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PubMed1992

Requirement of zymogen modification for activation of porcine plasminogen.

In physiological salt solutions, porcine plasminogen is refractory to activation by urokinase or trypsin and to proteolysis at Lys77 by plasmin or trypsin. Plasminogen becomes a substrate for urokinase (at Arg560), plasmin (at Lys77), and trypsin (at both bonds) if chloride ion is removed or if 6-aminohexanoate (2.5 mmol/L) is added. Irrespective of salts, activation of des(1-77)plasminogen is as efficient as activation of des(kringle1-4)plasminogen and is inhibited 50% by 2.5 mmol/L 6-aminohexanoate. In solutions lacking chloride or containing 6-aminohexanoate, plasminogen, des(1-77)plasminogen, and des(kringle1-4)plasminogen show no tendency to saturate urokinase in physiologically relevant concentrations (10 mumol/L). The findings are interpreted as indicating that plasminogen requires modification, either by proteolysis or by ligands, for activation.

Amino Acid Sequence↗
PubMed1993

Comparison of the lysine binding functions of lipoprotein(a) and plasminogen.

Regions of apoprotein(a) of lipoprotein(a) [Lp(a)] exhibit striking primary sequence homology to the kringles of plasminogen. The kringles of plasminogen are lysine binding structures and mediate interactions of plasmin(ogen) with substrates and inhibitors. In the current study, the lysine binding properties of Lp(a) have been compared to those of plasminogen and isolated kringle 4 of plasminogen (K4). An analytical assay was implemented to quantitate the interaction of kringle-containing molecules with lysine-Sepharose beads. Radioiodinated ligands, Lp(a), plasminogen, and K4, bound to the beads, and their interactions were inhibited by lysine analogues in a dose-dependent fashion. A series of omega-aminocarboxylic acids inhibited Lp(a), plasminogen, and K4 binding to the lysine-Sepharose beads, but marked differences in the effectiveness of these compounds were observed with each ligand. In this series of compounds, 6-aminohexanoic acid was the most potent inhibitor of binding to lysine-Sepharose for all three ligands. The pH had little effect on the inhibition of plasminogen binding by these compounds. For Lp(a), a low pH caused a marked decrease in inhibition by the 5-carbon and 4-carbon omega-amino acids. In addition, tranexamic acid was 750-fold more potent than lysine in inhibiting plasminogen and 55-fold more potent for K4 binding to the beads. In contrast, the differential potency of these compounds on Lp(a) binding was only 3-fold. These results suggest that the kringles of Lp(a) possess lysine binding functions which are similar, but not identical, to those of plasminogen and its K4.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1994

Synthesis of L-thiocitrulline, L-homothiocitrulline, and S-methyl-L-thiocitrulline: a new class of potent nitric oxide synthase inhibitors.

Nitric oxide synthase catalyzes the NADPH- and O2-dependent conversion of L-arginine to L-citrulline and nitric oxide. L-Thiocitrulline, L-homothiocitrulline, and S-methyl-L-thiocitrulline, novel citrulline analogs, have been synthesized and are shown to be potent inhibitors of both the constitutive brain and the inducible smooth muscle isoforms of nitric oxide synthase. Although many N omega-monosubstituted arginine derivatives inhibit nitric oxide synthase, inhibitory citrulline derivatives have not previously been reported. S-Methyl-L-thiocitrulline is significantly more potent than N omega-methyl-L-arginine, the prototypic nitric oxide synthase inhibitor.

Amino Acid Oxidoreductases↗
PubMed1990

Inhibition of plasmin by fibrinogen.

The kinetics of inhibition of the amidolytic activity of plasmin on D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251) by fibrinogen and fibrin were determined. Reciprocal (1/v versus 1/[S]) plots of plasmin inhibition by 0.50 microM-fibrinogen showed a non-linear downward curve. The Hill coefficient (h) was 0.68, suggesting negative co-operativity. By contrast, fibrin produced a simple competitive inhibition of plasmin (Ki = 12 micrograms/ml). Addition of 0.1 mM-6-aminohexanoic acid shifted the non-linear curve obtained in the presence of fibrinogen to a straight line as for controls, indicating that 6-aminohexanoic acid abolishes the fibrinogen-induced inhibition. Transient exposure of the enzyme to pH 1.0 abrogates the ability of fibrinogen to inhibit plasmin activity. Acidification had no effect on the Vmax but increased the Km of plasmin. The present evidence for modulation of plasmin reveals a novel mechanism for control of fibrinolysis by fibrinogen, a component of the coagulation system and the precursor of the physiological substrate of plasmin.

