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

溶酶体半胱氨酸蛋白酶，组织蛋白B，S和L，已经从许多哺乳动物中纯化出来，并以多种形式存在。由于这些酶的不稳定和自然产生的抑制物的抑制，这些酶的定量是困难的。这项建议的目的是量化不同分子形式的组织蛋白在细胞中的相对浓度，并确定哪些参与内吞蛋白的降解，如白蛋白和2-巨球蛋白。实验程序将在细胞培养系统中使用一种特定的放射性标记的抑制物，其形式要么是游离膜，要么是与蛋白质结合。一种纯形式的Z-ı125I！TYR-ALA-CHN 2将与特定的抗体和十二烷基硫酸钠/聚丙烯酰胺凝胶电泳法/放射自显影技术结合使用，以定量测定活细胞中每种酶的不同分子形式。这些程序避免了分离过程中的失活或抑制问题，并具有能够对每种酶的不同分子形式进行定量的额外优势。每种酶的相对浓度将表明这些酶在特定细胞中蛋白质周转中的相对重要性。ı125I！Tyr-Ala-Chn2与一系列蛋白质结合，将作为一种独特的工具用于鉴定首先与内吞蛋白质相遇的酶。在完整的细胞中，首先与这些试剂反应的酶的类型将表明哪些与内吞蛋白的降解有关。众所周知，溶酶体蛋白是作为前体在内质网中合成的，当它们进入溶酶体时被加工成中间形式，最后在成熟的溶酶体中被加工成较低的M成熟形式。与蛋白质结合的ı125I！TYR-ALA-CHN_2反应的酶的分子形式将允许区分内体与早期溶酶体的融合和内体与晚期(成熟)溶酶体的融合。这种直接鉴定内体中的酶的方法需要明确地确定哪些酶参与内吞蛋白的分解，以及这些酶是通过什么途径包装到内吞体内的。这个项目的目的是使用一种新的技术来确定细胞中一些重要的蛋白水解酶的数量，该技术带有膜透射式放射性标记的抑制剂。这项研究的结果将确定哪四种溶酶体半胱氨酸蛋白酶在哪些细胞中占主导地位，并使我们能够确定这些酶在细胞功能中的意义。第二个目标是确定参与内吞蛋白降解的酶的分子形式。这部分研究的结果将显示哪些酶参与了被吸收到细胞中的蛋白质的降解，还将指出内吞蛋白在生物合成过程中第一次与溶酶体酶相遇的位置。即将开发的程序为研究溶酶体蛋白水解酶的生物学作用提供了一系列新的技术。

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.