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ISOLATION AND CHARACTERIZATION OF BOVINE BRAIN CATHEPSIN D
Author(s) -
Whitaker John N.,
Seyer Jerome M.
Publication year - 1979
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1979.tb00355.x
Subject(s) - pepstatin , edman degradation , biochemistry , cathepsin , chemistry , size exclusion chromatography , cathepsin d , chromatography , gel electrophoresis , enzyme , isoelectric focusing , polyacrylamide gel electrophoresis , isoelectric point , sodium dodecyl sulfate , cathepsin o , microbiology and biotechnology , amino acid , peptide sequence , biology , gene , protease
— Bovine brain cathepsin D was purified 1774‐fold with a 19% recovery by affinity chromatography on immobilized pepstatin. Approximately 2 mg of enzyme protein were isolated from 150 g (wet weight) of bovine brain. The enzyme eluted from gel filtration as a single peak with a molecular weight of 40,000–42,000. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the predominant band migrated with a molecular weight of 48,000: however, less distinct bands were also present in the molecular weight ranges of 31,000 and 13,000. The isolated enzyme had isoelectric points over a range of pH 5–7 with 3 major peaks occurring at pH 5.6, 6.1, and 6.6. The amino acid composition of brain cathepsin D showed substantial differences from that reported for cathepsin D isolated from bovine spleen. Amino‐terminal sequence analysis revealed an Asp‐Val‐lle sequence by Edman degradation. With hemoglobin as the substrate the enzyme had an apparent K, of 60mM.