| 其他摘要 | Amphibian skin is a morphologically, biochemically and physiologically complex organ that Mfills a wide range of functions necessary for amphibian survival. A serious of mutually compatible functions takes place in the skin, including respiration, water regulation, anti-predator, antimicrobial defense, temperature control, reproduction, etc. Although numerous studies have focused on the bioactive components existed in amphibian skin "secretions", from which lots of peptides with diverse biological activities have been isolated and characterized, the physiological and functional complexity and the relative biochemical mechanisms of amphibian skin are incompletely understood. Following determination of trypsin inhibitory activity, a novel serine protease inhibitor (BmA-skin) was purified and characterized from frog Bombina maxima skin by a three steps protocol including DEAE-Sephadex A-50, Sephadex G-75 gel filtration and the DEAE-Sephadex A-50 ion exchange. BmA-skin is composed by a single peptide chain as demonstrated by SDS-PAGE and native-PAGE. Under reducing conditions, its apparent molecular weight is 67 kDa. However, under non-reducing conditions, there are at lest three bands (50 kDa, 55 kDa and 110 kDa) with identical N-terminal amino acids are determined by N-terminal protein sequencing. Thus, the protein may exist in isomeric and polymeric forms. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains and its sequence exhibits 39% and 38% sequence identities with those of human and bovine serum albumins, respectively. Frog serum albumin (BmA-serum) was subsequently purified and its coding cDNA was further obtained by PCR-based cloning from frog liver. BmA-serum coding cDNA obtained from the frog liver is generally the same as that of BmA-skin from the skin, except two nucleotide mutations in protein coding region that resulted in Gly471 to Asn and Ser569 to Asn replacements, and an 8 base insertion in 3* untranslated region in the cDNA of BmA-serum. BmA-skin is characterized of binding of a haem b (0.95 mol/mol protein). However, BmA-senim is distinct from BmA-skin by a much lower content of haem b (0.05 mol/mol protein). Different from bovine serum albumin, both of them potently inhibited trypsin. No inliibitory effect on thrombin, chymotrypsin, elastase and subtilisin was observed under the assay conditions. The equilibrium dissociation constant (KD) with rypsin determined are around 2 x 10"9M by BIAcore 3000. It formed a stable non-covalent complex with trypsin at 1:1 moler ratio through an exposed loop formed by a disulfide bond (Cys53~Cys62), which comprises the seissile bond Arg58(Pi>His59(Pi')r Immunohistochemical study revealed that BmA-skin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in frog skin, suggesting that it plays important roles in skin physiological functions, like water economy, metabolite exchange and osrnoregulation, skin respiration, etc. BmA-skin possessed cytotoxic activity on H9, C8166 human T lymphoblastoid cells, but not on K562 erythroleukemia cells. Pretreatment K562 cells with hemin to induce erythroid differentiation, it inhibited the viability of them too. After treatment cells with BmA-skin for 72 h5 fifty percentage cytotoxic concentrations (CC50) of BmA-skin on H95 C8166, and hemin-treated K562 cells were 0.96, 1.05, and 1.27 fiM, respectively. . BmA-skin induced apoptosis of the cell lines, which was demonstrated by nuclear morphological changes, DNA fragmentation, and DNA subdiploidy of apoptosis cells. BmA-serum did not affect the growth of the three cell lines. These results indicated that bound heam in BmA-skin contributed importantly to its 'cytotoxic and apoptosis-inducing activity on the cell Ikes assayed. BmA-skin and serum labeled with cy3 incubated with H9 and C8166 cells at different time points, and determined by laser scanning confocal microscopy. The results showed that more BmA-skin enter cells than BmA-senim, suggesting that entry cells of BmA-skin is a way to induce apoptosis |
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