Isolation And Characterization Of Novel Bioactive Polypeptide Aglycin And Its Effect On Glucose Metabolism

In the present study, we have isolated and characterized a novel bioactive polypeptide from pea seeds, named as aglycin due to its N-terminal alanine, and C-terminal glycine. Aglycin is chemically characterized as a single chain consisted of 37 amino acid residues with 6 cysteines at 3, 7, 15, 20, 22 and 32 of chain positions. The molecular mass of aglycin determined by MALDI-TOF-MS is 3742.3Da, which is consensus with the calculated mass (3743.424 Da) from the sequence by software Antheprot 5.0. With surface plasmon resonance (SPR) biosensor analysis, it has been clearly found that there is a binding protein existing in mammalian pancreatic cell membrane, and this binding protein was purified from porcine pancreas by affinity chromatography on a CNBr-activated Sepharose 4B column. The interaction between the purified porcine pancreas proteins and aglycin was further verified by ELISA in vitro. After separated by SDS-PAGE and stained with Coomassie Blue G-250, the protein band in the stained gel was excised and digested in gel by trypsin and the peptide mass fingerprint was analyzed by MALDI-TOF-MS. The resultant peptide mass fingerprint was compared with the data from database (http://www.matrixscience.com) to identify the protein using Mascot search, which clearly showed that this protein is the voltage-dependent anion channel 1 (VDAC 1) and its mass is 30737Da calculated from its amino acid sequence.We have found that aglycin and its isoforms can intensely resist hydrolysis by trypsin, pepsin and Glu-C proteases in vitro, it is a high stable peptide because it belongs to the cystine-knot motif peptide family in tertiary structure. We have found that aglycin can regulate glucose metabolism, after subcutaneous injection at a dose of 2.5ug/g, no obvious change of blood glucose concentration was observed from normal mice, the diabetes type I and II mice. When injected at a dose of 5ug/g or more than 5ug/g, aglycin is capable of elevating blood glucose concentration of normal mice and the diabetes type II mice, but the effect of enhancing blood glucose on type I diabetes mice was not obvious compared with normal and type II diabetes mice, which might be due to the damage of pancreas by STZ. All the results show that the capability of regulatory function of aglycin on glucose metabolism and the mechanism of regulation probably is via VDAC 1.In conlusion, four main results were drawn from this study, a novel bioactive polypeptide named as aglycin was isolated and characterized, and the method for largly preparation aglycin was confirmed; an interaction protein with aglycin was purified from mammalian pancreatic cell membrane, and characterized as the voltage-dependent anion channel 1 (VDAC 1);And found that aglycin is stale to heat and proteases; aglycin can regulate glucose metabolism. The innovation of this dissertation is that found the interaction protein with aglycin in mammalian pancreatic cell membrane and discovered that aglycin can regulate glucose metabolism.