Amino Acid Sequence↗
PubMed1998

Evidence that the conformation of unliganded human plasminogen is maintained via an intramolecular interaction between the lysine-binding site of kringle 5 and the N-terminal peptide.

Human Glu-plasminogen adopts at least three conformations that provide a means for regulating the specificity of its activation in vivo. It has been proposed previously that the closed (alpha) conformation of human Glu-plasminogen is maintained through physical interaction of the kringle 5 domain and a lysine residue within the N-terminal peptide (NTP). To examine this hypothesis, site-directed mutagenesis was used to generate variant proteins containing substitutions either for aspartic acid residues within the anionic centre of the kringle 5 domain or for conserved lysine residues within the NTP. Size-exclusion HPLC and rates of plasminogen activation by urokinase-type plasminogen activator were used to determine the conformational states of these variants. Variants with substitutions within the kringle 5 lysine-binding site demonstrated extended conformations, as did variants with alanine substitutions for Lys50 and Lys62. In contrast, molecules in which NTP residues Lys20 or Lys33 were replaced were shown to adopt closed conformations. We conclude that the lysine-binding site of kringle 5 is involved in maintaining the closed conformation of human Glu-plasminogen via an interaction with the NTP, probably through Lys50 and/or Lys62. These conclusions advance the current model for the initial stages of fibrinolysis during which fibrin is thought to compete with the NTP for the kringle 5 lysine-binding site.

Amino Acid Substitution↗
PubMed2005

X-ray crystallographic analysis of 6-aminohexanoate-dimer hydrolase: molecular basis for the birth of a nylon oligomer-degrading enzyme.

6-Aminohexanoate-dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII') that has only approximately 0.5% of the specific activity toward the 6-aminohexanoate-linear dimer, are encoded on plasmid pOAD2 of Arthrobacter sp. (formerly Flavobacterium sp.) KI72. Here, we report the three-dimensional structure of Hyb-24 (a hybrid between the EII and EII' proteins; EII'-level activity) by x-ray crystallography at 1.8 A resolution and refined to an R-factor and R-free of 18.5 and 20.3%, respectively. The fold adopted by the 392-amino acid polypeptide generated a two-domain structure that is similar to the folds of the penicillin-recognizing family of serine-reactive hydrolases, especially to those of d-alanyl-d-alanine-carboxypeptidase from Streptomyces and carboxylesterase from Burkholderia. Enzyme assay using purified enzymes revealed that EII and Hyb-24 possess hydrolytic activity for carboxyl esters with short acyl chains but no detectable activity for d-alanyl-d-alanine. In addition, on the basis of the spatial location and role of amino acid residues constituting the active sites of the nylon oligomer hydrolase, carboxylesterase, d-alanyl-d-alanine-peptidase, and beta-lactamases, we conclude that the nylon oligomer hydrolase utilizes nucleophilic Ser(112) as a common active site both for nylon oligomer-hydrolytic and esterolytic activities. However, it requires at least two additional amino acid residues (Asp(181) and Asn(266)) specific for nylon oligomer-hydrolytic activity. Here, we propose that amino acid replacements in the catalytic cleft of a preexisting esterase with the beta-lactamase fold resulted in the evolution of the nylon oligomer hydrolase.

Amidohydrolases↗
PubMed1990

The synthesis of polyamide-oligonucleotide conjugate molecules.

We have developed methods for the synthesis of peptide-oligodeoxyribonucleotide conjugate molecules in particular, and polyamide-oligonucleotide conjugates in general. Synthesis is carried out by a solid-phase procedure and involves the assembly of a polyamide on the solid support, conversion of the terminal amino group to a protected primary aliphatic hydroxy group by reaction with alpha, omega-hydroxycarboxylic acid derivatives, and finally oligonucleotide synthesis using phosphoramidite chemistry. The conjugate molecules can be used as DNA probes, with the polyamide component carrying one or more non-radioactive markers. These conjugates also have the potential to be used as anti-sense inhibitors of gene expression, with the peptide segment acting as a targeting moiety.

Amino Acid Sequence↗
PubMed1995

Domain-domain interactions in hybrids of tissue-type plasminogen activator and urokinase-type plasminogen activator.

