Stopped-flow kinetic analysis of long-chain fatty acid dissociation from bovine serum albumin

2002 ◽  
Vol 363 (3) ◽  
pp. 809-815 ◽  
Author(s):  
Erland J.F. DEMANT ◽  
Gary V. RICHIERI ◽  
Alan M. KLEINFELD
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Time Course ◽  
Acid Dissociation ◽  
Fatty Acid Transport ◽  
Acid Transport ◽  
Fatty Acid Binding ◽  
Kinetics Of ◽  
Initial Fatty Acid ◽  
Long Chain

The kinetics of the interaction of long-chain fatty acids (referred to as fatty acids) with albumin is critical to understanding the role of albumin in fatty acid transport. In this study we have determined the kinetics of fatty acid dissociation from BSA and the BSA-related fatty acid probe BSA-HCA (BSA labelled with 7-hydroxycoumarin-4-acetic acid) by stopped-flow methods. Fatty acid—albumin complexes of a range of natural fatty acid types and albumin molecules (donors) were mixed with three fatty acid-binding acceptor proteins. Dissociation of fatty acids from the donor was monitored by either the time course of donor fluorescence/absorbance or the time course of acceptor fluorescence. The results of these measurements indicate that fatty acid dissociation from BSA as well as BSA-HCA is well described by a single exponential function over the entire range of fatty acid/albumin molar ratios used in these measurements, from 0.5:1 to 6:1. The observed rate constants (kobs) for the dissociation of each fatty acid type reveal little or no dependence on the initial fatty acid/albumin ratio. However, dissociation rates were dependent upon the type of fatty acid. In the case of native BSA with an initial fatty acid/BSA molar ratio of 3:1, the order of kobs values was stearic acid (1.5s−1)<oleic acid<palmitic acid≅linoleic acid<arachidonic acid (8s−1) at 37°C. The corresponding values for BSA-HCA were about half the values for BSA. The results of this study show that the rate of fatty acid dissociation from native BSA is more than 10-fold faster than reported previously and that the off-rate constants for the five primary fatty acid-binding sites differ by less than a factor of 2. We conclude that for reported rates of fatty acid transport across cell membranes, dissociation of fatty acids from the fatty acid—BSA complexes used in the transport studies should not be rate-limiting.

scholarly journals New insights into long-chain fatty acid uptake by heart muscle: a crucial role for fatty acid translocase/CD36

2002 ◽  
Vol 367 (3) ◽  
pp. 561-570 ◽  
Author(s):  
Joep F.F. BRINKMANN ◽  
Nada A. ABUMRAD ◽  
Azeddine IBRAHIMI ◽  
Ger J. vanderVUSSE ◽  
Jan F.C. GLATZ
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Heart Muscle ◽  
Fatty Acid Transport ◽  
Acid Uptake ◽  
Membrane Fatty Acid ◽  
Acid Transport ◽  
Fatty Acid Binding ◽  
Fatty Acid Translocase ◽  
Long Chain

Long-chain fatty acids are an important source of energy for several cell types, in particular for the heart muscle cell. Three different proteins, fatty acid translocase (FAT)/CD36, fatty acid transport protein and plasma membrane fatty acid binding protein, have been identified as possible membrane fatty acid transporters. Much information has been accumulated recently about the fatty acid transporting function of FAT/CD36. Several experimental models to study the influence of altered FAT/CD36 expression on fatty acid homoeostasis have been identified or developed, and underscore the importance of FAT/CD36 for adequate fatty acid transport. These models include the FAT/CD36 null mouse, the spontaneously hypertensive rat and FAT/CD36-deficient humans. The fatty acid transporting role of FAT/CD36 is further demonstrated in mice overexpressing muscle-specific FAT/CD36, and in transgenic mice generated using a genetic-rescue approach. In addition, a wealth of information has been gathered about the mechanisms that regulate FAT/CD36 gene expression and the presence of functional FAT/CD36 on the plasma membrane. Available data also indicate that FAT/CD36 may have an important role in the aetiology of cardiac disease, especially cardiac hypertrophy and diabetic cardiomyopathy. This review discusses our current knowledge of the three candidate fatty acid transporters, the metabolic consequences of alterations in FAT/CD36 levels in different models, and the mechanisms that have been identified for FAT/CD36 regulation.

scholarly journals Transmembrane Movement of Exogenous Long-Chain Fatty Acids: Proteins, Enzymes, and Vectorial Esterification

2003 ◽  
Vol 67 (3) ◽  
pp. 454-472 ◽  
Author(s):  
Paul N. Black ◽  
Concetta C. DiRusso
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Molecular Genetics ◽  
Fatty Acyl ◽  
Model Systems ◽  
Fatty Acid Transport ◽  
Long Chain Fatty Acids ◽  
Acid Transport ◽  
Long Chain ◽  
Chain Fatty Acids

