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Purification and characterization of fatty acid-binding protein from human placenta
T Das1, G Sa, M Mukherjea
1Department of Biochemistry, University College of Science, Calcutta, India.
Insights
Cytosolic fatty acid-binding protein (FABP) from human placenta was purified and found to modulate glucose-6-phosphate dehydrogenase (G6PD) activity. FABP protects G6PD from inhibition by palmitoyl-CoA (PAL-CoA) by binding the inhibitor.
Area of Science:
- Biochemistry
- Molecular Biology
- Human Physiology
Background:
- Cytosolic fatty acid-binding proteins (FABPs) are involved in lipid metabolism.
- Human placental glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in glucose metabolism.
- Palmitoyl-CoA (PAL-CoA) is known to inhibit G6PD activity.
Purpose of the Study:
- To purify cytosolic FABP from human placenta.
- To investigate the role of placental FABP in modulating G6PD inhibition by PAL-CoA.
Main Methods:
- DEAE cellulose chromatography for FABP purification.
- Thin layer chromatography (TLC) and gas liquid chromatography (GLC) for lipid analysis.
- Ouchterlony double immunodiffusion for immunochemical identity confirmation.
- Enzyme activity assays to study G6PD inhibition and FABP modulation.
Main Results:
- FABP was resolved into three fractions (DE-I, DE-II, DE-III) with identical molecular weights (14,200 Daltons) but different binding affinities.
- DE-II fraction non-specifically binds long-chain fatty acids, while DE-III primarily binds arachidonic acid.
- Placental G6PD was inhibited by PAL-CoA, and the DE-II FABP fraction protected the enzyme from this inhibition.
- FABP's protective effect is attributed to its binding of acyl-CoA, not a direct interaction with G6PD.
Conclusions:
- Human placental FABP exists in multiple forms with distinct lipid-binding properties.
- FABP plays a crucial role in regulating G6PD activity by sequestering inhibitory acyl-CoA molecules.
- This interaction highlights FABP's significance in maintaining metabolic homeostasis in the human placenta.
Abstract:
Purification of a cytosolic fatty acid-binding protein (FABP) from developing human placenta has been achieved, and its role in modulating the inhibition of human placental glucose-6-phosphate dehydrogenase (G6PD) by palmitoyl-CoA (PAL-CoA) has been studied. FABP was resolved into three peaks, viz. DE-I, DE-II and DE-III, by DEAE cellulose chromatography. DE-I was almost lipid-free. Presence of endogenous fatty acids in DE-II and DE-III was detected by thin layer chromatography (TLC). Fatty acids were the only detectable lipid component in these fractions. Gas liquid chromatography (GLC) analysis revealed that DE-II binds long chain saturated and unsaturated fatty acids nonspecifically, whereas DE-III is mainly an arachidonic acid carrier. Each of these fractions, viz. DE-I, DE-II and DE-III, has a molecular weight of 14,200 Daltons. Ouchterlony double immunodiffusion studies have confirmed the immunochemical identity of these three fractions of placental FABP. Separation in ion exchanger may be due to their different isoelectric points and varied types of binding affinities. Human placental G6PD was inhibited 50% by 0.03 mM PAL-CoA. The DE-II fraction of FABP enhanced the activity of G6PD in the absence of added PAL-CoA and protected against PAL-CoA inhibition of the enzyme. Such a modulating effect of FABP in this inhibition is attributable to binding of long chain acyl-CoA rather than to a direct effect of FABP on the enzyme itself.