Fibrin-dependent plasminogen activation by tissue-type plasminogen activator (t-PA) is in part associated with the presence of the kringle 2 domain in t-PA. Within this kringle 2 domain a lysyl-binding site has been described. The plasminogen to plasmin conversion by urokinase-type plasminogen activator (u-PA), in contrast to that of t-PA, is not enhanced in the presence of fibrin. Within the u-PA kringle domain no lysyl-binding site is found. To study whether introduction of a lysyl-binding site in the u-PA kringle domain will make u-PA a fibrin-dependent plasminogen activator, three stretches of amino acid residues of the u-PA kringle domain (A28-Q33, D55-N57 and G67-V72) were substituted by three stretches of amino acids from the corresponding positions of the kringle 2 domain of t-PA (M28-K33, D55-D57 and N67-W72). These changes resulted in the creation of the lysyl-binding site consensus of the kringle 2 domain (K33, D55, D57, W62 and W72) in the u-PA kringle. However, the resulting u-PA mutant did not interact with lysyl-Sepharose, nor did it display fibrin-enhanced plasminogen activation in the presence of soluble fibrin mimic. When the kringle domain of u-PA was replaced by the kringle 2 domain of t-PA, similar results were obtained. The hybrid protein hardly interacted with lysyl-Sepharose and the plasminogen activation was not enhanced in the presence of fibrin mimic. However, the N-terminal fragment isolated from this hybrid molecule (consisting of growth factor domain and kringle 2 domain) did interact with lysyl-Sepharose, suggesting that in the hybrid molecule a functional lysyl-binding site is present but not operational. Indeed, lysine analogue (epsilon-amino-caproic acid) sensitive binding of isolated t-PA kringle 2 domain to u-PA could be observed. The modified u-PA kringle, the wild type u-PA kringle and the kringle 2 of the u-PA hybrid were also placed N-terminal of the protease domain of t-PA. As expected, the t-PA mutant consisting of the kringle 2 domain and the protease domain bound to lysyl-Sepharose and showed fibrin-dependent plasminogen activation. Further, the hybrid molecule consisting of the u-PA kringle placed N-terminal of the t-PA protease domain did not display these features. Introduction of the modified u-PA kringle N-terminal of the t-PA protease domain resulted in a very weak interaction with lysyl-Sepharose. Despite the high overall similarity in primary structure of the modified u-PA kringle and t-PA kringle 2 (68%), no fibrin-dependent plasminogen activation of this hybrid molecule was observed. The above-mentioned results question the concept that the structural auto-nomous domains within hybrid plasminogen activators t-PA and u-PA function as autonomous domains and suggest that interactions between the kringle and the protease domain in hybrid molecules strongly influences their functional features.

Amino Acid Sequence↗
PubMed1980

Localization of individual lysine-binding regions in human plasminogen and investigations on their complex-forming properties.

After partial digestion of human plasminogen with elastase, followed by chymotryptic cleavage of one of the fragments produced, two polypeptides with molecular weights of approximately 10 000 and with lysine-binding sites still intact were isolated by means of affinity chromatography and gel filtration. One fragment, which was completely sequenced (88 residues), was identified as the fourth kringle, whereas the other, according to partial sequence analysis represented the first kringle. Equilibrium dialysis against 6-aminohexanoic and yielded for the first kringle one high-affinity binding site (Ka = 60 mM-1) and for the fourth kringle one single low-affinity binding site (Ka = 28 mM-1). Moreover, interactions were detected between the first kringle and the N-terminal CNBr fragment of plasminogen and also fibrin. In these cases an additional lysine-binding site, though of low affinity, appears to be involved. Thus, the first kringle seems to play important roles, structurally by contributing to the maintenance of a compact structure of plasminogen through an intramolecular interaction with its N-terminal polypeptide region, and functionally by increasing the fibrin affinity of Lys-plasminogen (plasminogen lacking the first 76 residues) and plasmin.

Amino Acid Sequence↗
PubMed1989

Baclofen and phaclofen modulate GABA release from slices of rat cerebral cortex and spinal cord but not from retina.

