Highly purified calf brain cathepsin D (EC 3.4.23.5) selectively splits the LeuO-Phe78 and Ala3-Ala37 peptide bonds of human P-lipotropin. It is suggested that the formation of human "jB-melanotropin" from y-lipotropin, and that of -y-endorphin from f.endorphin, is due to the action of cathepsin D during isolation procedures.Since the discovery of 3-lipotropin (3-LPH) and its isolation from ovine pituitaries (1), our knowledge of the chemistry of lipotropins has steadily grown. The complete amino acid sequences (91 amino acids) of ovine (2-4), porcine (5), human (6), and bovine (7) (14) and subsequently identified as a 22-residue peptide containing four more amino acids than certain other species homologs (8, 15), might have been artifactually formed from human f-LPH during the extraction procedure.The present paper provides evidence for the proposition that lysosomal cathepsin D (EC 3.4.23.5) is one of the proteinases involved in the generation of human ",-MSH" from human 3-LPH. EXPERIMENTAL Isolation of cathepsin D from bovine brain was carried out by a modification of the procedure of Benuck et al. (16). The brains were removed within 1 hr of killing and homogenized in 2 vol of 0.05 M sodium citrate, pH 2.7, in an Ultraturrax homogenizer (Janke-Kunkel KG) at 4°C for 40 s. The homogenate was centrifuged at 25,000 X g for 20 min at 4°C and the supernatant fraction was stored at -20°C for later purification.Pepstatin-Sepharose was prepared as follows: 0.7 mg of pepstatin (The Protein Research Foundation, Osaka, Japan), 0.5 g of AH-Sepharose 4B (Pharmacia), and 16.5 mg of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide meto-p-toluenesulfonate (Fluka, Buchs, Switzerland) were mildly agitated in 10 ml of dimethylformamide/water, 1:3 (vol/vol), pH 6, for 16 hr at room temperature. The product was filtered and washed with 50-ml portions of dimethylformamide/water, 1:1 (vol/vol); dimethylformamide/dioxane, 1:1 (vol/vol); 4 M urea; and 0.1 M sodium citrate, pH 3.9/1 M NaCl in this sequence. The pepstatin-Sepharose column (5 cm X 0.7 cm) was prepared and washed alternately with 0.1 M sodium bicarbonate, pH 7.6/1 M NaCl and 0.1 M sodium citrate, pH 3.9/1 M NaCI, equilibrated with the latter solution, and subsequently loaded with 25 ml of the brain extract previously dialyzed against the pH 3.9 solution for 10 hr. After the column was washed with the latter solution, cathepsin D was eluted with 0.1 M sodium bicarbonate, pH 7.6/1 M NaCl, and the pH of the enzyme solution was adjusted to about 4 by addition of 1 M sodium citrate, pH 3.9. Enzyme solutions were kept frozen for later use.Cathepsin D was assayed on porcine f3-LPH (,Bp-LPH) as described (17). Op-LPH and human 3-LPHs (3b-LPHs) were prepared according to Graf and Cseh (18) and Cseh et al. (19,20), respectively.Digestion of f3-LPH with purified cathepsin D was performed in 0.1 M ammonium formate, pH 4, at a ratio of 1 to 1000 (wt/wt) of enzyme to peptide at 37°C. Digestion was terminated by addition of 1 ,uM pepstatin to the incubation mixture.The 13h-LP...