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-?125iodine!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. ?125iodine!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 ?125iodine!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.
