PROTEINASE, INHIBITOR & CRYSTALLIN FRAGMENTS IN CATARACT

本补助金提案的总体目标是确定透镜的作用 蛋白酶、它们的同源抑制剂和蛋白水解产物 晶体蛋白在高钼形成中的作用 重量 (HMW)蛋白质在 老年性白内障的发展 在这项研究中，人类晶状体的不同 将使用年龄和白内障晶状体。 以下四个具体 目标将是： (1)透镜中蛋白水解的调节： 将确定酶和蛋白质底物水平的透镜， 即通过寻找两种酶的酶原和/或蛋白酶-抑制剂复合物， 透镜蛋白酶，即25 kDa和膜蛋白酶，并检查 氧化作用对内源性蛋白酶抑制剂的影响 天然与氧化修饰的晶状体蛋白的优先蛋白水解 内源性蛋白酶 (2)晶体蛋白降解多肽的来源： “母体晶体蛋白”，在体内发现这三种晶体蛋白降解 人晶状体的多肽，即5.5kDa、9 kDa和15 kDa，源自 将通过比较它们的氨基酸序列来确定。 透镜 晶体蛋白将在体外用两种透镜蛋白酶进行蛋白水解（25 kDa和膜蛋白酶）和蛋白水解产物， 在体内发现的类似多肽的Mr和部分N-末端 氨基酸序列。 此外，降解多肽的来源 通过非酶促反应（芬顿反应）从晶状体蛋白中 考察 (3)降解多肽交联形成HMW蛋白： 体外降解形成HMW蛋白的交联机制 多肽本身或这些多肽与个体之间的差异。 将测定晶体蛋白。 (4)降解的交联产物的搜索和定量 正常老化的HMW和水不溶性蛋白质中的多肽， 白内障晶状体 针对特定交联的 降解的多肽将用于鉴定和定量 通过Western印迹分析测定这些多肽的聚集体水平， 放射免疫法测定。 上述研究将显示：（i）透镜蛋白水解过程是如何 体内调节;（ii）晶体蛋白在体内的降解是否受 酶促或非酶促的，和（iii）如果降解的多肽 本身交联并与单个晶体蛋白交联以形成HMW蛋白 存在于老化和白内障晶状体中的水平增加。

The overall goals of this grant proposal is to determine role(s) of lens proteinases, their cognate inhibitors and proteolytic products of crystallins in the formation of high mo. wt. (HMW) proteins during the development of senile cataract. In this study, human lenses of different ages and cataractous lenses will be utilized. The following four specific aims will be pursued: (1) Regulation of proteolysis in lens: The regulation of proteolysis in the lens at the enzyme and protein substrate levels will be determined, i.e. by searching for zymogens and/or proteinase-inhibitor complexes of two lens proteinases, i.e.a 25 kDa and a membrane proteinase, and examining the effect of oxidation on endogenous proteinase inhibitors and the preferential proteolysis of native vs. oxidatively modified crystallins by endogenous proteinases. (2) Origin of degraded polypeptides from crystallins: The identity of "parent crystallins" from which the three in vivo found degraded polypeptides of human lenses, i.e. 5.5 kDa, 9 kDa and 15 kDa, originated will be established by comparing their amino acid sequences. Lens crystallins will be proteolyzed in vitro with the two lens proteinases (25 kDa and a membrane proteinase) and proteolysis products compared with similar polypeptides found in vivo for their Mr's and partial N-terminal amino acid sequences. In addition, the origin of the degraded polypeptides from crystallins by a non-enzymatic reaction (Fenton reaction) will also be examined. (3) Cross-linking of degraded polypeptides to form HMW proteins: The mechanism of cross-linking to form HMW proteins in vitro by degraded polypeptides per se or between these polypeptides and individual crystallins will be determined. (4) Search for and quantitation of cross-linked products of degraded polypeptides in HMW and water insoluble proteins of normal aging and cataractous lenses. Antibodies raised against the specific cross-linked degraded polypeptides will be utilized to identify and quantitate the levels of aggregates of these polypeptides by Western blot analysis and a radioimmunoassay method respectively. The above studies will show: (i) how the lens proteolytic process is regulated in vivo; (ii) whether the degradation of crystallins in vivo is enzymatic or non-enzymatic, and (iii) if the degraded polypeptides cross-link per se and with individual crystallins to form the HMW proteins that exist in increasing levels in aging and cataractous lenses.