1. The effects of (-)-baclofen, muscimol and phaclofen on endogenous gamma-aminobutyric acid (GABA) release from rat cortical slices, spinal cord slices and entire retinas were studied. 2. The spontaneous resting release of GABA from the three tissues was 3 to 6 pmol mg-1 wet wt 10 min-1. Depolarization of cortical slices with KCl (50 mM) (high-K) produced an 8 fold increase in GABA release but high-K did not evoke an increased release of GABA from spinal slices or retinas. 3. When rats were injected with gamma-vinyl-GABA (250 mg kg-1 i.p.) (GVG) 18 h before death, the tissue GABA stores were increased 3 to 6 fold and high-K then evoked striking Ca-dependent releases of GABA from all three tissues. Thus, in subsequent experiments, unless otherwise stated, the nervous tissues were taken from GVG-treated rats. 4. (-)-Baclofen (10 microM) significantly reduced the K-evoked release of GABA from cortical and spinal slices but retinal release was not affected, even at a concentration of (+/-)-baclofen of 1 mM. For cortical slices, the IC50 for baclofen was approximately 5.2 microM. The inhibitory effect of baclofen on GABA release from cortical slices also occurred in slices prepared from saline-injected rats, indicating that GVG treatment did not qualitatively affect the results. 5. The inhibitory effect of (-)-baclofen on the K-evoked release of GABA from cortical and spinal slices was antagonised by phaclofen (500 microM), confirming that baclofen was producing its effects by acting at the GABAB-receptor. 6. Phaclofen (500 microM) increased the spontaneous resting release of GABA from cortical slices taken from GVG-treated rats but not from saline-injected rats. Phaclofen did not increase GABA release from spinal slices or retinas taken from GVG-treated rats. 7. Baclofen (10 microM) significantly reduced the K-evoked release from cortical slices of glutamate, aspartate, glycine and taurine. 8. Muscimol (10 microM) and delta-aminolaevulinic acid (10 microM) had no effect on either the resting or Kevoked release of GABA from cortical slices prepared from saline-injected or GVG-treated rats. 9. The results obtained with cortical and spinal slices are consistent with the presence of inhibitory GABAB-autoreceptors. The phaclofen-induced increase in GABA release from cortical slices taken from GVG-treated rats, but not from saline-injected rats, implies that under conditions of high GABA release, considerable feedback inhibition is occurring via activation of the GABAB inhibitory autoreceptors. No evidence was found for GABAB-autoreceptors on retinal GABAergic amacrine cells or for GABAA-autoreceptors in cortical slices or spinal cord slices.

Amino Acids↗
PubMed2001

Insertion of an N-terminal 6-aminohexanoic acid after the 7 amino acid position of glucagon-like peptide-1 produces a long-acting hypoglycemic agent.

The use of glucagon-like peptide-1 (GLP-1) as a routine treatment for type 2 diabetes mellitus is undermined by its short biological half-life. A cause of degradation is its cleavage at the N-terminal HAE sequence by the enzyme dipeptidyl peptidase IV (DPP IV). To protect from DPP IV, we have studied the biological activity of a GLP-1 analog in which 6-aminohexanoic acid (Aha) is inserted between histidine and alanine at positions 7 and 8. We have compared the biological activity of this new compound, GLP-1 Aha(8), with the previously described GLP-1 8-glycine (GLP-1 Gly(8)) analog. GLP-1 Aha(8) (10 nM) was equipotent with GLP-1 (10 nM) in stimulating insulin secretion in RIN 1046-38 cells. As with GLP-1 Gly(8), the binding affinity of GLP-1 Aha(8) for the GLP-1 receptor in intact Chinese hamster ovary (CHO) cells expressing the human GLP-1 receptor (CHO/GLP-1R cells) was reduced (IC(50): GLP-1, 3.7 +/- 0.2 nM; GLP-1 Gly(8), 41 +/- 9 nM; GLP-1 Aha(8), 22 +/- 7 nM). GLP-1 Aha(8) was also shown to stimulate intracellular cAMP production 4-fold above basal at concentrations as low as 0.5 nM. However, it exhibited a higher ED(50) when compared to GLP-1 and GLP-1 Gly(8) (ED(50): GLP-1, 0.036 +/- 0.002 nM, GLP-1 Gly(8), 0.13 +/- 0.02 nM, GLP-1 Aha(8), 0.58 +/- 0.03 nM). A series of D-amino acid-substituted GLP-1 compounds were also examined to assess the importance of putative peptidase-sensitive cleavage sites present in the GLP-1 molecule. They had poor binding affinity for the GLP-1 receptor, and none of these compounds stimulated the production of intracellular cAMP in CHO/GLP-1R cells or insulin secretion in RIN 1046-38 cells. GLP-1 Aha(8) (24 nmol/kg) administered sc to fasted Zucker (fa/fa) rats (mean blood glucose, 195 +/- 32 mg/dl) lowered blood glucose levels to a nadir of 109 +/- 3 mg/dl, and it remained significantly lower for 8 h. Matrix-assisted linear desorption ionization-time of flight mass spectrometry of GLP-1 Aha(8) incubated with DPP IV (37 C, 2 h) did not exhibit an N-terminal degradation product. Taken together, these results show that insertion of Aha after the 7 position in GLP-1 produces an effective, long-acting GLP-1 analog, which may be useful in the treatment of type 2 diabetes mellitus.

