Immunochemical Characterization of Bovine Brain Cathepsin D

Publisher Website
Google Scholar
Abstract
Antibodies to bovine brain cathepsin D were produced in rabbits and guinea pigs and utilized to characterize this enzyme immunochemically. By immunodiffusion, broad organ–but limited species–cross‐reactivity was demonstrated with antisera to the brain enzyme. On immunoelectrophoresis the immunoreactive material migrated as a single are in the beta‐gamma region. In a double antibody radioimmunoassay, which had a sensitivity for cathepsin D of 1 ng of immunoreactive enzyme per assay tube, no inhibition was produced by pepstatin, bovine myelin basic protein, or bovine basic protein peptides 1‐42, 43‐88 or 89‐169. Comparative studies of enzymatic and immunoreactive activity of bovine brain cathepsin D demonstrated a close correlation, but the immunoassay was much more sensitive in detecting the presence of enzyme protein. Immunoreactivity persisted unaltered under conditions that abolished or reduced enzymatic activity. Antibodies to brain cathepsin D inhibited the activity of the enzyme on a hemoglobin substrate at pH 5. On gel filtration of bovine brain homogenate, the immunoreactivity, measured by either radial immunodiffusion or radioimmunoassay, and enzymatic activity of cathepsin D coeluted, indicating a molecular weight of approximately 42,000. However, immunofixation of enzyme on polyacrylamide slab gels subjected to electrophoresis in the presence of sodium dodecyl sulfate demonstrated bands of immunoreactivity in purified enzyme in the molecular weight ranges of 42,000, 31,000 and 13,000 and two very faint bands in the range of 32,000‐36,000. Immunofixation also revealed antigen in 42,000 and 31,000 molecular weight species of homogenates of bovine brain, liver, spleen, and kidney.