PURIFICATION AND CHARACTERIZATION OF HEPSIN FROM RAT-LIVER MICROSOMES

Hepsin, a putative cell-surface serine proteinase, has been isolated f rom the microsomal membranes of rat liver and purified to homogeneity by hydroxyapatite, DEAE-Sepharose, and benzamidine-Sepharose chromatog raphy. The course of purification was monitored using antibodies raise d against a 20-mer peptide at the C-terminus of rat hepsin, and the id entity of the purified protein was confirmed by partial amino-acid seq uencing. A single-chain precursor of ca. 50 kDa found in the microsome s underwent spontaneous maturation in the course of purification so th at the last, affinity chromatography, step recovered only the mature f orm which dissociated to subunits of 31 and 19 kDa under reducing SDS- PAGE. Proteinase digestion experiments with microsomal vesicles are co nsistent with the luminal orientation of the precursor C-terminus, whi ch would result in its extracellular orientation upon transportation t o the cell surface. [H-3]diisopropylfluorophosphate covalently binds t o the large subunit showing it to be the catalytic one. The N-terminal sequencing of this subunit demonstrates that the zymogen is converted to the active serine proteinase by cleavage at the Arg(161)-Ile(162) site. Activity measurements with short synthetic peptides show that th e enzyme cleaves after basic amino-acid residues, Arg being preferable to Lys. The inhibition pattern is typical of trypsin-like serine prot einases. The pH-dependence of activity within the range pH 6-9 has no maximum, the activity increasing continuously with pH. These results a re consistent with the earlier predictions based on hepsin amino-acid sequence and elucidate the specificity and other earlier unknown enzym atic and molecular properties of the enzyme.