Isolation of cathepsin D from human leucocytes.

Acid and neutral protease activities were determined in the granule fractions of polymorpho and mononuclear leucocytes, separated from human blood by means of a discontinuous density gradient centrifugation. The mononuclear leucocytes contained only acid protease while preparations from polymorphonuclear leucocytes showed a predominant activity at neutral pH with a small peak in the acid range. A separation of the acid from the neutral enzyme could be obtained in the granule fraction of polymorphonuclear leucocytes by means of DEAE chomatography. The acid enzyme was then purified from a mixture of leucocytes, more than 400 times, by means of gel chromatography with Sephadex G-200 superfine. The purified acid protease showed an optimum pH of 3.6, had a molecular weight at 42 000 and was characterized by a single protein band (Rf = 0.31) by disc-gel electrophoresis. With all probability this enzyme can be classified as cathepsin D (EC 3.4.4.23).