid metabolism and membrane structure.

Animals↗

PubMed1990

Asymmetric distribution of a fluorescent sterol in synaptic plasma membranes: effects of chronic ethanol consumption.

Ethanol-induced structural changes in membranes have in some studies been attributed to an increase in total membrane cholesterol. Consistent changes in cholesterol content, however, have not been observed in membranes of ethanol consuming animals and alcoholic patients. This study examined the hypotheses that cholesterol was asymmetrically distributed in synaptic plasma membranes (SPM) and that chronic ethanol consumption alters the transbilayer distribution of cholesterol. Dehydroergosterol, a fluorescent cholesterol analogue was used to examine sterol distribution and exchange in chronic ethanol-treated and pair-fed control groups. The cytofacial leaflet was found to have significantly more dehydroergosterol as compared to the exofacial leaflet. This asymmetric distribution was significantly reduced by chronic ethanol consumption as was sterol transport. Total cholesterol content did not differ between the two groups. Chronic ethanol consumption appeared to alter transbilayer sterol distribution as determined by the incorporation and distribution of dehydroergosterol in SPM. The changes in transbilayer sterol distribution are consistent with recent reports on the asymmetric effects of ethanol in vitro ((1988) Biochim. Biophys. Acta 946, 85-94) and in vivo ((1989) J. Neurochem. 52, 1925-1930) on membrane leaflet structure. The results of this study also underscore the importance of examining membrane lipid domains in addition to the total content of different lipids.

Alcoholism↗

PubMed1990

Acidic phospholipids strikingly potentiate sterol carrier protein 2 mediated intermembrane sterol transfer.

A liposomal membrane model system was developed to examine the mechanism of spontaneous and protein-mediated intermembrane cholesterol transfer. Rat liver sterol carrier protein 2 (SCP2) and fatty acid binding protein (FABP, also called sterol carrier protein) both bind sterol. However, only SCP2 mediates sterol transfer. The exchange of sterol between small unilamellar vesicles (SUV) containing 35 mol % sterol was monitored with a recently developed assay [Nemecz, G., Fontaine, R. N., & Schroeder, F. (1988) Biochim. Biophys. Acta 943, 511-541], modified to continuous polarization measurement and not requiring separation of donor and acceptor membrane vesicles. As compared to spontaneous sterol exchange, 1.5 microM rat liver SCP2 enhanced the initial rate of sterol exchange between neutral zwwitterionic phosphatidylcholine SUV 2.3-fold. More important, the presence of acidic phospholipids (2.5-30 mol %) stimulated the SCP2-mediated increase in sterol transfer approximately 35-42-fold. Thus, acidic phospholipids strikingly potentiate the effect of SCP2 by 15-18 times as compared to SUV without negatively charged lipids. Rat liver FABP (up to 60 microM) was without effect on sterol transfer in either neutral zwitterionic or anionic phospholipid containing SUV. The potentiation of SCP2 action by acidic phospholipids was suppressed by high ionic strength, neomycin, and low pH. The results suggest that electrostatic interaction between SCP2 and negatively charged membranes may play an important role in the mechanism whereby SCP2 enhances intermembrane cholesterol transfer.

Carrier Proteins↗

PubMed1990

Synthesis and characterization of the dansyltyrosine derivatives of porcine pancreatic colipase.

Steady-state and time-resolved fluorescence techniques were used to study dansyltyrosine derivatives of porcine pancreatic colipase. Nitration, reduction, acylation, and dansylation reactions were utilized to synthesize two fluorescently labeled colipases: (o-aminodansyltyrosine 55 porcine colipase) (DNStyr55PC) and o-aminodansyltyrosine 59 porcine colipase (DNStyr59PC). DNStyr55PC was 200% active, while the DNStyr59 derivative maintained 80% activity in a pH stat assay. Emission spectra, lifetime analysis, acrylamide quenching, polarization, and anisotropy decay studies indicated that Tyr55 was located on the solvent-exposed surface of the protein, where the fluorophore experienced free rotation. Identical experiments done on DNStyr59PC indicated that Tyr59 was in a partially buried environment and the motion of the dansyl tyrosine group was hindered. The double-exponential decay of the fluorescence emission of N-acetyl-o-aminodansyltyrosine ethyl ester (DNStyr) and the DNStyr derivatives of colipase was investigated with pH, temperature, solvent, and emission-resolved-lifetime experiments. The existence of excited-state processes was eliminated in both pH and emission-resolved-lifetime experiments, whereas temperature studies indicated either a rotational isomer or a differential solvent quenching mechanism for multiple decay kinetics. These experiments also showed that DNStyr was a sensitive probe of solvent polarity and viscosity, but not of pH.