SUMMARY The processes that govern the regulated transport of long-chain fatty acids across the plasma membrane are quite distinct compared to counterparts involved in the transport of hydrophilic solutes such as sugars and amino acids. These differences stem from the unique physical and chemical properties of long-chain fatty acids. To date, several distinct classes of proteins have been shown to participate in the transport of exogenous long-chain fatty acids across the membrane. More recent work is consistent with the hypothesis that in addition to the role played by proteins in this process, there is a diffusional component which must also be considered. Central to the development of this hypothesis are the appropriate experimental systems, which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both (i) exhibit saturable long-chain fatty acid transport at low ligand concentrations, (ii) have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus, and (iii) can be easily manipulated using the tools of molecular genetics. In both systems, central players in the process of fatty acid transport are fatty acid transport proteins (FadL or Fat1p) and fatty acyl coenzyme A (CoA) synthetase (FACS; fatty acid CoA ligase [AMP forming] [EC 6.2.1.3]). FACS appears to function in concert with FadL (bacteria) or Fat1p (yeast) in the conversion of the free fatty acid to CoA thioesters concomitant with transport, thereby rendering this process unidirectional. This process of trapping transported fatty acids represents one fundamental mechanism operational in the transport of exogenous fatty acids.

scholarly journals Deletion of fatty acid transport protein 2 (FATP2) in the mouse liver changes the metabolic landscape by increasing the expression of PPARα-regulated genes

2020 ◽  
Vol 295 (17) ◽  
pp. 5737-5750 ◽  
Author(s):  
Vincent M. Perez ◽  
Jeffrey Gabell ◽  
Mark Behrens ◽  
Nishikant Wase ◽  
Concetta C. DiRusso ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Transport Protein ◽  
Transcriptomic Analysis ◽  
Fatty Acid Transport ◽  
Long Chain Fatty Acids ◽  
Acid Transport ◽  
Fatty Acid Transport Protein ◽  
Long Chain ◽  
Chain Fatty Acids

Fatty acid transport protein 2 (FATP2) is highly expressed in the liver, small intestine, and kidney, where it functions in both the transport of exogenous long-chain fatty acids and the activation of very-long-chain fatty acids. Here, using a murine model, we investigated the phenotypic impacts of deleting FATP2, followed by a transcriptomic analysis using unbiased RNA-Seq to identify concomitant changes in the liver transcriptome. WT and FATP2-null (Fatp2−/−) mice (5 weeks) were maintained on a standard chow diet for 6 weeks. The Fatp2−/− mice had reduced weight gain, lowered serum triglyceride, and increased serum cholesterol levels and attenuated dietary fatty acid absorption. Transcriptomic analysis of the liver revealed 258 differentially expressed genes in male Fatp2−/− mice and a total of 91 in female Fatp2−/− mice. These genes mapped to the following gene ontology categories: fatty acid degradation, peroxisome biogenesis, fatty acid synthesis, and retinol and arachidonic acid metabolism. Targeted RT-quantitative PCR verified the altered expression of selected genes. Of note, most of the genes with increased expression were known to be regulated by peroxisome proliferator–activated receptor α (PPARα), suggesting that FATP2 activity is linked to a PPARα-specific proximal ligand. Targeted metabolomic experiments in the Fatp2−/− liver revealed increases of total C16:0, C16:1, and C18:1 fatty acids; increases in lipoxin A4 and prostaglandin J2; and a decrease in 20-hydroxyeicosatetraenoic acid. We conclude that the expression of FATP2 in the liver broadly affects the metabolic landscape through PPARα, indicating that FATP2 provides an important role in liver lipid metabolism through its transport or activation activities.

scholarly journals New concepts of cellular fatty acid uptake: role of fatty acid transport proteins and of caveolae

2004 ◽  
Vol 63 (2) ◽  
pp. 259-262 ◽  
Author(s):  
Jürgen Pohl ◽  
Axel Ring ◽  
Thomas Herrmann ◽  
Wolfgang Stremmel
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Lipid Rafts ◽  
Transport Proteins ◽  
Fatty Acid Transport ◽  
Long Chain Fatty Acids ◽  
Acid Transport ◽  
Fatty Acid Transport Proteins ◽  
Lcfa Uptake ◽  
Long Chain

