Development of a direct assay for alpha-amylase.

Winn-Deen, Emily S.; David, H; Sigler, Gerald F.; Chavez, Rodrigo

doi:10.1093/clinchem/34.10.2005

Cited by 105 publications

(46 citation statements)

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“…The inhibitory potencies of these peptides for porcine pancreatic α-amylase were determined with CNPG 3 as substrate [45]. All peptides were studied at a single concentration of 840 µM with preincubation with the enzyme for 5 min or 1 h. The results were similar under both conditions.…”

Section: α-Amylase Assaymentioning

confidence: 98%

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

Larregola

Lequin

Karoyan

et al. 2011

Journal of Peptide Science

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The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β-turn stabilization of different analogs designed as mimics of the β-turn structure found in tendamistat. The β-turn conformation of linear β-amino acid-containing peptides and triazole-cyclized analogs were compared to 'conventional' lactam- and disulfide-bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β-turns in solution, and for those not structured in solution in the presence of α-amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac-Ser-Trp-Arg-Tyr-NH(2) and both the amide bond-cyclized, c[Pro-Ser-Trp-Arg-Tyr-D-Ala] and the disulfide-bridged, Ac-c[Cys-Ser-Trp-Arg-Tyr-Cys]-NH(2) hexapeptides adopt dominantly in solution a β-turn conformation closely related to the one observed in tendamistat. On the contrary, the β-amino acid-containing peptides such as Ac-(R)-β(3) -hSer-(S)-Trp-(S)-β(3) -hArg-(S)-β(3) -hTyr-NH(2) , and the triazole cyclic peptide, c[Lys-Ser-Trp-Arg-Tyr-βtA]-NH(2) , both specifically designed to mimic this β-turn, do not adopt stable structures in solution and do not show any characteristics of β-turn conformation. However, these unstructured peptides specifically interact in the active site of α-amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α-amylase inhibitor. Thus, in contrast to amide-cyclized or disulfide-bridged hexapeptides, β-amino acid-containing peptides and click-cyclized peptides may not be regarded as β-turn stabilizers, but can be considered as potential β-turn inducers.

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Section: α-Amylase Assaymentioning

confidence: 98%

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

Larregola

Lequin

Karoyan

et al. 2011

Journal of Peptide Science

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“…Such assays use an exohydrolase such as α-glucosidase as an auxiliary enzyme to release chromophores from mono-or disaccharides produced by amylase. Recently, 2-chloro-p-nitrophenyl-maltotrioside (G3ClpNP) was introduced as a "direct" amylase substrate that releases a chromophore without auxillary enzymes (12). Measurement of amylase activity in serum assists in the diagnosis and treatment of pancreatitis (11)(12)(13) but is not specific for pancreatitis.…”

Section: Introductionmentioning

confidence: 99%

“…Recently, 2-chloro-p-nitrophenyl-maltotrioside (G3ClpNP) was introduced as a "direct" amylase substrate that releases a chromophore without auxillary enzymes (12). Measurement of amylase activity in serum assists in the diagnosis and treatment of pancreatitis (11)(12)(13) but is not specific for pancreatitis. Measuring the pancreatic isoenzyme improves specificity in the diagnosis of pancreatic disorders, and many methods have been applied to selectively measure the pancreatic isoenzyme (11,14,15).…”

