Purification and characterization of active recombinant human napsin A

Recombinant human napsin A expressed in human embryonic kidney 293 cells wa s purified to homogeneity by a single-step procedure using part of napsin A propeptide as affinity ligand. N-Terminal amino-acid sequencing of the pur ified enzyme identified the mature form of napsin A. Treatment of purified napsin A with endoglycosidases F and H resulted in a decrease in its molecu lar mass from 39 kDa to approximate to 37 kDa, confirming that napsin A is glycosylated. The kinetic properties were analyzed by using two fluorogenic synthetic substrates K(Dabsyl)-TSLLMAAPQ-Lucifer yellow (DS1) and K(Dabsyl )-TSVLMAAPQ-Lucifer yellow (DS3). The K-m values obtained were 1.7 mu M and 6.2 mu M, respectively. A substrate-specificity study using a napsin A-tar geted peptide library confirmed the preference of napsin A for hydrophobic residues at positions P1 and P1'. Adjacent positions, P2-P4 and P2'-P4', ap peared less restricted in distribution of amino acids. A pH optimum between 4.0 and 5.5 at room temperature was determined. The purified enzyme was fu lly active for more than 10 h at pH 5.0 and 6.0, while a half-life of 4 h w as determined at pH 7.0 and 37 degrees C.