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Published on: December 24, 2009
Identification, isolation, and partial characterization of a fatty acid binding protein from rat jejunal microvillous
Abstract:
The mechanisms by which FFA are absorbed by the gut are unclear. To examine these processes, binding of [14C]oleate to isolated rat jejunal microvillous membranes (MVM) was studied in vitro. When [14C]oleate alone or compounded with bovine serum albumin at various molar ratios was incubated with MVM aliquots, binding was time- and temperature-dependent, inhibitable by addition of excess cold oleate, and decreased by heat denaturation or trypsin digestion of the membranes. When [14C]oleate binding to heat denatured MVM, which increased continuously as a function of the free oleate concentration and was taken as a measure of nonspecific binding, was subtracted from total binding to native MVM, a curve suggestive of saturable specific binding was observed. In contrast to fatty acids, there was no specific binding of [14C]taurocholate or [35S]sulfobromophthalein to jejunal MVM. After MVM solubilization with 1% Triton X-100, affinity chromatography over oleate-agarose and elution with 7 M urea yielded a single 40,000-mol-wt protein. This Sudan Black/periodic acid-Schiff-stain-negative protein co-chromatographed on Sephadex G-100 with [14C]oleate, [14C]palmitate, [14C]arachidonate, and [14C]linoleate, but not with the [14C]oleate ester of cholesterol, [14C]phosphatidylcholine, [14C]taurocholate, or [35S]sulfobromophthalein. A rabbit antibody to the previously reported hepatic membrane fatty acid binding protein (FABP) gave a single line of immunologic identity between the FABPs of rat jejunum and rat liver membrane. It inhibited the binding of [14C]oleate to native MVM but not heat denatured MVM, and, in immunohistochemical studies, demonstrated the presence of the FABP in the apical and lateral portions of the brush border cells of the jejunum, but not on the luminal surface of esophagus or colon. These data are compatible with the hypothesis that a specific FABP plays a role in fatty acid absorption from the gut.
Insights
A specific fatty acid-binding protein (FABP) in the rat jejunum plays a role in absorbing dietary fatty acids. This protein was identified and localized to the brush border cells, supporting its function in intestinal fat absorption.
Area of Science:
- Gastroenterology
- Molecular Biology
- Biochemistry
Background:
- The absorption mechanisms of free fatty acids (FFA) in the gastrointestinal tract remain incompletely understood.
- Investigating the molecular players involved in FFA uptake is crucial for understanding nutrient absorption and metabolic processes.
Purpose of the Study:
- To elucidate the mechanisms of free fatty acid absorption in the rat jejunum.
- To identify and characterize proteins involved in the binding and transport of fatty acids across the intestinal brush border membrane.
Main Methods:
- In vitro binding assays using radiolabeled oleate and isolated rat jejunal microvillous membranes (MVM).
- Protein isolation via affinity chromatography and characterization using gel filtration and SDS-PAGE.
- Immunological studies using antibodies against hepatic fatty acid-binding protein (FABP) and immunohistochemistry.
Main Results:
- Specific, saturable binding of oleate to jejunal MVM was observed, dependent on membrane integrity and temperature.
- A 40,000-mol-wt protein was isolated, which specifically bound various fatty acids but not other lipids or bile salts.
- Immunological identity was established between jejunal and hepatic FABPs, and the antibody inhibited oleate binding to MVM.
Conclusions:
- A specific fatty acid-binding protein (FABP) is present in the rat jejunal microvillous membrane.
- This FABP likely plays a significant role in the absorption of free fatty acids from the gut.
- The findings provide molecular insight into the process of intestinal fatty acid uptake.
