Fatty acid transfer between multilamellar liposomes and fatty acid-binding proteins.

Brecher, Peter; Saouaf, Rola; Sugarman, Jacqueline M.; Eisenberg, Dan T. A.; LaRosa, Karen

doi:10.1016/s0021-9258(18)90708-8

Cited by 82 publications

(26 citation statements)

References 33 publications

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“…However, it is not proper to make this assumption, and recalculating our data using the present formalism gave a K0 5 value of 60 nM. By calculation of the binding affinity using total ligand concentration minus bound ligand concentration, the K05 values are reproducible and independent of protein concentration but are greater than those reported by Brecher et al (1984) or Bass (1985), who assumed total ligand concentration equals free ligand concentration. However, when the ligand in question is relatively insoluble in aqueous solution and can exist in a variety of dispersed forms, the true free ligand concentration is not easily determined.…”

Section: Resultsmentioning

confidence: 55%

“…To determine the ligand-binding properties of H-ALBP, two assays were utilized; the liposome-binding assay for fatty acids and the fluorescence quenching assay for retinoids. Using the liposome assay of Brecher et al (1984), human ALBP binds [3H]oleic acid saturably, with a K0i value of 60 mM. Scatchard analysis indicated that H-ALBP binds approximately 1 mol of fatty acid per mole of ALBP (Figure 3).…”

Section: Resultsmentioning

confidence: 99%

“…Oleic Acid Binding. The fatty acid binding properties of H-ALBP were determined by using the liposome-binding assay developed by Brecher et al (1984), as described by Matarese and Bernlohr (1988). Briefly, stock liposomes were prepared containing 40 mM phosphatidylcholine, 13 mM cholesterol, and varying concentrations of [3H]oleate, ranging from 0.15 to 3.0 mM.…”

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confidence: 99%

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Human adipocyte lipid-binding protein: purification of the protein and cloning of its complementary DNA

Baxa¹,

et al. 1989

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Human adipocyte lipid-binding protein (H-ALBP) was purified from normal subcutaneous adipose tissue to greater than 98% homogeneity, utilizing a combination of acid fractionation, gel filtration, covalent chromatography on activated thiol-Sepharose 4B, and anion-exchange chromatography. Human ALBP comprised about 1% of total cytosolic protein in human adipose tissue, had a relative molecular mass of about 15 kDa, and existed as a monomer in solution. The amino terminus of H-ALBP was blocked to sequencing. When a liposome ligand delivery assay was used, H-ALBP saturably bound oleic acid with about 1 mol of ligand bound per mole of protein. Additionally, H-ALBP saturably bound retinoic acid as determined by the quenching of intrinsic tryptophan fluorescence. A full-length H-ALBP cDNA has been cloned; the sequence predicts a 649-base mRNA comprised of a 62-base 5'-noncoding region containing an 18S ribosome-binding site, a single 396-base open-reading frame, and a 191-base 3'-noncoding region. Comparative sequence analysis indicated that the 132 amino acid H-ALBP is a member of a multigene family of intracellular lipid-binding proteins and contains the consensus substrate phosphorylation sequence for tyrosyl kinases.

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Section: Resultsmentioning

confidence: 55%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Human adipocyte lipid-binding protein: purification of the protein and cloning of its complementary DNA

Baxa¹,

et al. 1989

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“…Despite such effects, properties of the fluorescent analogues used here are in fact similar to those of ffa. For example, Brecher et al (1984) have reported that at pH 4, the transfer of [14C] 18:1 from multilamellar egg PC-cholesterol vesicles to fatty acid binding proteins was negligible compared to transfer at pH 8. Thus, while there is little doubt that the presence of a relatively large hydrophobic group imparts properties different from the native ffa, the observed relative relationships are likely to hold for unmodified molecules.…”

