Purification and partial characterization of human placental particle-bound aminopeptidase.

Particle-bound aminopeptidase was purified from the human term placenta by separation of the particles from the soluble fraction by centrifugation and solubilization of the particle fraction with 0.5 per cent (v/v) Triton X-100 followed by CM-cellulose chromatography, Sepharose 6B gel filtration, DE-cellulose and Con A-Sepharose 4B affinity chromatography. Polyacrylamide gel electrophoresis indicated enzymatic and protein homogeneity of the purified enzyme. The enzyme seemed to be a glycoprotein with a molecular weight of 320 000. The purified enzyme did not endure freezing but was fairly stable at 4 degrees C. At 60 degrees C more than half and at 65 degrees C all enzyme activity disappeared in 15 min. The pH dependence of the purified enzyme varied between 6.75 and 7.5, and 6.0 and 6.5, depending on the substrate used. The enzyme hydrolysed most rapidly LeuNA (Km 0.095 +/- 0.008 mmol) followed by beta-naphthylamide derivatives of alanine (Km 0.222 +/- 0.02 mmol), arginine (Km 0.041 +/- 0.002 mmol), lysine (Km 0.084 +/- 0.005 mmol), methionine (Km 0.085 +/- 0.004 mmol) and cystine (Km 0.048 +/- 0.004 mmol). Thus LeuNA was the most specific among the substrates for the enzyme. The purification process revealed, however, that CysNA was the most selective substrate for the particulate enzyme, which readily hydrolysed also Leu-p-na (Km 0.865 +/- 0.023 mmol) and Bz-Cys-p-na (Km 0.248 +/- 0.034 mmol).