Amino Acid Sequence↗
PubMed2001

Binding of recombinant apolipoprotein(a) to human platelets and effect on platelet aggregation.

The interaction of lipoprotein(a) [Lp(a)] with platelets is not well defined, particularly with regards to the individual contribution of the protein components of Lp(a), the apo B-100 and the apolipoprotein apo(a). This study investigated the binding of different recombinant apo(a) [r-apo(a)] isoforms, to human platelets and its effect on platelet aggregation. Scatchard analysis of saturation binding experiments demonstrated that human platelets display a single class of high affinity r-apo(a) binding sites (71 +/- 46 molec./platelet, Kd = 5.6 +/- 2.0 nmol/L). Platelet activation with strong agonists (thrombin, arachidonic acid) increased 2- to 10-fold the r-apo(a) binding, without affecting the affinity. Competition assays showed that the binding sites are highly specific for r-apo(a) and Lp(a). At high concentration t-PA could also bind to the r-apo(a) binding sites. By contrast, neither fibrinogen nor plasminogen inhibited to the r-apo(a) binding. The lysine analogue EACA inhibits the binding of r-apo(a) to platelets, thus suggesting the involvement of lysine residues in that interaction. Moreover, the r-apo(a) binding to platelets is unlikely mediated by GPIIb/IIIa-attached fibrin since it is not affected by platelet treatment with either LJ-CP8, a monoclonal antibody that specifically blocks fibrinogen binding to GPIIb/IIIa, nor GPRP, an inhibitor of fibrin polymerisation. Finally, we show that the distinct recombinant apo(a) proteins, as well as native Lp(a), promote an aggregation response of platelets to otherwise subaggregant doses of arachidonic acid. This proaggregant effect of r-apo(a) is dependent on its binding to platelets since it requires a minimum incubation time, and it is prevented by EACA at concentration inhibiting the r-apo(a)-platelet interaction. These results suggest that the prothrombotic action of Lp(a) may be in part mediated by modulating the platelet function through the interaction of its apo(a) subunit with a specific receptor at the platelet surface.

Adenosine Diphosphate↗
PubMed1998

[Host-guest molecule interaction mechanism of hemostatics with liposomes and red blood cells studied with fluorescence polarimetric method].

The supermolecule compounds of adrenobazone, p-aminomethylbenzoic acid, vitamin K1, 6-amino caproic acid with liposomes and red blood cells were studied by fluorescence polarimetric method. The mechanisms of formation of the supermolecule compounds were examined by fluorescence probe of the link of 1,6-dipheny-1,3,5-hexatriene (DPH) with liposomes which were taken as a model of blood cells. The interaction mechanism of hemostatics with red blood cells was described according the quantitative relationship between polarization value (P) and the microviscosity [formula: see text]. The result showed that the acting force between hemostatics and liposomes or that between hemostatics and red blood cells were mainly supermolecular acting force. The acting force between vitamin K1 and cytomembrane is hydrophobic force and those between adrenobazone, p-aminomethylbenzoic acid, or 6-amino caproic acid and cytomembrane are hydrogen bond or electrostatic force. Under the same drug concentration, all of the four haemostatics can reduce the fluidity of the cell membrane, which benefits blood coagulation. The binding ways of hemostatics with red blood cells was also discussed.

4-Aminobenzoic Acid↗
PubMed1992

Comparison of recombinant plasminogen activator inhibitor-1 and epsilon amino caproic acid in a hemorrhagic rabbit model.

A rabbit ear model of blood loss was developed to compare the effects of an active form of recombinant plasminogen activator inhibitor-1 (rPAI-1) with epsilon amino caproic acid (EACA) in antagonizing tissue-type plasminogen activator (r-tPA)-induced blood loss. The antagonism of both rebleeding, which occurs as a result of hemostatic plug degradation, and r-tPA-induced hemorrhage, where rabbits lose approximately 30% of their blood volume, was studied. rPAI-1 (1 mg/kg i.v.) or EACA (70 mg/kg i.v.) antagonized the rebleeding induced by r-tPA (10 micrograms kg-1 min-1) to a similar extent. In the hemorrhagic studies, rPAI-1 effectively antagonized the r-tPA-induced hemorrhage with an ED50 of 3 mg/kg i.v., while the ED50 obtained for EACA was 230 mg/kg i.v. rPAI-1 may be of value in reversing r-tPA-induced blood loss during thrombolytic therapy or in clinical situations where excessive fibrinolysis contributes to bleeding.

Amino Acid Sequence↗