Substrate specificity and inhibitors of a capillary injury-related protease from sheep lung lymph.
A serine protease (Mr 70,000 to 75,000) appearing in sheep lung lymph after capillary damage induced by Escherichia coli endotoxin, oleic acid, or air emboli, was studied for its specificity toward a series of synthetic peptide and thioester substrates containing an Arg residue in the P1 position. High specificity constants (kcat/Km) were generally obtained with substrates having two or more basic amino acid residues, and with those having a Gln residues in the P2 position. Secondary enzyme-substrate interactions at sites more removed from the scissile bond are of importance, since a few peptides with two basic residues were hydrolyzed slowly, and the site of cleavage of natural peptides was influenced by the amino acid sequence beyond the immediate vicinity of the hydrolyzed bond. The properties of the enzyme and its pattern of specificity distinguish it from enzymes of the clotting cascade, from components of the complement system, and from lung and skin tryptase. The enzyme was inactivated by p-amidinophenylmethanesulfonyl fluoride and by a series of mechanism-based isocoumarin derivatives, the most potent inhibitor being 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Enzyme solutions inactivated by reaction with isocoumarin inhibitors could be completely reactivated after 30 h by treatment with hydroxylamine at neutral pH. Formation of a stable sheep lymph acyl enzyme--in contrast to thrombin and other trypsin-like enzymes--is not followed by alkylation of an active site nucleophile that leads to irreversible enzyme inactivation. The high activity toward substrates with two basic residues suggests that the enzyme may potentially function in processing of precursors of bioactive peptides.
Orlowski
M
M
Department of Pharmacology and Medicine, Mount Sinai School of Medicine, New York 10029.
Lesser
M
M
Ayala
J
J
Lasdun
A
A
Kam
C M
CM
Powers
J C
JC
D000818:Animals; D000937:Antigen-Antibody Reactions; D002196:Capillaries / Q000201:enzymology / Q000503:physiopathology*; D006868:Hydrolysis; D007106:Immune Sera; D007700:Kinetics; D008168:Lung / Q000098:blood supply / Q000201:enzymology* / Q000503:physiopathology; D008196:Lymph / Q000201:enzymology* / Q000276:immunology / Q000503:physiopathology; D011817:Rabbits; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors; D012756:Sheep; D012967:Sodium Dodecyl Sulfate; D013379:Substrate Specificity
D007106:Immune Sera; D015842:Serine Proteinase Inhibitors; D012967:Sodium Dodecyl Sulfate; D012697:Serine Endopeptidases