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

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

#### **Figure 2.**

*New Insights into Metabolic Syndrome*

manner, even at the tissue level.

**BBM that bind pancreatic α-amylase**

hematoxylin.

respectively.

**Identified proteins**

202 SGLT1, SI

46 Epoxide

50–57 SGLT1 Na+

hydrase

*dipeptidylpeptidase IV; ACE 2, angiotensin-converting enzyme 2.*

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

mannan, indicating that α-amylase binds to duodenal BBM in a mannose-specific

One-centimeter duodenum sections from fasted pigs were incubated with pancreatic α-amylase (10 μM) in PBS (pH 7.2) including phenylmethylsulfonyl fluoride (final concentratation 1 mM) at 4°C for 30 min, then fixed and paraffinembedded. The paraffin sections were immunostained with rabbit anti-α-amylase IgGs-HRP. The color was developed with DAB/H2O2 and then counterstained with

**2.2 Separation and identification of glycoproteins from the small intestinal** 

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,

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

144–156 VLA-2 Aminopeptidases, Enterokinase


Aminopeptidase N

**MW (kDa) Group 1 Group 2 Group 3**

110–122 SGLT1 DPP-IV, Integrin β1, ACE2 DPP-IV

receptor

/K+

CD Man-6-P receptor, Aminopeptidase N

*SGLT1, sodium glucose co-transporter 1; SI, sucrose-isomaltase; VLA-2, integrin very late antigen-2; DPP-IV,* 

78 SGLT1 Transferrin, Transferrin

99 Aminopeptidases

**202**

**Table 1.**

*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].*

deficiency and remarkable fluctuations in enzymatic activity are thought to have a significant effect on starch digestion and glucose absorption. In this study, a method was established for measuring SI activity using BBM that evaluates the effects of pancreatic α-amylase on SI activity as an α-glucosidase [14].

The effect of pancreatic α-amylase on SI activity was investigated with and without CaCl2 and NaCl. As a comparison with α-amylase, the effects of concanavalin A (ConA), a lectin that recognizes α-mannose and α-glucose, were also measured because α-amylase shows a high affinity to α-mannose. In maltose degradation activity by SI, the α-amylase showed enhanced activity only in the presence of 5 mM CaCl2 and 0.15 M NaCl, while no effect of α-amylase was shown in the absence of CaCl2 and NaCl (**Figure 2**, left). ConA had no effect on the maltose degradation activity by SI. On the other hand, α-amylase did not affect the sucrose degradation activity, and ConA inhibited it by about 20% in the presence and absence of 5 mM CaCl2 and 0.15 M NaCl, respectively (**Figure 2**, right).
