Cathepsins D from rhesus monkey lung. Purification and characterization.:Purification and Characterization

Two types of cathepsin D (cathepsins D-I and D-II) were purified from rhesus monkey lung to homogeneity as judged from disc gel electrophoresis. Cathepsin D-I was purified about 2, 000-fold with a 5.1% yield while cathepsin D-II was purified about 2, 300-fold with a 14.3% yield. Both cathepsins D were rich in the lysosome fraction of the lung, but appeared to be present in part extracellularly. Both showed a molecular weight of about 35, 000 on Sephadex G-100 chromatography, and about 41, 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cathepsin D-I showed the maximal activity on bovine hemoglobin and albumin at pH 3.4 and 4.0, respectively. It was most stable in the pH range of 5 to 7, but was rather unstable outside this pH range. Cathepsin D-II was quite similar in properties to that from Japanese monkey lung (Moriyama, A. & Takahashi, K. (1978) J. Biochem. 83, 441-451), and was remarkably stable in the pH range of 1-9. Under the conditions used, it retained at least 80% of the original activity when incubated at 37°C for 20 h in this pH range. This stability seems to allow cathepsin D-II to be fairly active even at pH 1.0. Both cathepsins D acted on protein substrates fairly similarly and hydrolyzed hemoglobin most rapidly among the proteins tested. They did not hydrolyze N-acetyl-L-phenylalany1-3, 5-di-iodotyrosine. Upon incubation with the oxidized B-chain of insulin, both cathepsins D hydrolyzed the ¹⁴Ala-¹⁵Leu, ¹⁵Leu-¹⁶Tyr, ¹⁶Tyr-¹⁷Leu, ²⁴Phe-²⁵Phe, and ²⁵Phe-²⁶Tyr bonds at both pH 3.0 and 5.0. In addition, cathepsin D-II hydrolyzed the ¹¹Leu-¹²Val and ²⁶Tyr-²⁷Thr bonds at pH 3.0 and the ²Val-³Asn bond at pH 5.0. Both cathepsins D were inactivated by acid proteasespecific inhibitors such as pepstatin, 1, 2-epoxy-3-(p-nitrophenoxy)propane, p-bromophenacyl bromide, and diazoacetyl-DL-norleucine methyl ester, although cathepsin D-II was much less susceptible to these reagents except p-bromophenacyl bromide.