The isolation and properties of a proteolytic enzyme, cathepsin D, from bovine spleen

Abstract

A proteolytic enzyme was isolated from bovine spleen, which accounts for 2/3 of the total proteolytic activity of the crude mince. It is present in at least 10 forms which differ from one to another in their charge at pH 8.4 or 5.5. The specific activity of the different forms of the enzyme is the same and their purity was established by starch-gel electrophoresis at pH 9.1 and 5.5, by ultracentrifugal analysis and determination of N-terminal amino acid, which is glycine. Cathepsin D has a pH optimum of 3.0 with acid-denatured hemoglobin and 4.2 with acid-denatured albumin. It will not hydrolyze the synthetic substrates described by Fruton (1957-58) for cathepsins A, B and C nor will it hydrolyze the pepsin substrates N-acetyl-DL-phenylalanyl-L-diiodotyrosine, L-cysteinyl-L-tyrosine or L-tyrosyl-L-cysteine. The specificity o''f action on the B chain of oxidized insulin is similar to that of pepsin but more restricted. No difference is specificity could be detected between the different forms of the enzyme. The proteolytic activity of cathepsin D is not affected by cysteine, iodoacetamide, p-chloro-mercuribenzoate, ethylenediaminetetraacetate or di-isopropyl phosphorofluoridate. Cathepsin D is heat-labile and activity is rapidly lost below pH 2.5 at room temperature.