Presence Of 6-aminohexanoic Acid Research Articles

Roles of Ruthenium–Support Interactions of Size-Controlled Ruthenium Nanoparticles for the Product Distribution of Fischer–Tropsch Synthesis

A simple aqueous-phase route to the synthesis of ruthenium (Ru) nanoparticles with size in the sub-2-nm regime was prepared by reducing RuCl3 with NaBH4 as a reducing agent in the presence of 6-aminohexanoic acid (AHA) as a capping agent. We found that AHA plays a key role in the formation and distribution of ultrasmall Ru nanoparticles on inorganic supports, with its amine and carboxyl groups serving as a surface-capping agent for the Ru nanoparticles and an anchor to inorganic supports, respectively. The catalytic activity of the size-controlled Ru nanoparticles with sub-2-nm size on various supports, such as TiO2, CeO2, and Vulcan carbon, revealed that a strong metal–support interaction of Ru nanoparticles, especially on carbon support, is responsible for a higher CH4 selectivity during Fischer–Tropsch synthesis.

Skizzle Is a Novel Plasminogen- and Plasmin-binding Protein from Streptococcus agalactiae That Targets Proteins of Human Fibrinolysis to Promote Plasmin Generation

Skizzle (SkzL), secreted by Streptococcus agalactiae, has moderate sequence identity to streptokinase and staphylokinase, bacterial activators of human plasminogen (Pg). SkzL binds [Glu]Pg with low affinity (K(D) 3-16 mum) and [Lys]Pg and plasmin (Pm) with indistinguishable high affinity (K(D) 80 and 50 nm, respectively). Binding of SkzL to Pg and Pm is completely lysine-binding site-dependent, as shown by the effect of the lysine analog, 6-aminohexanoic acid. Deletion of the COOH-terminal SkzL Lys(415) residue reduces affinity for [Lys]Pg and active site-blocked Pm 30-fold, implicating Lys(415) in a lysine-binding site interaction with a Pg/Pm kringle. SkzL binding to active site fluorescein-labeled Pg/Pm analogs demonstrates distinct high and low affinity interactions. High affinity binding is mediated by Lys(415), whereas the source of low affinity binding is unknown. SkzL enhances the activation of [Glu]Pg by urokinase (uPA) approximately 20-fold, to a maximum rate indistinguishable from that for [Lys]Pg and [Glu]Pg activation in the presence of 6-aminohexanoic acid. SkzL binds preferentially to the partially extended beta-conformation of [Glu]Pg, which is in unfavorable equilibrium with the compact alpha-conformation, thereby converting [Glu]Pg to the fully extended gamma-conformation and accelerating the rate of its activation by uPA. SkzL enhances [Lys]Pg and [Glu]Pg activation by single-chain tissue-type Pg activator, approximately 42- and approximately 650-fold, respectively. SkzL increases the rate of plasma clot lysis by uPA and single-chain tissue-type Pg activator approximately 2-fold, confirming its cofactor activity in a physiological model system. The results suggest a role for SkzL in S. agalactiae pathogenesis through fibrinolytic enhancement.

Identification of amino acids in antiplasmin involved in its noncovalent 'lysine-binding-site'-dependent interaction with plasmin.

The lysine-binding-site-mediated interaction between plasmin and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulating the fibrinolytic enzyme system. To identify structures important for its noncovalent interaction with plasmin, we constructed seven single-site mutants of antiplasmin by modifying charged amino acids in the C-terminal part of the molecule. All the variants were expressed in the Drosophila S2 cell system, purified, and shown to form stable complexes with plasmin. A kinetic evaluation revealed that two mutants of the C-terminal lysine (K452E or K452T) did not differ significantly from wild-type antiplasmin in their reactions with plasmin, in either the presence or absence of 6-aminohexanoic acid, suggesting that this C-terminal lysine is not important for this reaction. On the other hand, modification of Lys436 to Glu decreased the reaction rate about fivefold compared with wild-type. In addition, in the presence of 6-aminohexanoic acid, only a small decrease in the reaction rate was observed, suggesting that Lys436 is important for the lysine-binding-site-mediated interaction between plasmin and antiplasmin. Results from computerized molecular modelling of the C-terminal 40 amino acids support our experimental data.

