Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli

Journal Article · Sat Aug 25 04:00:00 EDT 1990 · Journal of Biological Chemistry; (USA)

OSTI ID:6530470

Wilhelm, O G; Jaskunas, S R; Vlahos, C J; Bang, N U ^[1]

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1. Eli Lilly and Company, Indianapolis, IN (USA)

The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. (35S)Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis.

• OSTI ID: 6530470
• Journal Information: Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 265:24; ISSN JBCHA; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CHEMICAL COMPOSITION
CHROMATOGRAPHY
CLONING
COAGULANTS
CYSTEINE
DAYS LIVING RADIOISOTOPES
DRUGS
ESCHERICHIA COLI
EVEN-ODD NUCLEI
FIBRIN
FIBRINOLYTIC AGENTS
FRACTIONATION
GENE REGULATION
HEMATOLOGIC AGENTS
HEMOSTATICS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LIPOTROPIC FACTORS
LYSINE
METHIONINE
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PEPTIDES
PLASMINOGEN
POLYPEPTIDES
PROTEINS
RADIOISOTOPES
SCLEROPROTEINS
SEPARATION PROCESSES
STRUCTURE-ACTIVITY RELATIONSHIPS
SULFUR 35
SULFUR ISOTOPES
THIOLS
TRACER TECHNIQUES