Purification and Properties of Cathepsin D from Bovine Uterus

Cathepsin D was prepared from bovine uterus by homogenization, acid dialysis, (NH4)2SO4 concentration, dioxane fractionation, and chromatography on Sephadex G-100 and DEAE-Sephadex. A purification of 1000-fold has been achieved and the recovery exceeds 40%. The resulting product contains a large number of distinct forms of cathepsin D as shown by disc electrophoresis. The preparation appears to be free of contaminating acid proteases and peptidases as shown by the failure to digest 16 synthetic peptides commonly used as substrates for these enzymes. The specificity of the enzyme was studied with the B chain of insulin as a substrate. Major points of cleavage were found at Leu15-Tyr16, Phe24-Phe25, and Phe25-Tyr26. Additional points of cleavage included Phe1-Val2, Glu13-Ala14, Ala14-Leu15, Tyr16-Leu17. This specificity indicates that this enzyme from the bovine uterus belongs to the cathepsin D family of proteases. The enzyme showed maximum stability in the pH range 5 to 7. Activity was rapidly lost when the enzyme was heated to 60° at pH 6.3. The apparent molecular weight was estimated to be 43,000 based on molecular sieving on Sephadex G-100 and G-75 columns. The hydrolysis of denatured hemoglobin by cathepsin D is inhibited by high concentrations (0.01 m) of heavy metal ions and by high salt concentrations (ionic strength = 0.3). Dithiothreitol (1 mm), 3-p-hydroxyphenylpyruvate (5 mm), and vitamin A (1 mm) are moderately inhibitory.