Characterization of the activation of pro-urokinase by thermolysin

The bacterial metalloproteinase thermolysin catalyzes the efficient activation of pro-urokinase to an active high-molecular-weight form of the protein. Thermolysin and plasmin convert pro-urokinase to enzymes of essentially equal activities in amidolytic assays, but with different molecular structures. The B-chains of the proteins produced by thermolysin and plasmin are of the same size (33 kDa) and have the same amino-terminal sequences, demonstrating that the cleavage of the Lys¹⁵⁸-Ile¹⁵⁹ bond of pro-urokinase is catalyzed by both enzymes. However, thermolysin also reacts at additional sites in the growth factor domain of the A-chain at nearly the same rate as that of the activation reaction. Polypeptides derived from hydrolyses of the Glu³-Leu⁴, Tyr²⁴-Phe²⁵, Asn²⁷-Ile²⁸ and Lys³⁶-Phe³⁷ bonds are recovered after reduction of the activated protein. The carboxy-terminus of the A-chain has been shown to be Arg-156, a consequence of proteolysis of the Arg¹⁵⁶-Phe¹⁵⁷ bond. In contrast to plasmin, thermolysin activates thrombin-inactivated pro-urokinase nearly as rapidly as it does the native zymogen. Thermolysin provides a useful alternative to plasmin for the catalytic activation and analysis of pro-urokinase, since the bacterial metalloproteinase is stable in solution and not susceptible to inhibition by aprotinin and other serine proteinase inhibitors.