Human cathepsin D.

Cathepsin D was purified from human liver by a procedure involving autolysis, acetone fractionation, and chromatography on ion-exchange media and organomercurial-sepharose. Multiple forms of the enzyme were then separated by preparative isoelectric focusing. The molecular weight of the protein was found to be 43,000. Its amino acid composition was determined and it was shown to be a glycoprotein. When treated with sodium dodecyl sulphate or chaotropic agents (without reduction) all forms of the enzyme tested gave components of about 28,000 and 14,000 molecular weight. Specific antisera were raised against the enzyme, and the characteristics of immunoinhibition were investigated. Immuno-inhibition of rabbit cathepsin D within living macrophages was shown to interfere with degradation of some proteins endocytosed by the cells. The antisera against human and rabbit cathepsin D were used in immunofluorescent localization of the enzyme in sites of tissue damage in which cathepsin D might be implicated. The characteristics of inhibition of human cathepsin D by pepstatin were established. At pH values below 5, KD values of 5 x 10(-10)M were determined and pepstatin was shown to be an excellent titrant for cathepsin D. In the range pH 5-6.4 DK increased steeply and it was concluded that the binding site for substrate and inhibitor was abolished by a conformational change in the enzyme molecule in which three protons are lost.