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Arginine is essential for the alpha-amylase inhibitory activity of the alpha-amylase/subtilisin inhibitor (BASI) from barley seeds.

ABSTRACT: Treatment of barley alpha-amylase/subtilisin inhibitor (BASI) with reagents specific for arginine, histidine, methionine and tyrosine residues and amino and carboxyl groups indicates that an arginine residue(s) is essential for its action on the target enzyme barley alpha-amylase 2. Phenylglyoxal modified eight out of 12 arginine residues in BASI. Kinetic analysis shows that the inactivation of BASI follows a pseudo-first-order reaction and is due to reaction with one molecule of phenylglyoxal; the second-order rate constant is determined to be 2.95 M-1.min-1. At pH 8.0, BASI and barley alpha-amylase 2 form an inactive 1:1 complex. The Ki value of this association is 2.2 x 10(-10) M. The alpha-amylase protects four arginine residues and also the alpha-amylase inhibitory activity of BASI against phenylglyoxal. When BASI from the phenylglyoxal-modified target enzyme-inhibitor complex is isolated and subjected to a second treatment with phenylglyoxal, four additional arginine residues are modified, with concomitant loss of the inhibitory activity. These results are discussed in relation to a three-dimensional model of BASI based on the known structure of the corresponding inhibitor from wheat.

SUBMITTER: Abe J

PROVIDER: S-EPMC1134333 | biostudies-other | 1993 Jul

REPOSITORIES: biostudies-other

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Project description:BackgroundIndian medicinal plants used in the Ayurvedic traditional system to treat diabetes are a valuable source of novel anti-diabetic agents. Pancreatic ?-amylase inhibitors offer an effective strategy to lower the levels of post-prandial hyperglycemia via control of starch breakdown. In this study, seventeen Indian medicinal plants with known hypoglycemic properties were subjected to sequential solvent extraction and tested for ?-amylase inhibition, in order to assess and evaluate their inhibitory potential on PPA (porcine pancreatic ?-amylase). Preliminary phytochemical analysis of the lead extracts was performed in order to determine the probable constituents.MethodsAnalysis of the 126 extracts, obtained from 17 plants (Aloe vera (L.) Burm.f., Adansonia digitata L., Allium sativum L., Casia fistula L., Catharanthus roseus (L.) G. Don., Cinnamomum verum Persl., Coccinia grandis (L.) Voigt., Linum usitatisumum L., Mangifera indica L., Morus alba L., Nerium oleander L., Ocimum tenuiflorum L., Piper nigrum L., Terminalia chebula Retz., Tinospora cordifolia (Willd.) Miers., Trigonella foenum-graceum L., Zingiber officinale Rosc.) for PPA inhibition was initially performed qualitatively by starch-iodine colour assay. The lead extracts were further quantified with respect to PPA inhibition using the chromogenic DNSA (3, 5-dinitrosalicylic acid) method. Phytochemical constituents of the extracts exhibiting? 50% inhibition were analysed qualitatively as well as by GC-MS (Gas chromatography-Mass spectrometry).ResultsOf the 126 extracts obtained from 17 plants, 17 extracts exhibited PPA inhibitory potential to varying degrees (10%-60.5%) while 4 extracts showed low inhibition (< 10%). However, strong porcine pancreatic amylase inhibitory activity (> 50%) was obtained with 3 isopropanol extracts. All these 3 extracts exhibited concentration dependent inhibition with IC50 values, viz., seeds of Linum usitatisumum (540 ?gml-1), leaves of Morus alba (1440 ?gml-1) and Ocimum tenuiflorum (8.9 ?gml-1). Acarbose as the standard inhibitor exhibited an IC50 (half maximal inhibitory concentration)value of 10.2 ?gml-1. Phytochemical analysis revealed the presence of alkaloids, tannins, cardiac glycosides, flavonoids, saponins and steroids with the major phytoconstituents being identified by GC-MS.ConclusionsThis study endorses the use of these plants for further studies to determine their potential for type 2 diabetes management. Results suggests that extracts of Linum usitatisumum, Morus alba and Ocimum tenuiflorum act effectively as PPA inhibitors leading to a reduction in starch hydrolysis and hence eventually to lowered glucose levels.

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Arginine is essential for the alpha-amylase inhibitory activity of the alpha-amylase/subtilisin inhibitor (BASI) from barley seeds.

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