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Fatty acid binding protein: stimulation of microsomal phosphatidic acid formation
C A Jolly1, T Hubbell, W D Behnke
1Department of Physiology and Pharmacology, Texas A & M University 77843-4466, USA.
Abstract:
The effect of fatty acid binding proteins (FABPs) on two key steps of microsomal phosphatidic acid formation was examined. Rat liver microsomes were purified by size-exclusion chromatography to remove endogenous cytosolic fatty acid and fatty acyl-CoA binding proteins while recombinant FABPs were used to avoid cross-contamination with such proteins from native tissue. Neither rat liver (L-FABP) nor rat intestinal fatty acid binding protein (I-FABP) stimulated liver microsomal fatty acyl-CoA synthase. In contrast, L-FABP and I-FABP enhanced microsomal conversion of [14C]oleoyl-CoA and glycerol 3-phosphate to [14C]phosphatidic acid by 18- and 7-fold, respectively. The mechanism for this stimulation, especially by I-FABP, is not known. However, several observations presented here suggest that, like L-FABP, I-FABP may interact with fatty acyl-CoA and thereby stimulate enzyme activity. First, I-FABP decreased microsomal membrane-bound oleoyl-CoA. Second, oleoyl-CoA displaced I-FABP bound fluorescent fatty acid, cis-parinaric acid, with Ki of 5.3 microM and 1.1 sites. Third, oleoyl-CoA decreased I-FABP tryptophan fluorescence with a Kd of 4.2 microM. Fourth, oleoyl-CoA red shifted emission spectra of acrylodated I-FABP, a sensitive marker of I-FABP interactions with ligands. In summary, the results demonstrate for the first time that both L-FABP and I-FABP stimulate liver microsomal phosphatidic acid formation by enhancing synthesis of phosphatidate from fatty acyl-CoA and glycerol 3-phosphate.
Insights
Fatty acid binding proteins (FABPs), including liver (L-FABP) and intestinal (I-FABP), significantly enhance phosphatidic acid formation in liver microsomes. This occurs by boosting the conversion of fatty acyl-CoA and glycerol 3-phosphate to phosphatidate.
Area of Science:
- Biochemistry
- Cell Biology
- Lipid Metabolism
Background:
- Fatty acid binding proteins (FABPs) are involved in lipid transport and metabolism.
- Microsomal phosphatidic acid formation is a crucial step in phospholipid synthesis.
- The specific roles of L-FABP and I-FABP in this process require further elucidation.
Purpose of the Study:
- To investigate the effect of L-FABP and I-FABP on key steps of microsomal phosphatidic acid formation.
- To determine whether FABPs influence fatty acyl-CoA synthase activity.
- To characterize the mechanism by which FABPs enhance phosphatidic acid synthesis.
Main Methods:
- Purification of rat liver microsomes using size-exclusion chromatography.
- Utilized recombinant FABPs to prevent cross-contamination.
- Enzyme assays measuring fatty acyl-CoA synthase and phosphatidic acid formation.
- Fluorescence spectroscopy to study FABP-ligand interactions.
Main Results:
- Neither L-FABP nor I-FABP stimulated liver microsomal fatty acyl-CoA synthase.
- Both L-FABP and I-FABP significantly enhanced the conversion of fatty acyl-CoA and glycerol 3-phosphate to phosphatidic acid (18-fold and 7-fold, respectively).
- Evidence suggests I-FABP interacts with fatty acyl-CoA, potentially mediating the observed stimulation.
Conclusions:
- L-FABP and I-FABP directly stimulate liver microsomal phosphatidic acid formation.
- The stimulation is primarily due to enhanced synthesis of phosphatidate from fatty acyl-CoA and glycerol 3-phosphate.
- FABP interaction with fatty acyl-CoA is implicated in the mechanism of stimulation.