Section: Discussionmentioning

confidence: 99%

Transfer of long-chain fluorescent free fatty acids between unilamellar vesicles

Storch¹,

Kleinfeld²

1986

Biochemistry

166

170

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Movement of free fatty acids (ffa) between small unilamellar vesicles (SUV) was studied by measuring the transfer of fluorescent n-(9-anthroyloxy)-labeled analogues (AOffa) between donor and acceptor vesicles. Donors were composed of egg phosphatidylcholine (PC) loaded with 1-2 mol % AOffa, and acceptors were egg PC containing 10-12 mol % N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE). The fluorescence of AO added directly to acceptor SUV was greater than 98% quenched by energy transfer to NBD. Thus, AOffa movement from donor to acceptor was monitored by the time-dependent decrease in AO fluorescence. The transfer of the short-chain AOffa, although too fast to be resolved by the methods used here, is consistent with studies that find transfer rates on the order of milliseconds and kinetics which are first order. In contrast, transfer rates for the long-chain AOffa are more than 2 orders of magnitude slower, and the kinetics of the transfer process are best described by the sum of two exponentials plus a constant. The ffa ionization state was also found to be an important determinant of transfer rate. The charged species transferred an average of 10-fold faster than the protonated ffa. The ffa pKa in the membrane is 9, as calculated from the pH dependence of transfer. Similar to results found for other lipids, long-chain AOffa are transferred via water rather than a collision-mediated process. The aqueous phase route of AOffa intermembrane transfer is indicated by the lack of effect on transfer of large alterations in the product of donor and acceptor phospholipid concentrations. Moreover, the transfer rate is decreased as [NaCl] is increased from 0.1 to 4 M. This effect of ionic strength is probably due not only to a decrease in the aqueous phase partition of the AOffa but also to an alteration in bilayer structure, as measured by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The observed kinetics are consistent with a model in which the transfer involves two steps: transbilayer movement between the inner and outer bilayer leaflets, followed by transfer from the outer leaflet to the aqueous phase (off rate). Within the framework of this model, the observed slow rate is primarily determined by the rate of transbilayer movement, and the observed fast rate is approximately equal to the off rate. The off rate is about 10-fold faster than the rate of transbilayer movement.

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“…The key physical chemistry questions are whether i ) the affinity of albumin for FA is too high compared to a simple phospholipid bilayer and/or ii ) the rate of desorption from albumin is too slow to allow appreciable transfer of FA. While net transfer of FA to phospholipid bilayers (17) or to cells (18,19) can be measured by methods that involve separation of donor and acceptor (possibly affecting the binding equilibrium), it is not trivial to make such a demonstration in a complex mixture without separation procedures.…”

Section: Adsorptionmentioning

confidence: 99%

Fatty acid transport: difficult or easy?

Hamilton

1998

Journal of Lipid Research

291

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Transport of unesterified fatty acids (FA) into cells has been viewed either as a simple diffusion process regulated mainly by lipid physical chemistry or as a more complex process involving protein catalysis. In this review FA transport in cell membranes is broken down into three essential steps: adsorption, transmembrane movement, and desorption. The physical properties of FA in aqueous, membrane, and protein environments relevant to transport mechanisms are discussed, with emphasis on recent information derived from NMR and fluorescence studies. Because of their low solubility in water and high hydrophobicity, FA bind rapidly and avidly to model membranes (phospholipid bilayers); if albumin is a donor, FA desorb rapidly to reach their equilibrium distribution between the membrane and albumin. The ionization properties of FA in a phospholipid bilayer result in a high population of the un-ionized form ( ϳ 50%) at pH 7.4, which diffuses across the lipid bilayer (flip-flops) rapidly (t 1/2 Ͻ 1 sec). Desorption of FA from a phospholipid surface is slower than transmembrane movement and dependent on the FA chain length and unsaturation, but is rapid for typical dietary FA. These physical properties of FA in model systems predict that proteins are not essential for transport of FA through membranes. The only putative FA transport protein to be purified and reconstituted into phospholipid bilayers, the mitochondrial uncoupling protein (UCP1), was shown to transport the FA anion in response to FA flip-flop. New experiments with cells have found that FA movement into cells acidifies the cytosol, as predicted by the flip-flop model.-

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Fatty acid transfer between multilamellar liposomes and fatty acid-binding proteins.

Cited by 82 publications

References 33 publications

Human adipocyte lipid-binding protein: purification of the protein and cloning of its complementary DNA

Human adipocyte lipid-binding protein: purification of the protein and cloning of its complementary DNA

Transfer of long-chain fluorescent free fatty acids between unilamellar vesicles

Fatty acid transport: difficult or easy?

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