Section: Introductionmentioning

confidence: 99%

Effect of substrate size on immunoinhibition of amylase activity

Warshawsky

Hortin

2001

Clinical Laboratory Analysis

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Immunoinhibition assays are hypothesized to work by antibodies blocking substrate access to enzyme active sites. To test this hypothesis, the inhibition of amylase isoenzymes by monoclonal and polyclonal antisera was assessed using substrates of varying sizes: chromogenic sustrates 3, 5, or 7 glucose units in length, novel synthetic macromolecular substrates, and starch. The synthetic macromolecular substrates consisted of small oligosaccharide substrates linked to an inert polymer that conferred a large size to substrate molecules as determined by gel filtration chromatography. When substrate size increased, amylase activity could be inhibited equivalently by antibody concentrations that are 10-fold lower. Progressively less polyclonal serum was required to inhibit amylase activity as substrate length increased from 3 to 5 to 7 glucose units and as size was increased by linkage to a polymer. Different effects of substrate size were observed with two monoclonal antibodies. One monoclonal antibody blocked amylase activity independent of substrate size, while another monoclonal antibody had little inhibitory effect except using starch as substrate. We conclude that use of larger substrates can expand the repertoire of inhibitory epitopes on enzymes and convert a noninhibitory antibody into an inhibitory one.

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“…Inhibition of α-amylase activity was determined according to an established method (Foo & Bais, 1998;Okutan, Kongstad, Jager, & Staerk, 2014;Winn-Deen, David, Sigler, & Chavez, 1988) with slight modifications. In a 96-well plate, 50 µL aliquots of the extract or acarbose (Wako, Osaka, Japan) at different concentrations (0.001-10 mg/mL) were incubated with 50 µL of 0.6 U/mL α-amylase derived from porcine pancreas (Sigma-Aldrich, St. Louis, MO, USA) in 100 mM phosphate buffer (pH 6.8, 60 mM NaCl) at 37°C for 10 min.…”

Section: α-Amylase Inhibition Assaymentioning

confidence: 99%

Isolation and Characterization of Antihyperglycemic Compounds from Vigna angularis Extracts

Kuriya

Nishio

Ono

et al. 2019

Journal of Food Science

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Functional foods that inhibit α‐amylase and α‐glucosidase activity are effective for regulating the blood glucose level and preventing hyperglycemia. Extracts of adzuki beans (ABs, Vigna angularis), widely eaten in East Asia, can inhibit α‐amylase and α‐glucosidase activity. In this study, we identified and evaluated the components in an AB water extract (ABWE) after boiling, which is an essential process for cooking ABs. The ABWE before boiling inhibited α‐amylase and α‐glucosidase activity and the boiled ABWE showed slightly stronger inhibitory effects. High‐performance liquid chromatography, liquid chromatography‐mass spectrometry, and nuclear magnetic resonance analyses identified (+)‐catechin 7‐O‐β‐d‐glucopyranoside (C7G), (+)‐epicatechin 7‐O‐β‐d‐glucopyranoside (E7G), and (+)‐catechin as the bioactive components in boiled ABWE. Interestingly, the quantity of E7G significantly increased after boiling (from 0% to 17.1 ± 1.3%). E7G showed stronger inhibition of α‐amylase and α‐glucosidase than C7G; the IC50 values for α‐amylase were 0.74 ± 0.04 mg/mL (C7G) and 0.40 ± 0.09 mg/mL (E7G), and for α‐glucosidase the IC50 values were 0.085 ± 0.032 mg/mL (C7G) and 0.051 ± 0.007 mg/mL (E7G). Our findings suggest that C7G and E7G are the main active components in ABWE as they inhibit α‐amylase and α‐glucosidase and their inhibitory effect is not lost after boiling. These results support the effectiveness of boiled ABs in the promotion of health. Practical Application We identified (+)‐catechin 7‐O‐β‐d‐glucopyranoside (C7G), (+)‐epicatechin 7‐O‐β‐d‐glucopyranoside (E7G), and (+)‐catechin in adzuki bean extracts and commercially available boiled adzuki bean products. Interestingly, the E7G content was increased by boiling, and this compound showed strong inhibitory activity toward α‐amylase and α‐glucosidase. These results support the consumption of boiled adzuki beans to prevent acute rises in blood glucose level.

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Development of a direct assay for alpha-amylase.

Cited by 105 publications

References 0 publications

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

Effect of substrate size on immunoinhibition of amylase activity

Isolation and Characterization of Antihyperglycemic Compounds from Vigna angularis Extracts

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