Isolation and characterization of a novel large protease accumulated in mammalian cells in the presence of inhibitors.
We have isolated and characterized a novel, large, multicatalytic protease from mammalian cells. This protease was designated PABI (protease accumulated by inhibitors). When baby hamster kidney (BHK) cells were grown in medium containing leupeptin, a potent serine-cysteine protease inhibitor, the trypsin-like protease activity (PABI) in the cells increased its level more than 100-fold over the control. This increase was also observed in other cultured cells such as COS, HepG2, and skin fibroblast cells. The activity was also elevated by treatment with other protease inhibitors including chymostatin or trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane. Immunoblot analysis, by employing antisera prepared against the purified PABI, also showed a concomitant increase of this protein in BHK, COS, and HepG2 cells on leupeptin treatment. PABI was purified to a homogeneous state from leupeptin-treated BHK cells. PABI is a glycoprotein of molecular weight 700,000. PABI was found to be a multimer of a major subunit of apparent Mr of 84,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopic analysis. PABI dissociates into subunits only under reducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PABI has both trypsin-like and chymotrypsin-like protease activities toward synthetic substrates. Both activities were inhibited by phenylmethanesulfonyl fluoride, aprotinin, bovine pancreas trypsin inhibitor, and chymostatin. Leupeptin inhibited only the trypsin-like activity of PABI. p-Chloromercuribenzoate had no effect on either activity. Furthermore, PABI degraded collagen type I and fibronectin. These results indicate that PABI is a novel protease which differs from any known proteases including cytosolic high molecular weight proteases. The physiological function of PABI is yet to be determined.
Tsuji
A
A
Department of Human Genetics, University of Michigan, Ann Arbor 48109-0618.
Kurachi
K
K
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D006224:Cricetinae; D007668:Kidney; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D000596:Amino Acids; D011480:Protease Inhibitors; D010447:Peptide Hydrolases