Animals↗

PubMed1990

Interaction of fluorescent delta 5,7,9(11),22-ergostatetraen-3 beta-ol with sterol carrier protein-2.

The fluorescent sterol delta 5,7,9(11)-dehydroergostatetraen-3 beta-ol (dehydroergosterol) was used as an analogue of cholesterol to examine the molecular interaction of purified rat liver sterol carrier protein-2 (SCP-2) with sterol. The binding of dehydroergosterol to SCP-2 was evidenced by light scatter and by fluorescence polarization, lifetime, limiting anisotropy, and rotational relaxation time of dehydroergosterol. In addition, energy transfer efficiency from SCP-2 tryptophan to dehydroergosterol was 96%, indicating that the apparent distance, R, between the SCP-2 tryptophan (energy donor) and the dehydroergosterol (energy acceptor) was 13.7 A. Scatchard binding analysis of light scatter, lifetime, and energy transfer data all indicated a 1:1 molar stoichiometry with Kd = 1.2, 1.6, and 1.3 microM, respectively. SCP-2 enhanced the activity of microsomal acyl-CoA:cholesterol acyltransferase through transfer of [3H]cholesterol from donor palmitoyloleoyl phosphatidylcholine/cholesterol small unilamellar vesicles to rat liver microsomes containing the enzyme. A recently developed fluorescence assay utilizing dehydroergosterol fluorescence polarization (Nemecz, G., Fontaine, R. N., and Schroeder, F. (1988) Biochim. Biophys. Acta 948, 511-521; Nemecz, G., and Schroeder, F. (1988) Biochemistry 27, 7740-7749) was applied to examine the effect of SCP-2 on sterol exchange. In the absence of SCP-2, two spontaneously exchangeable sterol domains were observed in palmitoyloleoyl phosphatidylcholine/sterol (65:35 molar ratio) small unilamellar vesicles. SCP-2 enhanced the rate of exchange of the faster exchanging domain 2-fold. The transfer rate of the more slowly exchangeable sterol domain and the fraction of cholesterol represented by each domain were not affected. These results demonstrate the utility of dehydroergosterol to probe SCP-2 interactions with sterols and are indicative of a physiological role for SCP-2 as a soluble sterol carrier.

Animals↗

PubMed1990

Intermembrane cholesterol transfer: role of sterol carrier proteins and phosphatidylserine.

The effect of phosphatidylserine and sterol carrier proteins on cholesterol exchange was determined using an assay not requiring separation of donor and acceptor membrane vesicles. Sterol carrier protein-2 (SCP2, also called nonspecific lipid transfer protein), but not fatty acid binding protein (FABP, also called sterol carrier protein), enhanced the initial rate of sterol exchange between neutral zwitterionic phosphatidylcholine small unilamellar vesicles (SUV) 2.3-fold. Phosphatidylserine at 10 mol% increased the initial rate of spontaneous and of SCP2-mediated (but not FABP-mediated) sterol exchange by 22% and 44-fold, respectively. The SCP2 potentiation of sterol transfer was dependent on SCP2 concentration and on phosphatidylserine concentration. The SCP2-mediated sterol transfer was inhibited by a variety of cations including KCl, divalent metal ions, and neomycin. The data suggest that SCP2 increase in activity for sterol transfer may be partly ascribed to charge on the phospholipid.

Biological Transport↗