**2.2 Separation and identification of glycoproteins from the small intestinal BBM that bind pancreatic α-amylase**

It was shown that pig duodenal BBM contains glycoproteins that bind to pancreatic α-amylase having high mannose-type and complex-type *N*-glycans. Therefore, we separated and identified the glycoproteins binding α-amylase from fasted pig duodenal BBM [14]. The binding glycoproteins were separated from BBM solubilized with 1% TritonX-100 by using affinity chromatography with an α-amylase-Sepharose 6B column. The α-amylase-binding fractions were eluted by methyl-D-mannopyranoside and separated by molecular weight using SDS-PAGE. Seven main bands stained by SYPRO Ruby protein staining were cut out, treated with pepsin, and analyzed by LC–MS/MS for identification of the proteins in the bands. The major α-amylase-binding glycoproteins identified are shown in **Table 1**. The binding glycoproteins were grouped by functions and localizations. Group 1 consisted of membrane glycoproteins involved in α-glucosidase and sugar absorption, which function in glucose assimilation after starch digestion by α-amylase in the small intestine. Groups 2 and 3 consisted of membrane glycoproteins involved in transcytosis and proteolysis, respectively.

All seven bands were stained by lectins with ConA, GNA, and L-PHA, indicating the presence of *N*-glycans. ConA (concanavalin A), GNA (snowdrop lectin), and L-PHA (*Phaseolus vulgaris* lectin type L) bind specifically to *N*-glycans, highmannose structures, and complex-type *N*-glycans, respectively. The identification of these α-amylase-binding proteins indicates that the *N*-glycan-binding activity


*SGLT1, sodium glucose co-transporter 1; SI, sucrose-isomaltase; VLA-2, integrin very late antigen-2; DPP-IV, dipeptidylpeptidase IV; ACE 2, angiotensin-converting enzyme 2.*

**203**

**Figure 2.**

*Regulatory Functions of α-Amylase in the Small Intestine Other than Starch Digestion…*

α-glucosidase/sugar absorption, transcytosis, and proteolysis.

**3.1 Effects of pancreatic α-amylase on α-glucosidase activity**

of pancreatic α-amylase in the duodenum may play a regulatory role involved in

**3. Interaction between pancreatic α-amylase and binding glycoproteins** 

First, effects of pancreatic α-amylase on the activities of glycoproteins in Group 1 were investigated. Group 1 consists of SI and SGLT1. SI, a membrane glycoprotein, digests sucrose, isomaltose, and maltose on the membrane as α-glucosidases. SGLT1 is also a membrane glycoprotein and plays an essential role in glucose

SI plays an important role in the end digestion of starch because SI breaks down the maltose produced by α-amylase from starch into glucose as an α-glucosidase on the small intestinal membrane. SI is essential for the end digestion of starch, and its

*Effects of pancreatic α-amylase and ConA on maltose- and sucrose-degrading activity of pig duodenum BBM vesicles. A BBM vesicle suspension (50 μl) was pre-incubated with various concentrations of pancreatic α-amylase or ConA (0–50 μM) for 15 min at 37°C, and incubated with 0.056 M maltose (left) or 0.056 M sucrose (right) as substrate (50 μl) for 30 min (left) or 60 min (right) at 37°C. The maltose or sucrose hydrolysis was measured in the presence of 5 mM CaCl2 and 0.15 M NaCl (upper) or in the absence of CaCl2 and NaCl (lower). Phlorizin (0.5 mM), a glucose transporter inhibitor, was included in the substrate solution. The degrading activity by the production of glucose was measured using a glucose C-II test Wako (Wako, FUJIFILM). ●: α-amylase, ○: ConA. Results are given as means±SE; n = 6. \**p *< 0.05; \*\**p *< 0.01 compared with* 

*the absence of α-amylase or ConA by Student's* t*-test [14].*

*DOI: http://dx.doi.org/10.5772/intechopen.92660*

**in small intestinal BBM**

absorption in the small intestine.

#### **Table 1.**

*Identification of α-amylase-binding proteins in intestinal BBM.*

*Regulatory Functions of α-Amylase in the Small Intestine Other than Starch Digestion… DOI: http://dx.doi.org/10.5772/intechopen.92660*

of pancreatic α-amylase in the duodenum may play a regulatory role involved in α-glucosidase/sugar absorption, transcytosis, and proteolysis.
