Active site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone demonstrates the full activity of the refolded and purified tissue-type plasminogen activator variant BM 06.022.
BM 06.022 is a tissue-type plasminogen activator deletion variant that is comprised of the kringle 2 and the protease domain of the native molecule. BM 06.022 is expressed as inactive inclusion bodies in E. coli and transferred into the active enzyme by an in vitro folding process. Active site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone provides evidence that the purified BM 06.022 is fully active and that misfolded species are completely removed by affinity chromatography on ETI-Sepharose. The comparison of the kinetics of the inhibition of BM 06.022 with that of CHO-t-PA indicates that the active centers of both enzymes are rather similar. The further evaluation of the site of interaction of BM 06.022 and DnsEGRck by mass spectroscopy and amino acid sequence analysis revealed that the inhibitor is bound selectively to His322, which is part of the catalytic triad of this serine protease.
D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002846:Chromatography, Affinity; D003619:Dansyl Compounds / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002479:Inclusion Bodies / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D008970:Molecular Weight; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; C030824:dansylglutamyl-glycyl-arginine chloromethyl ketone; D010959:Tissue Plasminogen Activator