Purification and characterization of hepsin from rat liver microsomes.
Hepsin, a putative cell-surface serine proteinase, has been isolated from the microsomal membranes of rat liver and purified to homogeneity by hydroxyapatite, DEAE-Sepharose, and benzamidine-Sepharose chromatography. The course of purification was monitored using antibodies raised against a 20-mer peptide at the C-terminus of rat hepsin, and the identity of the purified protein was confirmed by partial amino-acid sequencing. A single-chain precursor of ca. 50 kDa found in the microsomes underwent spontaneous maturation in the course of purification so that the last, affinity chromatography, step recovered only the mature form which dissociated to subunits of 31 and 19 kDa under reducing SDS-PAGE. Proteinase digestion experiments with microsomal vesicles are consistent with the luminal orientation of the precursor C-terminus, which would result in its extracellular orientation upon transportation to the cell surface. [3H]diisopropylfluorophosphate covalently binds to 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 Arg161-Ile162 site. Activity measurements with short synthetic peptides show that the enzyme cleaves after basic amino-acid residues, Arg being preferable to Lys. The inhibition pattern is typical of trypsin-like serine proteinases. The pH-dependence of activity within the range pH 6-9 has no maximum, the activity increasing continuously with pH. These results are consistent with the earlier predictions based on hepsin amino-acid sequence and elucidate the specificity and other earlier unknown enzymatic and molecular properties of the enzyme.
Zhukov
A
A
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Hellman
U
U
Ingelman-Sundberg
M
M
D000595:Amino Acid Sequence; D000818:Animals; D006863:Hydrogen-Ion Concentration; D008297:Male; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D008565:Membrane Proteins; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; C055645:hepsin