Purification and partial characterization of an ATP-hydrolyzing serine protease from lettuce leaves

Abstract

Abstract A novel ATP-hydrolyzing protease has been purified from lettuce leaves by combination of (NH 4 ) 2 SO 4 fractionation, gel filtration and anionic exchange chromatography. The purified enzyme is made up of a single subunit with an apparent molecular weight of 40 000, even though a possible higher molecular organization might occur in vivo induced by the presence of ATP or protease's substrate. The lettuce protease showed caseinolytic, trypsin-like and, to a smaller extent, peptidyl glutamyl hydrolase activities. It is a serine protease as both peptidase activities are highly sensitive to tosyl- l -lysinechloromethylketone (TLCK) and leupeptin. The trypsin-like activity of the enzyme was not affected by MgATP complex or ATP alone. Peptidyl glutamyl hydrolase activity, instead, and ATP hydrolysis were strictly correlated as incubation of the enzyme with MgATP, but not with ATP alone, stimulated the peptidase activity of the enzyme, while peptide substrate as well as TLCK enhanced ATPase activity. Moreover, the ATPase inhibitor vanadate, which also blocked the peptidyl glutamyl hydrolase activity, caused a strong activation of the trypsin-like activity of the enzyme. Therefore, these studies could indicate the existence of multiple functional states of the enzyme achieved in vivo by ATP hydrolysis. The cytosolic localization of the enzyme is finally discussed.