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

1979; Elsevier BV; Volume: 254; Issue: 15 Linguagem: Inglês

10.1016/s0021-9258(18)50268-4

1083-351X

John N. Whitaker, Jerome M. Seyer,

Protein Hydrolysis and Bioactive Peptides

Bovine myelin basic protein (BP) microheteroge- neous components were isolated and exposed to ho- mogeneous bovine brain cathepsin D purified by affin- ity chromatography on immobilized pepstatin. The ex- tent of the reaction was followed by polyacrylamide gel electrophoresis at pH 8.8, and the reaction products were separated by column chromatography on car- boxymethyl-cellulose and Sephadex following which the BP peptides were characterized by amino acid anal- ysis and partial sequence analysis. Components one and three were degraded with the initial site of cleav- age at the Phe-Phe bond at residues 42 and 43 to gen- erate 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 in- hibited the initial cleavage of BP by cathepsin D. Mi- croheterogeneous 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 micro- heterogeneous 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. Myelin encephalitogenic or basic protein, which accounts for approximately 30% of myelin proteins in the central nerv- ous system (CNS) (l), has a monomeric molecular weight of 18,500 and consists of 169 amino acid residues (2,3). The total amino acid sequence has been determined for human (4) and bovine (2, 3) BP’, 2 and the small BP of the rat (5), and the