THIOL PROTEASE INHIBITORY ACTIVITY OF SALIVARY CYSTATINS

存在多种细菌和哺乳动物衍生的蛋白酶 在人的嘴里。 这些蛋白水解酶影响 几种唾液和薄膜成分的结构和活性。 这些蛋白水解酶中的许多是巯基蛋白酶。 唾液 分泌物含有含半胱氨酸的磷蛋白家族， 其中一些至少是“半胱氨酸蛋白酶抑制剂”家族的成员， 巯基蛋白酶抑制剂。 这些蛋白质已经被证明 是薄膜蛋白酶;因此有助于维持 唾液和表膜保护作用的完整性。 这项工作的直接具体目标是：（1）确定 唾液中主要胱抑素的磷酸化状态 影响其作为巯基蛋白酶抑制剂的功能 或与金属离子和羟基磷灰石相互作用，以及（2） 确定巯基蛋白酶抑制活性是否 与羟基磷灰石结合的胱抑素相比， 在溶液中显示。 总体实验设计是将3个主要班级隔离 用凝胶过滤和离子交换法测定唾液胱抑素 色谱法，并从每一个纯化主要蛋白质组分 类，使用反相HPLC。 对于每个主要组件 每个分子中共价结合的磷酸基团的数量 将被确定。 磷酸盐含量将与 巯基蛋白酶抑制活性，在溶液中测定 使用荧光巯基蛋白酶测定，以及 结合金属离子的组分，使用荧光测定 滴定和竞争结合研究。 每个人的约束力 羟基磷灰石的成分也将与 磷酸化状态 巯基蛋白酶抑制活性 将测定羟基磷灰石结合的半胱氨酸蛋白酶抑制剂。 这些目标的实现将表明唾液是否 半胱氨酸蛋白酶抑制剂可以在体内发挥巯基蛋白酶抑制作用， 将提供必要的材料和信息， 项目的长期目标。 这些目标是 探索磷酸化在合成中的作用， 功能和各种唾液腺的体内定位 胱抑素。

A variety of bacterial and mammalian derived proteases are present in the human mouth. These proteolytic enzymes influence the structure and activity of several salivary and pellicle components. Many of these proteolytic enzymes are thiol proteases. Salivary secretions contain a family of cysteine containing phosphoproteins, some of which at least are members of the "cystatin" family of thiol protease inhibitors. These proteins, which ave been shown to be pellicle proteases; and hence serve to help maintain the integrity of the protective roles of saliva and pellicle. The immediate specific aims of this work are: (1) to determine whether the phosphorylation state of the major salivary cystatins influences their ability to function as thiol protease inhibitors or to interact with metal ions and hydroxyapatite, and (2) to establish whether the thiol protease inhibitory activity of hydroxyapatite-bound cystatins is altered compared to that exhibited in solution. The overall experimental design is to isolate the 3 major classes of salivary cystatins using gel filtration and ion exchange chromatography, and to purify the major protein component from each class using reversed phase HPLC. For each major component isolated, the number covalently bound phosphate groups per molecule will be determined. The phosphate content will be correlated with the thiol protease inhibitory activity, determined in solution using a fluorescent thiol protease assay, and the ability of the component to bind metal ions, determined using fluorescence titrations and competition binding studies. The binding of each component to hydroxyapatite will also be correlated to the phosphorylation state. The thiol protease inhibitory activity of the hydroxyapatite-bound cystatins will be determined. The achievement of these aims will indicate whether salivary cystatins may play a thiol protease inhibitory role in vivo and will provide the material and information necessary for the anticipated long term goals of the project. These goals are to explore the role phosphorylation may play in the synthesis, function, and in vivo localization of the various salivary cystatins.