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

溶酶体半胱氨酸蛋白酶，组织蛋白酶B， S和L，已经从许多哺乳动物物种中纯化出来，并且已知以多种形式存在。由于它们的不稳定性和天然抑制剂的抑制作用，这些酶的定量是困难的。这项提议的目的是定量组织蛋白酶在细胞中不同分子形式的相对浓度，并确定哪些分子形式参与了内吞蛋白（如白蛋白和0.2巨球蛋白）的降解。实验程序将采用特定的放射性标记抑制剂，要么是自由膜渗透形式，要么是与细胞培养系统中的蛋白质结合。一个纯形式的Z-ı125iodine！Tyr-Ala-CHN2将与特异性抗体和SDS/聚丙烯酰胺凝胶电泳/放射自显影相结合，定量测定活细胞中每种酶的不同分子形式。这些方法避免了在分离过程中失活或抑制的问题，并且具有能够定量每种酶的不同分子形式的额外优势。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。ı125碘!与一系列蛋白质结合的Tyr-Ala-CHN2将被用作鉴定首先与内吞蛋白相遇的酶的独特工具。在完整细胞中首先与这些试剂反应的酶的类型将表明哪些酶参与了内吞蛋白的降解。众所周知，溶酶体蛋白酶在内质网中作为前体合成，进入溶酶体后加工成中间形式，最后在成熟溶酶体中加工成低M成熟形式。酶的分子形式与ı125iodine！与蛋白质结合的Tyr-Ala-CHN2可以区分核内体与早期溶酶体的融合和核内体与晚期（成熟）溶酶体的融合。这种直接识别内体酶的方法需要明确地确定哪些酶参与了内吞蛋白水解，以及酶通过哪种途径被包装到内吞室中。该项目旨在使用膜渗透放射性标记抑制剂的新技术确定细胞中一些重要蛋白酶的数量。这项研究的结果将确定四种溶酶体半胱氨酸蛋白酶中哪一种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重要性。第二个目的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进入细胞的蛋白质的降解，并将表明内吞蛋白质在其生物合成过程中首先与溶酶体酶相遇的位置。所开发的程序为研究溶酶体蛋白酶的生物学作用提供了一系列新技术。

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.