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.
Marcotte
P A
PA
Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064-3500.
Henkin
J
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D004800:Enzymes, Immobilized; D005341:Fibrinolysin / Q000494:pharmacology; D008969:Molecular Sequence Data; D009842:Oligopeptides; D011994:Recombinant Proteins; D013056:Spectrophotometry, Ultraviolet; D013820:Thermolysin / Q000494:pharmacology*; D014568:Urokinase-Type Plasminogen Activator / Q000302:isolation & purification / Q000378:metabolism*
D004800:Enzymes, Immobilized; D009842:Oligopeptides; D011994:Recombinant Proteins; C028190:5-oxo-prolyl-glycyl-arginine-4-nitroanilide; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D013820:Thermolysin; C067551:saruplase