Characterization of Binding Between the Rat Small Intestinal Brush-border Membrane and Dietary Proteins in the Sensory Mechanism of Luminal Dietary Proteins

Abstract

Dietary proteins are recognized by the gastrointestinal tract to display physiological functions, however, the sensory mechanism of the intestinal mucosa is not known. We examined binding properties between the rat small intestinal brush-border membrane (BBM) and proteins by using a surface plasmon resonance biosensor. BBM and solubilized BBM prepared from the rat jejunum bound to casein immobilized on the sensor surface, but not to bovine serum albumin. The ileal BBM showed less binding to casein than the jejunal BBM. Solubilized BBM binding to immobilized a-casein was slightly inhibited by aminopeptidase inhibitors, but still more inhibited by addition of casein with the inhibitors. Guanidinated casein inhibited the solubilized BBM binding to a-casein more strongly than casein (casein sodium and a-casein) inhibited. Trypsinization of solubilized BBM abolished its binding activity to a-casein. These results indicate that some membrane protein, but not aminopeptidases, contained in BBM interacts with dietary proteins, and that guanidinated casein has a higher affinity for BBM than intact casein. These binding intensities for proteins were closely correlated to physiological responsiveness, and are possibly involved in a sensory system for dietary protein in the intestine.

Casein Binds to the Cell Membrane and Induces Intracellular Calcium Signals in the Enteroendocrine Cell: A Brief Communication

“…These results suggest that casein and ␣-casein bind to STC-1 cell membrane components and that ␣-casein binds more readily than casein sodium. The difference between BSA and casein is consistent with previous observations with rat small intestinal brushborder membranes (20).…”

“…The supernatant was divided into aliquots and stored at −80°C. When the biosensor analysis was conducted, the aliquots were diluted 1:10 with HBS-E buffer before binding analysis (final concentration of Triton X-100 was 0.01%) (20).…”

“…Binding between dietary proteins and rat small intestinal brush-border membranes was recently demonstrated using a surface plasmon resonance sensor (SPR sensor) (20). In that study, the binding between dietary proteins and the brush-border membranes correlated with the physiological activities of the proteins in the rat small intestine.…”

Pork Peptone Stimulates Cholecystokinin Secretion from Enteroendocrine Cells and Suppresses Appetite in Rats

“…13,14) It has been suggested that binding of dietary proteins or peptides to the rat small intestinal brush-border membrane (BBM) is an integral part of a common sensory mechanism to release CCK. 15) We found that appetite suppression by BconP corresponds to the binding ability of this peptone to the rat small intestinal BBM as well as stimulation of CCK release directly from rat isolated dispersed intestinal mucosal cells. 14) Cellular preparations from rat isolated dispersed intestinal mucosa might not consist solely of CCKproducing I-cells due to cell heterogeneity in the intestinal mucosa.…”

“…19) Briefly, the soluble components of rat intestinal BBM were prepared by solubilization with Triton X-100; they were immobilized as ligand over a flow cell surface (a test flow cell) of the CM5 sensor chip by an amine-coupling procedure. 15) Ethanolamine was immobilized onto another flow cell as a reference. Test peptone was dissolved (100 mg/ml) in HBS-E buffer containing 10 mM HEPES, 150 mmol/l NaCl, and 3 mmol/l EDTA, pH 7.4, and injected over both the test and reference flow cells at a rate of 10 ml/min for 2 min as the analyte.…”