ISOLATION AND CHARACTERIZATION OF BOVINE BRAIN CATHEPSIN D |
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Authors: | John N Whitaker Jerome M Seyer |
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Institution: | Research and Neurology Services, Memphis Veterans Hospital and the Departments of Neurology and Biochemistry, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38104, U.S.A. |
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Abstract: | 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. |
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