Dataset Information

Purification, developmental expression, and in silico characterization of ?-amylase inhibitor from Echinochloa frumentacea.

ABSTRACT: Barnyard (Echinochloa frumentacea) and finger (Eleusine coracana) millet growing at northwestern Himalaya were explored for the ?-amylase inhibitor (?-AI). The mature seeds of barnyard millet variety PRJ1 had maximum ?-AI activity which increases in different developmental stage. ?-AI was purified up to 22.25-fold from barnyard millet variety PRJ1. Semi-quantitative PCR of different developmental stages of barnyard millet seeds showed increased levels of the transcript from 7 to 28 days. Sequence analysis revealed that it contained 315 bp nucleotide which encodes 104 amino acid sequence with molecular weight 10.72 kDa. The predicted 3D structure of ?-AI was 86.73% similar to a bifunctional inhibitor of ragi. In silico analysis of 71 ?-AI protein sequences were carried out for biochemical features, homology search, multiple sequence alignment, phylogenetic tree construction, motif, and superfamily distribution of protein sequences. Analysis of multiple sequence alignment revealed the existence of conserved regions NPLP[S/G]CRWYVV[S/Q][Q/R]TCG[V/I] throughout sequences. Superfam analysis revealed that ?-AI protein sequences were distributed among seven different superfamilies.

SUBMITTER: Panwar P

PROVIDER: S-EPMC5924430 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Purification, developmental expression, and in silico characterization of α-amylase inhibitor from Echinochloa frumentacea.

Panwar Priyankar P Verma A K AK Dubey Ashutosh A

3 Biotech 20180427 5

Barnyard (Echinochloa frumentacea) and finger (Eleusine coracana) millet growing at northwestern Himalaya were explored for the α-amylase inhibitor (α-AI). The mature seeds of barnyard millet variety PRJ1 had maximum α-AI activity which increases in different developmental stage. α-AI was purified up to 22.25-fold from barnyard millet variety PRJ1. Semi-quantitative PCR of different developmental stages of barnyard millet seeds showed increased levels of the transcript from 7 to 28 ...[more]

PMID: 29719769

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Project description:alpha-Amylase was purified to apparent homogeneity from normal pancreas and a transplantable pancreatic acinar carcinoma of the rat by affinity chromatography on alpha-glucohydrolase inhibitor (alpha-GHI) bound to aminohexyl-Sepharose 4B. Recovery was 95-100% for both pancreas and tumour alpha-amylases. They were monomeric proteins, with Mr approx. 54000 on SDS/polyacrylamide-gel electrophoresis. Isoelectric focusing of both normal and tumour alpha-amylases resolved each into two major isoenzymes, with pI 8.3 and 8.7. Tumour-derived alpha-amylase contained two additional minor isoenzymes, with pI 7.6 and 6.95 respectively. All four tumour isoenzymes demonstrated amylolytic activity when isoelectric-focused gels were treated with starch and stained with iodine. Two-dimensional electrophoresis, on SDS/10-20%-polyacrylamide-gradient gels after isoelectric focusing, separated each major isoenzyme into doublets of similar Mr values. Pancreatic and tumour-derived alpha-amylases had similar Km and Ki (alpha-GHI) values, but the specific activity of the tumour alpha-amylase was approximately two-thirds that of the normal alpha-amylase. Although amino acid analysis and peptide mapping with the use of CNBr, N-chlorosuccinimide or Staphylococcus aureus V8 proteinase gave comparable profiles for the two alpha-amylases, tryptic-digest fingerprint patterns were different. Antibodies raised against the purified pancreatic alpha-amylase and tumour alpha-amylase respectively showed only one positive band on immunoblotting after gel electrophoresis of crude extracts of rat pancreas and carcinoma, at the same position as that of the purified enzyme. More than 95% of the alpha-amylase activity in the pancreas and in the tumour was absorbed by an excess amount of either antibody, indicating that normal and tumour alpha-amylases are immunologically identical. The presence of additional isoenzymes in the carcinoma, and dissimilarity of tryptic-digest patterns, may reflect an alteration in gene expression or in the post-translational modification of this protein in this heterogeneously differentiated transplantable pancreatic acinar carcinoma.

Dataset Information

Purification, developmental expression, and in silico characterization of ?-amylase inhibitor from Echinochloa frumentacea.

Publications

Purification, developmental expression, and in silico characterization of α-amylase inhibitor from <i>Echinochloa frumentacea</i>.

Similar Datasets

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