Fig. 6. Cathepsin D activity is required in the conditioned medium for an efficient cleavage of rPRL. (A) Proteolytic effect of purified thrombin and cathepsin D on rPRL. Immunoblotting analysis of rPRL forms when 1.5 µg/ml rPRL was incubated in Tris-buffer pH 7.4 for 60 minutes at 37°C in the absence (lane 1) or presence of purified enzymes and their inhibitors: 12 U/ml thrombin (lane 2); 0.1 U/ml CD (lane 3); 0.1 U/ml CD and 25 µM pepstatin A (lane 4); or 12 U/ml thrombin and 15 U/ml hirudin (lane 5). (B) Effect of hirudin on the cleaving activity of cathepsin D in Tris-buffer at pH 7.4 for 60 minutes at 37°C. Immunoblotting analysis of rPRL forms in the presence of 0.1 U/ml CD alone (lane 2) or with 15 U/ml hirudin (lane 1). (C) Effect of hirudin on the cleaving activity of the conditioned medium. Immunoblotting analysis of rPRL forms when 1 µg/ml PRL was incubated for 60 minutes at 37°C in conditioned medium in the absence (lane 1) or presence of 15 U/ml hirudin. (lane 2). The doublet is detectable in both lanes 1 and 2. (D) Effect of pepstatin A on the cleaving activity of the conditioned medium. Immunoblotting analysis of rPRL forms when 1 µg/ml rPRL was incubated for 60 minutes at 37°C in conditioned medium in the absence (lane 1) or presence of 10 µM pepstatin A (lane 2). The doublet is hardly detectable in lane 2. (E) Acini-conditioned medium was depleted of CD by immunoprecipitation. In native medium (lane 1) the following rat CD molecular forms can be detected: ProCD, the 51 kDa precursor form; Msc, the 44 kDa single-chain mature form; LM, the 31 kDa large chain of the double-chain mature form. Almost all molecular forms of CD were cleared from the medium after immunoprecipitation with anti-CD serum (lane 2). (F) Immunoblotting analysis of rPRL molecular forms obtained by incubation for 60 minutes at 37°C of 2.5 µg/ml rPRL in conditioned medium (lane 1) or CD-immunodepleted mammary acini-conditioned medium (lane 2). No cleaved forms of rPRL were detectable in the latter case. At the end of the incubation periods media were treated for immunoblotting as described in Materials and Methods.