Ligand preferences of kringle 2 and homologous domains of human plasminogen: canvassing weak, intermediate, and high-affinity binding sites by 1H-NMR.

The interaction of various small aliphatic and aromatic ionic ligands with the human plasminogen (HPg) recombinant kringle 2 (r-K2) domain has been investigated by 1H-NMR spectroscopy at 500 MHz. The results are compared against ligand-binding properties of the homologous, lysine-binding HPg kringle 1 (K1), kringle 4 (K4), and kringle 5 (K5). The investigated ligands include the omega-aminocarboxylic acids 4-aminobutyric acid (4-ABA), 5-aminopentanoic acid (5-APA), 6-aminohexanoic acid (6-AHA), 7-aminoheptanoic acid (7-AHA), lysine and arginine derivatives with free and blocked alpha-amino and/or carboxylate groups, and a number of cyclic analogs, zwitterions of similar size such as trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA), and the nonzwitterions benzylamine and benzamidine.

Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth.

Angiostatin is a potent angiogenesis inhibitor which has been identified as an internal fragment of plasminogen that includes its first four kringle modules. We have recently demonstrated that the anti-endothelial cell proliferative activity of angiostatin is also displayed by the first three kringle structures of plasminogen and marginally so by kringle 4 (Cao, Y., Ji, R.

Role of angiotensin II in the renal effects induced by nitric oxide and prostaglandin synthesis inhibition.

The objective of this study was to examine the renal effects of changes in intrarenal angiotensin II levels during the administration of a cyclooxygenase inhibitor, when nitric oxide synthesis is reduced. In the first group of dogs, the administration of meclofenamate and a subpressor dose of L-NAME induced an increase (P < 0.05) in arterial pressure (14 +/- 2 mm Hg), a decrease (P < 0.

Nitric oxide synthase activity in renal cortex and medulla of normotensive and spontaneously hypertensive rats.

The medullary portion of the kidney plays a crucial role in the control of sodium and water excretion and arterial pressure. This control is anomalous in hypertension and may be related to an impaired renal nitric oxide (NO) production. We have measured the activity of NO synthase (NOS) in the renal medulla, renal cortex, heart, and aorta from normotensive Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR).

Kringle domains of human angiostatin. Characterization of the anti-proliferative activity on endothelial cells.

Recently we have identified angiostatin, an endogenous angiogenesis inhibitor of 38 kDa which specifically blocks the growth of endothelial cells (O'Reilly, M. S., Holmgren, L.

Effects of verapamil on the renal actions induced by nitric oxide and prostaglandin synthesis inhibition.

This study was designed to determine the effects of a calcium antagonist (verapamil) on the renal actions induced by nitric oxide synthesis inhibition, with and without simultaneous prostaglandin synthesis inhibition. The renal effects of verapamil (2 micrograms/kg/min) were examined in anesthetized dogs before and after an increase of extracellular volume and during the reduction of nitric oxide synthesis (1 microgram/kg/min NG-nitro-L-arginine methyl ester [L-NAME]), with and without the administration of a cyclooxygenase inhibitor (5 micrograms/kg/min meclofenamate). Nitric oxide synthesis inhibition produced an increase in proximal sodium reabsorption (lithium clearance technique) and a decrease in the excretory response to volume expansion that was prevented by the administration of verapamil.

Complexation of the tissue plasminogen activator protease with benzamidine-type inhibitors: interference by the kringle 2 module.

Well-resolved high-field 1H NMR signals between -0.1 and -0.7 ppm afford convenient probes to monitor the conformational state of the tissue plasminogen activator (tPA) protease, modulated by covalent inhibitor binding or activation cleavage [Hu, C.

Recombinant gene expression and 1H NMR characteristics of the kringle (2 + 3) supermodule: spectroscopic/functional individuality of plasminogen kringle domains.

The plasminogen kringle 2 (K2HPg) and kringle 3 (K3HPg) modules occur in tandem within the polypeptide segment that affords the heavy chain of plasmin. The K2HPg and K3HPg are unique among the plasminogen kringle domains in that they also are linked to each other via the Cys169-Cys297 (Cys4 of K2HPg to Cys43 of K3HPg, kringle numbering convention) disulfide bridge, thus generating a K2HPg-K3HPg "supermodule". The kringle (2 + 3) sequence of human plasminogen (r-EE[K2HPgK3HPg]DS) was expressed in Escherichia coli, using an expression vector containing the phage T5 promoter/operator N250PSN250P29 and the codons for an N-terminal hexahistidine tag to ensure the isolation of the recombinant protein by affinity chromatography on Ni(2+)-nitrilotriacetic acid/agarose under denaturing and reducing conditions.

