| Summary: | 博士 === 國立臺灣大學 === 生化科學研究所 === 87 === Part I.
We present a systematic structure comparison of three major classes of postsynaptic snake toxins, which include short and long chain -type neurotoxins plus one angusticeps-type toxin of black mamba snake family. Two novel -type neurotoxins isolated from Taiwan cobra (Naja naja atra) possessing distinct primary sequences and different postsynaptic neurotoxicities were taken as exemplars for short and long chain neurotoxins and compared with the major lethal short-chain neurotoxin in the same venom, i.e., cobrotoxin, based on the derived three-dimensional structure of this toxin in solution by NMR spectroscopy. A structure comparison among these two -neurotoxins and angusticeps-type toxin (denoted as FS2) was carried out by the secondary-structure prediction together with computer homology-modeling based on multiple sequence alignment of their primary sequences and established NMR structures of cobrotoxin and FS2. It is of interest to find that upon pairwise superpositions of these modeled three-dimensional polypeptide chains, distinct differences in the overall peptide flexibility and interior microenvironment between these toxins can be detected along the three constituting polypeptide loops, which may reflect some intrinsic differences in the surface hydro-phobicity of several hydrophobic peptide segments present on the surface loops of these toxin molecules as revealed by hydropathy profiles. Construction of a phylogenetic tree for these structurally related and functionally distinct toxins corroborates that all long and short toxins present in diverse snake families are evolutionarily related to each other, supposedly derived from an ancestral polypeptide by gene duplication and subsequent mutational substitutions leading to divergence of multiple three-loop toxin peptides.
Part II.
The juice of the green wheat has been used as alternative folk medicine in place of insulin to treat diabetic patients in Taiwan. We suspect that the plant may contain insulin-like substances. Current studies are aimed to investigate which compounds in this natural plant are involved in decreasing blood glucose level. After extraction of the green wheat, TA1 and TA2 were demonstrated to cross-react with anti-insulin antiserum using native PAGE and Western blot analysis. Following ammonium sulfate precipitation, DEAE ion exchange chromatography and preparative gel electrophoresis were used for further purification. We obtained the hypoglycemic principles which consist of three proteins, TA1, TA2a and TA2b. The molecular size of TA1, TA2-a, TA2-b was determined to be 19, 21 and 22.8 kDa, respectively. We found that TA1 & 2 possess similar activity to insulin, which at physiological concentration can suppress hepatitis B surface antigen gene expression in cultured human hepatoma Hep3B/T2 cells, concomitant with the stimulation of cell proliferation. Additionally, intraperitoneal administration of insulin (2 IU/kg), TA1 & 2 (6, 10 mg/kg) exhibited significant hypoglycemic effect when compared to control group (p < 0.05) on alloxan-induced diabetic mice. Using TA1 digested in gel with endoproteinase Lys-C for sequence analysis, the results show that the primary sequence of TA1 is identical to a hypothetical protein in the Gene Data Bank whose function is still unknown to date.
|
|---|
