Characterization of cathepsin D in porcine adrenocortical lysosomes

More than 95% of the apparent cathepsin D activity in the lysosomes of porcine adrenal cortex was due to a genuine cathepsin D that has a molecular weight of 42,800 +/- 800 (mean +/- standard deviation of the mean in 5 runs) as determined by Sephadex G-100 column chromatography. On CM-Sephadex C-50 column chromatography at pH 6.9, the enzyme was resolved into five peaks which were termed Fractions D1 through D5 in order of their elution from the column. Fraction D4 and Fraction D5 together constituted more than 70% of the total cathepsin D, and were purified through chromatographic procedures to constant specific activities. The content of lysosomal cathepsin D was estimated to be about 1% of the total cellular protein. On isoelectric focusing, Fraction D4 was resolved into one major band with pI of 7.34 (termed Form D4-1) and one minor band of 7.20 (Form D4-2), while Fraction D5 gave one major band with pI of 7.50 (Form D5-1) and two minor ones of 7.37 (Form D5-2) and of 7.20 (Form D5-3). All of these 5 bands were enzymatically active. On SDS-polyacrylamide gel electrophoresis, both Form D4-1 and Form D5-1 dissociated into three subunits with molecular weights of 27,400 +/- 500 (termed Subunit A), 24,600 +/- 400 (Subunit B) and 13,800 +/- 600 (Subunit C) (mean +/- standard deviation of the mean in 16 determinations). The three subunits all contained carbohydrates.