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.

Interactions between angiotensin and nitric oxide in the renal response to volume expansion.

This study examined, in anesthetized dogs, the possible interactions between nitric oxide (NO) and angiotensin II (ANG II) in mediating the renal response to an extracellular volume expansion (ECVE). It was found that the intrarenal maintenance of ANG II levels (group 1) or the intrarenal NO synthesis inhibition (group 2) did not induce changes in renal hemodynamics but reduced (P < 0.05) the ECVE-induced increments in sodium excretion and fractional lithium excretion (FeLi).

Role of prostaglandins and nitric oxide in mediating renal response to volume expansion.

The objective of the present study was to examine, in anesthetized dogs, the possible interaction between prostaglandins (PG) and nitric oxide (NO) in mediating the renal response to an extracellular volume expansion (ECVE). The renal response to ECVE was examined during 1) intrarenal infusion of a PG synthesis inhibitor, 2) intrarenal administration of a NO synthesis inhibitor, and 3) simultaneous inhibition of PG and NO synthesis in the right kidney. Compared with the control group, the ECVE-induced increments in sodium excretion and fractional excretion of lithium were not affected by the PG synthesis inhibition.

Plasminogen kringle 4 binds the heptapeptide fragment 44-50 of the plasminogen N-terminal peptide.

The interaction between the plasminogen kringle 4 module and a synthetic peptide corresponding to the tryptic heptapeptide fragment Ala-Phe-Gln-Tyr-His-Ser-Lys (AFQYHSK), segment 44-50 of the plasminogen N-terminal peptide (Wiman and Wallén, Eur J Biochem 1975; 50:489-494), has been investigated by 1H-NMR spectroscopy. AFQYHSK, as well as the shorter fragments thereof, FQYHSK, QYHSK and YHSK, all bound to kringle 4 with equilibrium association constant (Ka) values ranging between 2.5 and 8.

Ligand binding to the tissue-type plasminogen activator kringle 2 domain: structural characterization by 1H-NMR.

Ligand binding to a recombinant human tissue-type plasminogen (tPA) kringle 2 domain has been characterized via 1H-NMR spectroscopy at 500 MHz. Seven omega-amino acid ligands were investigated: L-Lys, 6-aminohexanoic acid (6AHA), 7-aminoheptanoic acid (7AHA), trans-(aminomethyl)-cyclohexanecarboxylic acid (AMCHA), p-(aminomethyl)benzoic acid (PAMBA), p-(aminoethyl)benzoic acid (PAEBA), and p-benzylaminesulfonic acid (BASA). The interactions with two peptides containing a C-terminal lysyl residue, Tyr-Leu-Leu-Lys (YLLK) and Ala-Phe-Gln-Tyr-His-Ser-Lys (AFQYHSK), were also studied.

Tissue-type plasminogen activator domain-deletion mutant BM 06.022: modular stability, inhibitor binding, and activation cleavage.

Recombinant BM 06.022 (M(r) 39,589) is a domain-deletion mutant of the human tissue-type plasminogen activator (tPA) structured by the kringle 2 and protease modules. Unfolding under various conditions was investigated via 1H-NMR spectroscopy by monitoring the well-resolved high-field methyl resonances at approximately -0.

Solution structure of the epsilon-aminohexanoic acid complex of human plasminogen kringle 1.

The solution structure of the human plasminogen kringle 1 domain complexed to the antifibrinolytic drug 6-aminohexanoic acid (epsilon Ahx) was obtained on the basis of 1H-NMR spectroscopic data and dynamical simulated annealing calculations. Two sets of structures were derived starting from (a) random coil conformations and (b) the (mutated) crystallographic structure of the homologous prothrombin kringle 1. The two sets display essentially the same backbone folding (pairwise root-mean-square deviation, 0.

1H-NMR assignments and secondary structure of human plasminogen kringle 1.

The 1H-NMR spectrum of the kringle 1 domain of human plasminogen complexed with 6-aminohexanoic acid, an antifibrinolytic drug, has been assigned. Elements of secondary structure have been identified on the basis of sequential, medium and long-range dipolar interactions, back-bone amide spin-spin couplings (3JHN-H alpha) and 1H-2H exchange rates. The kringle contains scarcely any repetitive secondary structure: eight reverse turns and two short beta-sheets.

