ROBUST PROTEIN SEALANTS FROM MUSSEL BYSSUS

足丝是海洋贻贝的一种外部肌腱状结构， 至少有两个科学上有趣的特性：A。它调解强者 以及这些动物在宽表面上表现出的机会性粘连 在海洋中（金属、玻璃、矿物、聚甲基丙烯酸甲酯等），和B. 它具有物理和化学坚固性，超过了 其单个成分（如胶原蛋白和贻贝）的稳健性 粘附蛋白（MAP）。 这种健壮性显然是由 一种胶体密封剂的醌鞣制， 足丝在释放到海洋中之前被包覆（厚度10微摩尔 水 由于这种密封剂对微生物攻击的抵抗力， 对胶原组织的亲和力，以及对酸的广泛稳定性， 碱、有机物和热，更好地了解它的形成， 组成和稳定化可以大大有助于 医疗/牙科密封剂的灵感设计。 这是特别 适用于保护含胶原的组织， 牙本质、骨、关节软骨和肌腱，其经历各种 退化过程 该提案的实验目标是 从副腺中分离含有密封剂前体的颗粒 贻贝（Mytilus edulis）足。 这将通过以下方式完成： 诱导脱颗粒或组织均质化，然后进行分化， 离心法 将分离的颗粒溶解，并将内容物 通过酶（儿茶酚氧化酶、磷酸酶）、3，4- 二羟基苯丙氨酸（DOPA），通过聚丙烯酰胺凝胶电泳， 使用可获得的多克隆抗体的蛋白质印迹 （抗儿茶酚氧化酶和抗MAP）作为探针。 该组合物，N- 末端序列和每种颗粒的相对浓度 蛋白质将由传统的蛋白质化学测定。 单-或 制备每种新蛋白质的多克隆抗体， 这些蛋白质在附腺颗粒中的位置 如在实际的深渊密封剂中，将尝试通过免疫荧光或 胶体金技术 任何一种蛋白质保护 将使用含有推定的胶原的体外测定来评估胶原。 屏蔽剂，可溶性胶原蛋白（II型，小牛皮肤）和梭菌 胶原酶，pH 8.0（0.1 M硼酸盐）。 最后， 探讨了醌鞣法对胶态密封剂的稳定作用 通过加入强亲核试剂C-14甘氨酰甲酯， 预密封颗粒。 这样，常驻的儿茶酚氧化酶可以形成 MAP或一些类似蛋白质中的肽基邻醌， 交叉链接。 然后可以回收经修饰但仍可溶的MAP， 表征了

The byssus, an external tendon-like structure in marine mussels, has at least two scientifically intriguing properties: A. It mediates the strong and opportunistic adhesion that these animals exhibit on a wide surfaces in the sea (metal, glass, mineral, polymethylmethacrylate, etc), and B. It possesses a physical and chemical robustness that exceeds the robustness of its individual constituents such as collagen and mussel adhesive protein (MAP). This robustness is apparently imparted by the quinone tanning of a colloidal sealant with which every portion of the byssus is coated (thickness 10 micromoles) prior its release into the sea water. Because of this sealant's resistance to microbial attack, its affinity for collagenous tissues, and its broad stability to acids, bases, organics, and heat, a better understanding of its formation, composition and stabilization could contribute significantly to the inspired design of medical/dental sealants. This is particularly appropriate for the protection of collagen-containing tissues such as dentin, bone, articular cartilage, and tendon that undergo a variety of degenerative processes. The experimental goal of this proposal is to isolate granules containing sealant precursors from the accessory gland of the mussel (Mytilus edulis) foot. This will be done either by inducing degranulation or tissue homogenization followed by differential centrifugation. Isolated granules will be lysed and contents will be analyzed by assays for enzymes (catecholoxidase, phosphatase), 3,4- dihydroxyphenylalanine (DOPA), by polyacrylamide gel electrophoresis and western blotting using available polyclonal antibodies (anti-catecholoxidase and anti-MAP) as probes. The composition, N- terminal sequence, and relative concentration of each of the granule proteins will be determined by traditional protein chemistry. Mono- or polyclonal antibodies will be prepared to each of the novel proteins, and location of each of these proteins in the accessory gland granule as well as in the actual byssal sealant will be attempted by immunofluorescent or colloidal gold techniques. The ability of any of the proteins to protect collagen will be assessed using an in vitro assay containing the putative shielding agent, a soluble collagen (type II, calf skin) and clostridial collagenase at pH 8.0 (0.1 M borate). Finally, the mechanism of stabilization of the colloidal sealant by quinone-tanning will be probed by adding a strong nucleophile, C-14 glycylmethylester, to activated presealant granules. In this way, the resident catecholoxidase can form peptidyl o-quinones in MAP or some similar protein without leading to cross-links. Modified, but still soluble, MAP can then be recovered and characterized.