A sensitive procedure for determination of cathepsin D: activity in alveolar and peritoneal macrophages

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Abstract
Several new synthetic substrates fulfilling the specificity requirements of cathepsin D were synthesized. One of these D-Phe-Ser(O-CH2-C6H5)-Phe-Phe-Ala-Ala-pAB (pAB = p-aminobenzoate) proved to be highly sensitive and convenient for measuring activity. Enzyme determination was carried out in a two-step reaction. In the first step the enzyme hydrolyzes the Phe-Phe bond of the substrate at pH 3.4. In the second step aminopeptidase M (EC 3.4.11.2) degrades one of the products Phe-Ala-Ala-pAB at pH 7 to 8 with the release of free pAB, which is then determined by a diazotization procedure. Activity can be measured in as little as 1 to 5 µg of macrophage protein. The activity of cathepsin D in rat alveolar macrophages was almost ten times higher than in resident peritoneal macrophages, and more than 25 times higher than in blood monocytes. The data indicate that transformation of blood monocytes into macrophages is associated with a much greater increase of cathepsin D activity in alveolar than peritoneal macrophages.