A novel serine protease (IRCM-serine protease 1) from porcine neurointermediate and anterior pituitary lobes. Isolation, polypeptide chain structure, inhibitor sensitivity, and substrate specificity with fluorogenic peptide substrates.
A novel serine protease, which we have called IRCM-serine protease 1, was purified from both porcine neurointermediate and anterior pituitary lobes. The enzyme was inhibited by soybean trypsin inhibitor, pancreatic trypsin inhibitor, benzamidine, phenylmethyl-sulfonyl fluoride, and thiol reagents including HgCl2, p-chloromercuribenzoate, and 5,5'-dithiobis-(2-nitrobenzoic acid) and was resistant to lima bean trypsin inhibitor, alpha 2-macroglobulin, alpha 1-antitrypsin, and C1-esterase inhibitor. IRCM-serine protease 1 displayed "trypsin-like" specificity toward a number of tripeptide coumarin-containing substrates, with kcat/km values ranging from 10(4) to 10(6) M-1 S-1. The best substrate was benzyloxycarbonyl-L-Ala-L-Lys-L-Arg-4-methylcoumarin-7-amide with a kcat/Km value of 2.27 X 10(6) M-1 S-1. IRCM-serine protease 1, Mr = 169,000-190,000 determined by gradient gel electrophoresis and gel filtration, respectively, appears to be a homologous dimer. The monomeric subunits of the enzyme are composed of an Mr = 38,000 polypeptide chain which is modifiable by 125I-D-Tyr-Glu-Phe-Lys-Arg-CH2Cl, disulfide-linked to another polypeptide resulting in a subunit molecular weight of 88,000.
D000818:Animals; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010902:Pituitary Gland / Q000201:enzymology*; D010903:Pituitary Gland, Anterior / Q000201:enzymology*; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine; D014361:Trypsin Inhibitors / Q000494:pharmacology
D046911:Macromolecular Substances; D013439:Sulfhydryl Reagents; D014361:Trypsin Inhibitors; D010450:Endopeptidases; C048880:IRCM-serine protease 1; D012697:Serine Endopeptidases