The proteolytic activity and cleavage specificity of fibronectin-gelatinase and fibronectin-lamininase.
Human plasma fibronectin contains two latent aspartic proteinases, FN-gelatinase and FN-lamininase. Both enzymes can be generated and activated in the presence of Ca2+ from the purified cathepsin D-produced 190-kDa fibronectin fragment. We investigated the proteolytic activity and cleavage specificity of both enzymes in a range of pH from 3.5 to 9.0 using the B chain of oxidized bovine insulin and chromogenic peptides as substrates. The inhibition of the enzymes by several natural inhibitors from human plasma was also tested. The specificities of FN-gelatinase and FN-lamininase are similar to other major acidic proteinases, including pepsin, renin, cathepsin D, and HIV-proteinases. Both enzymes mainly hydrolyze three peptide bonds in the oxidized insulin B chain, namely Glu-Ala (residues 13-14), Tyr-Leu (residues 16-17), and Phe-Phe (residues 24-25). For the peptide substrates H-Pro-Thr-Glu-Phe-p-nitro-Phe-Arg-Leu-OH and H-Phe-Gly-His-p-nitro-Phe-Phe-Val-Leu-OMe that were cleaved the respective values of k(cat)/K(M) were 105.1 and 11.8 mM(-1) sec(-1) for cleavage by FN-gelatinase, and 123.2 and 15.5 mM(-1) sec(-1) for cleavage by FN-lamininase. The maximal activities of both enzymes were observed in a range between pH 5.6 and 6.3 and they became inactivated at a pH value above 8.4. Both FN-gelatinase and FN-lamininase were efficiently inhibited by alpha2-macroglobulin.
Unger
J
J
Lehrstuhl für Biochemie, Fakultät für Chemie, Universität Bielefeld, Germany.
Tschesche
H
H
D002118:Calcium / Q000494:pharmacology; D002863:Chromogenic Compounds / Q000378:metabolism; D003432:Cross-Linking Reagents; D003538:Cystamine / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005353:Fibronectins / Q000378:metabolism*; D018093:Gelatinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D000511:alpha-Macroglobulins / Q000494:pharmacology
D002863:Chromogenic Compounds; D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D005353:Fibronectins; D007328:Insulin; D010446:Peptide Fragments; D000511:alpha-Macroglobulins; D010450:Endopeptidases; D018093:Gelatinases; C079023:FN-gelatinase; C064016:fibronectin laminase; D003538:Cystamine; D002118:Calcium