Purification of Cathepsin D from Cartilage and Uterus and Its Action on the Protein-Polysaccharide Complex of Cartilage

Cathepsin D has been purified approximately 900-fold from two types of cartilage: elastic cartilage of young rabbit ears and hyaline cartilage from the legs of 12- to 13-day-old chick embryos. Cathepsin D accounts for most of the hemoglobin-digesting activity of chick cartilage; rabbit ear cartilage also contains a sulfhydryl-dependent acid cathepsin. The identification of the acid proteolytic activity of the cartilages as cathepsin D is based on the specificity of cleavage of the B chain of insulin. The molecular weight of the two enzymes, as estimated by molecular sieving, is about 40,000 to 43,000. The two cartilage enzymes and cathepsin D from bovine uterus all digested the protein-polysaccharide complex of bovine nasal cartilage at pH 5. The digestion could be followed viscosimetrically and it obeyed a second order kinetic expression. The protein-polysaccharide complex was completely degraded to fragments that were larger than single chains of chondroitin sulfate. Digestion at pH 5 was inhibited by e-aminocaproate and arginine (0.1 m), chloroquine and 3-indolepyruvate (0.02 m), and pepstatin (2 x 10-7 m). Activity curves were measured as a function of pH; all three preparations of cathepsin D digested protein-polysaccharide complex optimally at pH 4. On comparing one hemoglobin-digesting unit of each enzyme, it was found that the cathepsin D from bovine uterus digested protein-polysaccharide complex twice as rapidly as the rabbit ear enzyme and 5 times as rapidly as the chick cartilage enzyme. At pH 7.2 digestion of protein-polysaccharide complex was quite variable, the chick preparation was completely inactive, and the other preparations had variable activities that were not in proportion to their hemoglobin-digesting activity. It was finally shown that cathepsin D is completely inactive at pH 7.2 and that protein-polysaccharide complex digestion at this pH is due to the action of a contaminant neutral protease.