Quantitation of Cathepsins B, S, and L in Cells and Organelles

细胞和细胞器中组织蛋白酶 B、S 和 L 的定量

• 批准号: 9304109
• 负责人: Robert Mason
• 金额: $ 8.7万
• 依托单位: Virginia Polytechnic Institute and State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-06-15 至 1995-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9304109&HistoricalAwards=false
• 关键词: Quantitation; Cathepsins; Cells; Organelles

The lysosomal cysteine proteinases, cathepsins B, S, and L, have been purified from a number of mammalian species and are known to exist in multiple forms. Quantitation of these enzymes is difficult due to their instability and inhibition by naturally occurring inhibitors. The objectives of this proposal are to quantitate the relative concentrations of the different molecular forms of cathepsins in cells and to determine which ones are involved in the degradation of endocytosed proteins such a albumin and .2-macroglobulin. Experimental procedures will employ a specific radio-labeled inhibitor in either a free membrane-permeant form or conjugated to proteins in cell culture systems. A pure form of Z-ı125iodine!Tyr-Ala-CHN2 will be used in conjunction with specific antibodies and SDS/polyacrylamide gel electrophoresis/autoradiography to quantitate different molecular forms of each enzyme in living cells. These procedures circumvent the problems of inactivation or inhibition during isolation and have the added advantage of being able to quantitate different molecular forms of each of the enzymes. The relative concentrations of each enzyme will indicate the relative importance of these enzymes in protein turnover in a given cell. ı125iodine!Tyr-Ala-CHN2 conjugated to a range of proteins will be used as a unique tool for the identification of the enzymes that first meet endocytosed proteins. the types of enzymes that first react with such reagents in intact cells will indicate which are involved in the degradation of endocytosed proteins. It is known that lysosomal proteinases are synthesized as precursors in the endoplasmic reticulum and processed to intermediate forms as they enter the lysosome and finally are processed to lower M, mature forms in the mature lysosome. the molecular forms of the enzymes that react with ı125iodine!Tyr-Ala-CHN2 conjugated to proteins will permit the differentiation between fusion of endosomes with early lysosomes and fusion of endosomes with late (mature) lysosomes. Such a direct method of identifying the enzymes in endosomes is required to unequivocally determine which enzymes are involved in endocytic proteolysis, and by which route the enzymes are packaged into the endocytic compartment. %%% This project is aimed at determining the quantities of a number of important proteinases in cells using a novel technique with a membrane permeant radio-labeled inhibitor. Results from this study will identify which of the four lysosomal cysteine proteases predominate in which cells and enable us to determine the significance of these enzymes in cell function. A second aim is to determine the molecular forms of the enzymes that are involved in the degradation of endocytosed proteins. Results from this part of the study will show which enzymes are involved in the degradation of proteins taken up into the cell, and will also indicate the site at which endocytosed proteins first meet with lysosomal enzymes during their biosynthesis. The procedures to be developed provide a series of new techniques to study the biological roles of lysosomal proteases.

溶酶体半胱氨酸蛋白酶，组织蛋白酶B、S和L， 从许多哺乳动物物种中纯化，并且已知 以多种形式存在。 这些酶的定量是 由于它们的不稳定性和天然的抑制作用， 发生抑制剂。 本提案的目标是 定量不同分子的相对浓度 细胞中的组织蛋白酶的形式，并确定哪些是 参与内吞蛋白质如白蛋白的降解 和0.2-巨球蛋白。 实验程序将采用 特异性放射性标记的抑制剂， 在细胞培养系统中形成蛋白质或与蛋白质结合。 纯 Z-<$125碘的形式！Tyr-Ala-CHN 2将与 特异性抗体和SDS/聚丙烯酰胺凝胶 电泳/放射自显影以定量不同的分子 活细胞中每种酶的形式。 这些程序规避了 在分离过程中的失活或抑制问题， 具有能够定量不同的 每种酶的分子形式。 的相对 每种酶的浓度将表明相对重要性 这些酶在特定细胞中的蛋白质周转中的作用。 碘125！与一系列蛋白质缀合的Tyr-Ala-CHN 2将是 用作鉴定酶的独特工具， 首先遇到内吞蛋白质。 这些类型的酶首先 在完整细胞中与这些试剂反应将指示哪些是 参与内吞蛋白质的降解。 已知 溶酶体蛋白酶作为前体在细胞内合成， 内质网和加工成中间形式，因为它们 进入溶酶体，最后被加工成低M， 在成熟的溶酶体中形成。 酶的分子形式 与碘125反应！与蛋白质缀合的Tyr-Ala-CHN 2将 允许区分核内体与早期核内体融合 溶酶体和内体与晚期（成熟）溶酶体的融合。 这种鉴定内体中酶的直接方法是 需要明确确定哪些酶参与 内吞蛋白水解，以及通过何种途径包装酶 进入内吞区。 %%% 该项目旨在确定一些 细胞中重要的蛋白酶， 膜渗透性放射性标记抑制剂。 本研究结果 将鉴定出四种溶酶体半胱氨酸蛋白酶中 在哪些细胞中占主导地位，使我们能够确定 这些酶在细胞功能中的重要性。 第二个目标是 确定参与的酶的分子形式 内吞蛋白质的降解。 本部分的结果 这项研究将显示哪些酶参与了降解， 蛋白质进入细胞，也将表明网站 内吞蛋白质与溶酶体酶首次相遇的位置 在其生物合成过程中。 拟制定的程序规定， 一系列新技术来研究 溶酶体蛋白酶