PROTEINASE, INHIBITOR & CRYSTALLIN FRAGMENTS IN CATARACT

这项拨款提案的总体目标是确定镜头(S)的角色 蛋白水解酶及其同源抑制剂和蛋白水解物 结晶素在高钼形成中的作用。WT.(HMW)蛋白质 老年性白内障的发展。在这项研究中，人类不同的晶状体 年龄和白内障镜片将被使用。以下四个具体 我们将致力于以下目标： (1)晶状体中蛋白分解的调节：晶状体中蛋白质分解的调节 将确定酶和蛋白质底物水平上的晶状体， 即通过搜索发酵源和/或两个 晶状体蛋白水解酶，即25 kDa和一种膜蛋白水解酶，并检测 氧化对内源性蛋白水解酶抑制物的影响 天然晶体蛋白与氧化修饰晶体蛋白的优先蛋白水解性 内源性蛋白酶。 (2)晶体蛋白降解多肽的来源：同源性 三种在体内被发现降解的“母体晶体蛋白” 人类晶状体多肽，即5.5 kDa、9 kDa和15 kDa，起源于 将通过比较它们的氨基酸序列来确定。镜头 晶体蛋白将在体外与两种晶状体蛋白水解酶(25 KDA和膜蛋白水解酶)和蛋白水解物与 体内发现的类似多肽的MR‘s和部分N-末端 氨基酸序列。此外，还对降解多肽的来源进行了探讨 从晶体蛋白通过非酶反应(芬顿反应)也将 检查过了。 (3)降解多肽的交联形成HMW蛋白： 体外降解交联形成HMW蛋白的机理 多肽本身或在这些多肽和个体之间 晶状体蛋白将被测定。 (4)降解产物的寻找和定量 正常老化的HMW和水不溶蛋白中的多肽 白内障镜片。针对特定的交联物产生的抗体 将利用降解的多肽来鉴定和定量 用Western印迹分析和A_(？) 分别采用放射免疫测定法。 上述研究将显示：(I)晶状体的蛋白质分解过程是怎样的 体内调节；(Ii)体内晶体蛋白的降解是否 酶或非酶，以及(Iii)如果降解的多肽 本身和与单个晶体蛋白交联会形成HMW蛋白 在老化和白内障晶状体中存在的水平越来越高。

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.