Kinetic characterization of the proteinase binding defect in a reactive site variant of the serpin, antithrombin. Role of the P1' residue in transition-state stabilization of antithrombin-proteinase complex formation.
AuthorsOlson ST, Stephens AW, Hirs CH, Bock PE, Björk I
JournalJ Biol Chem
PubMed ID7730349
'To elucidate the role of the P1' residue of the serpin, antithrombin (AT), in proteinase inhibition, the source of the functional defect in a natural Ser-394-->Leu variant, AT-Denver, was investigated. AT-Denver inhibited thrombin, Factor IXa, plasmin, and Factor Xa with second order rate constants that were 430-, 120-, 40-, and ... More
Surface-induced alterations in the kinetic pathway for cleavage of human high molecular weight kininogen by plasma kallikrein.
AuthorsTayeh MA, Olson ST, Shore JD
JournalJ Biol Chem
PubMed ID8206938
'We have studied the cleavage of human high molecular weight kininogen (HK) by plasma kallikrein in the absence and presence of the surfaces, dextran sulfate (DxSO4) and sulfatides. Using a combination of SDS-polyacrylamide gel electrophoresis, Western blotting with polyclonal antibodies that specifically recognize the COOH terminus of the bradykinin moiety, ... More
Sensitive assay for plasminogen activator of transformed cells.
'A sensitive in situ assay for the plasminogen activator of transformed cells is described; it uses the fluorogenic molecule 3'',6''-bis(4-guanidinobenzoyloxy)-5-(N''-4-carboxylphenyl)thioureidospiro[isobenz ofuran-1(3H),9''-[9H]xanthen]-3-one. This fluorescein derivative is an excellent active-site titrant of the esterase activity of plasmin. When transformed cells are incubated with purified plasminogen and the resulting plasmin is titrated with ... More
A fluorogenic active-site titrant for serine proteases.
AuthorsGreen DP
JournalAnal Biochem
PubMed ID3893218
The synthesis of 3'-(4-guanidinobenzoyloxy)-spiro(isobenzofuran-1(3H),9H-[9H]xan then]-3-one or 3HFGB, an active-site titrant for serine proteases, is described. 3HFGB has two advantages over previously described titrants. It is very sensitive (it can measure serine proteases at the 10(-11)-10(-12) M level), and it can be used at stoichiometric ratios of titrant to protease of ... More
Further inhibition studies on guanidinobenzoatase, a trypsin-like enzyme associated with tumour cells.
AuthorsSteven FS, Griffin MM, Wong TL, Yasmin R, Mangel WF
JournalJ Enzyme Inhib
PubMed ID3334244
Guanidinobenzoatase is a proteolytic enzyme capable of degrading fibronectin and is a tumour associated enzyme. Guanidinobenzoatase has been shown to be an arginine selective protease and is distinct from trypsin, plasminogen activator, plasmin, thrombin and a newly described tumour associated enzyme specific for guanidino phenylalanine residues. These conclusions have been ... More
Isolation and characterization of gastric trypsin from the microsomal fraction of porcine gastric antral mucosa.
AuthorsJeohn GH, Serizawa S, Iwamatsu A, Takahashi K
JournalJ Biol Chem
PubMed ID7782340
A gastric serine protease(s) was found in porcine gastric antral mucosa and was shown to be distributed in the endoplasmic reticulum (ER)-microsome fraction and also in the vesicle fraction. Two forms of the protease were purified over 6,000-fold from the ER-microsome fraction. Analyses of various molecular and enzymatic characteristics including ... More
Synthesis and characterization of a new fluorogenic active-site titrant of serine proteases.
The molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-[N'-(4-carboxyphenyl)thioureido[spirop]isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue of p-nitrophenyl p-guanidino-benzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of trypsin in phosphate-buffered saline, ... More
Isolation of human blood coagulation alpha-factor Xa by soybean trypsin inhibitor-sepharose chromatography and its active-site titration with fluorescein mono-p-guanidinobenzoate.
AuthorsBock PE, Craig PA, Olson ST, Singh P
JournalArch Biochem Biophys
PubMed ID2774557
A method based on active-site affinity chromatography on soybean trypsin inhibitor (SBTI)-Sepharose was developed for isolation of human factor Xa in primarily the undergraded alpha-form. The chromatography procedure separated factor Xa from factor X, the Russel's viper venom proteinase used to activate factor X, and traces of contaminating thrombin. alpha-Factor ... More
Role of the catalytic serine in the interactions of serine proteinases with protein inhibitors of the serpin family. Contribution of a covalent interaction to the binding energy of serpin-proteinase complexes.
AuthorsOlson ST, Bock PE, Kvassman J, Shore JD, Lawrence DA, Ginsburg D, Björk I
JournalJ Biol Chem
PubMed ID8530403
The contribution of a covalent bond to the stability of complexes of serine proteinases with inhibitors of the serpin family was evaluated by comparing the affinities of beta-trypsin and the catalytic serine-modified derivative, beta-anhydrotrypsin, for several serpin and non-serpin (Kunitz) inhibitors. Kinetic analyses showed that anhydrotrypsin had little or no ... More
A binding site for heparin in the apple 3 domain of factor XI.
AuthorsHo DH, Badellino K, Baglia FA, Walsh PN
JournalJ Biol Chem
PubMed ID9632702
Since heparin potentiates activated factor XI (FXIa) inhibition by protease nexin-2 by providing a template to which both proteins bind (Zhang, Y., Scandura, J. M., Van Nostrand, W. E., and Walsh, P. N. (1997) J. Biol. Chem. 272, 26139-26144), we examined binding of factor XI (FXI) and FXIa to heparin. ... More