Solution structure of the kringle domain from urokinase-type plasminogen activator.

The solution structure of the kringle domain from urokinase-type plasminogen activator (u-PA) has been determined using 1H nuclear magnetic resonance spectroscopy and dynamical simulated annealing calculations. A total of 35 structures, 20 generated using a distance geometry method prior to simulated annealing and 15 generated using initial random phi, psi values, have been calculated based on 946 experimental nuclear Overhauser effect distance constraints and 48 dihedral angle constraints. Excluding the N- and C-terminal residues (-1 to 12, 77 to 82) and a number of surface residues (M18, G19, S42, D55 to R60, G67) that are disordered or flexible, the root mean square deviation values from the mean structure are 0.

Kringle solution structures via NMR: two-dimensional 1H-NMR analysis of horse plasminogen kringle 4.

The kringle 4 domain of equine plasminogen (ePgn/K4), a close variant of the human homolog (hPgn/K4), contains residues, such as Trp32, which also appear in human apolipoprotein(a) kringle 4-type modules. The ePgn/K4 was investigated as a complex with epsilon-aminocaproic acid, an antifibrinolytic drug, by two-dimensional 1H-NMR spectroscopy at 500 MHz. Secondary structure elements were recognized from sequential medium and long-range dipolar (proton Overhauser) interactions, as well as from the identification of resonances originating from backbone amide protons with slow 1H-2H exchange in 2H2O.

1H NMR characterization of the urokinase kringle module. Structural, but not functional, relatedness to homologous domains.

The human urokinase (uPA) kringle (K) domain has been characterized via high resolution NMR spectroscopy. The 1H spectrum is analogous to that of the K2 domain of tissue-type plasminogen activator (tPA) and other homologous domains from the plasminogen (Pgn) heavy chain. This indicates a similar folding for the uPA/K.

The two polypeptide chains in fibronectin are joined in antiparallel fashion: NMR structural characterization.

The fibronectin C-terminal interchain disulfide-linked heptapeptide dimer (Val-Asn-Cys-Pro-Ile-Glu-Cys)2 has been investigated via 1H NMR spectroscopy in both water and dimethyl sulfoxide (DMSO) solutions. Proton Overhauser experiments in DMSO indicate unambiguously that the two fibronectin polypeptide chains are linked head-to-tail (N-terminus to C-terminus), in an antiparallel fashion. It is found that the structure of the peptide is extended.

Heparin binding to the urokinase kringle domain.

The binding of urokinase to immobilized heparin and dextran sulfate was studied using activity assays of the bound urokinase. The markedly higher binding observed with high M(r) urokinase compared to low M(r) urokinase indicated a role for the amino-terminal fragment (ATF). This was confirmed by the use of inactive truncated urokinase and monoclonal antibodies specific for the ATF in competition assays of urokinase binding.

1H-n.m.r. studies of the fibronectin 13 kDa collagen-binding fragment. Evidence for autonomous conserved type I and type II domain folds.

A 1H-n.m.r.

Refined solution structure and ligand-binding properties of PDC-109 domain b. A collagen-binding type II domain.

We have determined, via 1H-n.m.r.

Solution structure of the tissue-type plasminogen activator kringle 2 domain complexed to 6-aminohexanoic acid an antifibrinolytic drug.

The solution structure of a recombinant tissue-type plasminogen activator kringle 2 domain, complexed with the antifibrinolytic drug 6-aminohexanoic acid (6-AHA) was determined via 1H nuclear magnetic resonance spectroscopy and dynamical simulated annealing calculations. The structure determination is based on 610 intramolecular kringle 2 and 14 intermolecular kringle 2-6-AHA interproton distance restraints, as well as on 82 torsion angle restraints. Three sets of simulated annealing structures were computed from three different classes of starting structures: (1) random conformations devoid of disulfide bridges; (2) random conformations that contain correct disulfide bonds; and (3) a folded conformation modeled after the homologous prothrombin kringle 1 X-ray crystallographic structure.

Ligand specificity of human plasminogen kringle 4.

The ligand specificity of the human plasminogen kringle 4 was characterized in terms of ligand size, aromatic/aliphatic character, and ionic charge distribution. The binding of the following ligands was investigated via 1H NMR spectroscopy, and their equilibrium association constants (Ka) were determined: (1) p-aminomethylbenzoic acid (Ka approximately 4.8 mM-1), (2) benzylamine (Ka approximately 0.