Isolation, purification, and characterization of bovine brain cathepsin D

Bovine brain cathepsin D was purified 1120-fold from enriched white matter with an overall yield of 46%. The enzyme is a single polypeptide chain with an apparent molecular weight of 42,000 as estimated by SDS-polyacrylamide gel electrophoresis and sephadex G-100 gel filtration. This is in sharp contrast to porcine spleen cathepsin D which consist of two polypeptide chains held together by noncovalent associations. Secretion of cathepsin D by cultured rat peritoneal macrophages was studied. These cells secreted the lysosomal marker enzyme β-glucuronidase and lactic acid when challenged with derivatized, noningestible central nervous system tissue in a dose- and time-dependent manner. Secreted cathepsin D levels, however, were insignificant when measured with 14C-hemoglobin substrate. It was observed, however, that certain monosaccharides such as mannose and N-acetyl glucosamine were able to release cell surface bound cathepsin D. The observed specificity and the calculated binding constant indicated that cathepsin D bound to the mannose/N-acetyl glucosamine receptor of macrophages