Efficient uptake and channelling of long-chain fatty acids (LCFA) are critical cell functions. Evidence is emerging that proteins are important mediators of LCFA-trafficking into cells and various proteins have been suggested to be involved in this process. Amongst these proteins is a family of membrane-associated proteins termed fatty acid transport proteins (FATP). So far six members of this family, designated FATP 1–6, have been characterized. FATP 1, 2 and 6 show a highly-conserved AMP-binding region that participates in the activation of very-long-chain fatty acids (VLCFA) to form their acyl-CoA derivatives. The mechanisms by which FATP mediate LCFA uptake are not well understood, but several studies provide evidence that uptake of LCFA across cellular membranes is closely linked to acyl-CoA synthetase activity. It is proposed that FATP indirectly enhance LCFA uptake by activating VLCFA to their CoA esters, which are required to maintain the typical structure of lipid rafts in cellular membranes. Recent work has shown that the structural integrity of lipid rafts is essential for cellular LCFA uptake. This effect might be exerted by proteins, e.g. caveolin-1 and FAT/CD36, that use lipid rafts as platforms and bind or transport LCFA. The proposed molecular mechanisms await further experimental investigation.

scholarly journals Placental transfer of long-chain polyunsaturated fatty acids (LC-PUFA)

2007 ◽  
Vol 35 (s1) ◽  
pp. S5-S11 ◽  
Author(s):  
Berthold Koletzko ◽  
Elvira Larqué ◽  
Hans Demmelmair
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Polyunsaturated Fatty Acids ◽  
Human Placenta ◽  
Placental Transfer ◽  
Fetal Brain ◽  
Fatty Acid Transport ◽  
Omega 3 ◽  
Fatty Acid Binding ◽  
Long Chain

AbstractConsiderable evidence exists for marked beneficial effects of omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) during pregnancy. The omega-3 LC-PUFA docosahexaenoic acid (DHA) is incorporated in large amounts in fetal brain and other tissues during the second half of pregnancy, and several studies have provided evidence for a link between early DHA status of the mother and visual and cognitive development of her child after birth. Moreover, the supplementation of omega-3 LC-PUFA during pregnancy increases slightly infant size at birth, and significantly reduces early preterm birth before 34 weeks of gestation by 31%. In our studies using stable isotope methodology in vivo, we demonstrated active and preferential materno-fetal transfer of DHA across the human placenta and found the expression of human placental fatty acid binding and transport proteins. From the correlation of DHA values with placental fatty acid transport protein 4 (FATP 4), we conclude that this protein is of key importance in mediating DHA transport across the human placenta. Given the great importance of placental DHA transport for infant outcome, further studies are needed to fully appreciate the effects and optimal strategies of omega-3 fatty acid interventions in pregnancy, dose response relationships, and the potential differences between subgroups of subjects such as women with gestational diabetes or other gestational pathology. Such studies should contribute to optimize substrate intake during pregnancy and lactation that may improve pregnancy outcome as well as fetal growth and development.

Transfer of fatty acids between intracellular membranes: roles of soluble binding proteins, distance, and time

2002 ◽  
Vol 282 (1) ◽  
pp. G105-G115 ◽  
Author(s):  
R. A. Weisiger ◽  
S. D. Zucker
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Binding Proteins ◽  
Specific Cell ◽  
Fatty Acid Transport ◽  
Membrane Separations ◽  
Acid Transport ◽  
Fatty Acid Binding ◽  
Intracellular Membranes ◽  
Rate Limiting

Soluble fatty acid binding proteins (FABPs) are thought to facilitate exchange of fatty acids between intracellular membranes. Although many FABP variants have been described, they fall into two general classes. “Membrane-active” FABPs exchange fatty acids with membranes during transient collisions with the membrane surface, whereas “membrane-inactive” FABPs do not. We used modeling of fatty acid transport between two planar membranes to examine the hypothesis that these two classes catalyze different steps in intracellular fatty acid transport. In the absence of FABP, the steady-state flux of fatty acid from the donor to the acceptor membrane depends on membrane separation distance (d) approaching a maximum value ( J max) as d approaches zero. J max is one-half the rate of dissociation of fatty acid from the donor membrane, indicating that newly dissociated fatty acid has a 50% chance of successfully reaching the acceptor membrane before rebinding to the donor membrane. For larger membrane separations, successful transfer becomes less likely as diffusional concentration gradients develop. The mean diffusional excursion of the fatty acid into the water phase (dm) defines this transition. For d≪dm, dissociation from the membrane is rate limiting, whereas for d≫dm, aqueous diffusion is rate limiting. All forms of FABP increase dmby reducing the rate of rebinding to the donor membrane, thus maintaining J max over larger membrane separations. Membrane-active FABPs further increase J max by catalyzing the rate of dissociation of fatty acids from the donor membrane, although frequent membrane interactions would be expected to reduce their diffusional mobility through a membrane-rich cytoplasm. Individual FABPs may have evolved to match the membrane separations and densities found in specific cell lines.

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