The sequential limited degradation of bovine myelin basic protein by bovine brain cathepsin D.

Bovine myelin basic protein (BP) microheterogeneous components were isolated and exposed to homogeneous bovine brain cathepsin D purified by affinity chromatography on immobilized pepstatin. The extent of the reaction was followed by polyacrylamide gel electrophoresis at pH 8.8, and the reaction products were separated by column chromatography on carboxymethyl-cellulose and Sephadex following which the BP peptides were characterized by amino acid analysis and partial sequence analysis. Components one and three were degraded with the initial site of cleavage at the Phe-Phe bond at residues 42 and 43 to generate peptides l-42 and 43-169. With more prolonged exposure to enzyme, peptide l-42 was degraded to form peptide l-36 and peptide 43-169 was degraded to form peptides 43-88, 89-169, and 92-169 as well as smaller amounts of peptides 43-89 and 43-91. Pepstatin inhibited the initial cleavage of BP by cathepsin D. Microheterogeneous components two, four, and five showed similar patterns of fragmentation to yield bands with the migration of peptides l-36, 43-88, and 89-169 or 92-169. Peptides 43-169, 89-169, and 92-169 had a decreasing cathodal migration progressing from components one to five. These findings demonstrate the sequential but limited cleavage of BP by brain cathepsin D. The effects of the enzyme on the microheterogeneous components of the molecule in forming fragments of different charge characteristics suggest that the processes regulating microheterogeneity may influence the outcome of degradation of BP by brain cathepsin D and possibly other proteinases.