Abstract
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
Highlights
From the Research and Neurology Services, Departments of Neurology and Biochemistry, Memphis Veterans Medical Center, Memphis, Tennessee University of Tennessee Center for the Health Sciences, 38104, and the Memphis, Tennessee
Electrophoresis at pH 10.6 in the presence of urea demonstrated the microheterogeneity of basic protein (BP) and that BP microheterogeneous components one and three, which were to be used for the preparation of BP peptides, did not contain other BP components
BP component one (I), BP component one incubated with brain cathepsin D at a protein to enzyme ratio of 200~1 for 120 min (2), peptide l-42 (Peak E in Fig. 4) (3), and peptide l-42 incubated with brain cathepsin D at a protein to enzyme ratio of 2oO:l for 30 (4) and 120 min (5)
Summary
Sephadex from Pharmacia, Bio-Gel P-2 from Bio-Rad, molecular weight markers (ovalbumin, chymotrypsinogen A, and cytochrome c) from Schwarz/Mann, and pepstatin A from Peptide. Digestion of BP with Brain Cathepsin D-For analytical polyacrylamide disc gel electrophoretic studies, 300 to 400 pg of BP or BP peptide were dissolved in 1 ml of 0.05 M ammonium acetate, pH 3.5, with or without varying amounts of enzyme and incubated at 37°C in a shaking water bath. Polyacrylamide disc gel electrophoretic study at pH 8.8 of the degradation of bovine BP microheterogeneous components three (a) and one (b), by brain cathepsin D with increasing lengths of incubation. NHdHC03 of 97 mg of bovine BP microheterogeneous component one digested with brain cathepsin D at a protein to enzyme ratio of 18O:l at 37°C for 30 min. NHdHC03 of 75 mg of bovine BP microheterogeneous component one digested with brain cathepsin D at a protein to enzyme ratio of 1200:. Cl8 using acetonitrile/sodium rate of 1.9 ml/min (34)
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