Effect of phospholipase A2 digestion on the conformation and lysine/fibrinogen binding properties of human lipoprotein[a

In vitro hydrolysis of human lipoprotein[a] (Lp[a]) by phospholipase A2 (PLA2) decreased the phosphatidylcholine (PC) content by 85%, but increased nonesterified fatty acids 3.2-fold and lysoPC 12.9-fold. PLA2-treated Lp[a] had a decreased molecular weight, increased density, and greater electronegativity on agarose gels. In solution, PLA2-Lp[a] was a monomer, and when assessed by sedimentation velocity it behaved like untreated Lp[a], in that it remained compact in NaCl solutions but assumed the extended form in the presence of 6-amino hexanoic acid, which was shown previously to have an affinity for the apo[a] lysine binding site II (LBS II) comprising kringles IV5–8. We interpreted our findings to indicate that PLA2 digestion had no effect on the reactivity of this site. This conclusion was supported by the results obtained from lysine Sepharose and fibrinogen binding experiments, in the presence and absence of Tween 20, showing that phospholipolysis had no effect on the reactivity of the LBS-II domain. A comparable binding behavior was also exhibited by the free apo[a] derived from each of the two forms of Lp[a]. We did observe a small increase in affinity of PLA2-Lp[a] to lysine Sepharose and attributed it to changes in reactivity of the LBS I domain (kringle IV10) induced by phospholipolysis. In conclusion, the extensive modification of Lp[a] caused by PLA2 digestion had no significant influence on the reactivity of LBS II, which is the domain involved in the binding of apo[a] to fibrinogen and apoB-100. These results also suggest that phospholipids do not play an important role in these interactions.—Fless, G. M., E. W. Kirk, O. Klezovitch, J. Y. Santiago, C. Edelstein, J. Hoover-Plow, and A. M. Scanu. Effect of phospholipase A2 digestion on the conformation and lysine/fibrinogen binding properties of human lipoprotein[a]. J. Lipid Res. 1999. 40: 583–592.

Lipoprotein(a) isoforms display differences in affinity for plasminogen-like binding to human mononuclear cells.

Binding of lipoprotein(a) (Lp(a)) to membrane proteins of the monocyte-macrophage cell lineage may be an important event in atheroma formation. Since Lp(a) with distinct apolipoprotein(a) (apo(a)) isoforms may show differences in their affinity with regard to fibrin binding, the existence of such a functional behavior in the interaction of apo(a) in Lp(a) with these cells was explored using the monocytic cell line THP-1. Lp(a) preparations containing small size apo(a) isoforms (M(r) = 450,000 to 550,000) and high molecular mass isoforms (M(r) > or = 700,000) were purified from plasmas containing > 0.35 g/L of Lp(a) obtained from subjects (n = 14) with cardiovascular atherosclerotic disease. Binding of plasminogen to THP-1 cells was performed using the method of radioisotopic dilution. For binding of Lp(a) to cells, the THP-1 monocytic cells were incubated with varying concentrations of the different Lp(a) preparations; cells were then washed and the amount of Lp(a) bound was detected with a radiolabeled polyclonal antibody directed against apo(a). Binding due to kringle interactions with lysine residues was calculated by subtracting from the total bound the amount of Lp(a) bound (approximately 10%) in the presence of 6-aminohexanoic acid. Analysis of data with the Langmuir equation indicated identical and independent (non-interacting) sites and allowed evaluation of the Kd. Binding isotherms of small size isoforms showed saturation and a high affinity (Kd = 25.8 +/- 19 nmol/L) relative to that of plasminogen (Kd = 1750 +/- 760 nmol/L). A similar difference (Kd = 17.5 +/- 7.9 nmol/L versus Kd = 600 +/- 220 nmol/L) was found when binding experiments were performed with a fibrin surface. In contrast, binding isotherms of the high molecular mass isoforms did not show saturation at the highest Lp(a) concentrations used, thus indicating a lower affinity. In conclusion, these results show that apo(a) isoforms may display polymorphism-linked functional heterogeneity with regard to cell binding, which may explain the higher association with cardiovascular risk of small size isoforms. These qualitative differences in the binding of apo(a) isoforms to fibrin or cells may modulate the cardiovascular risk associated with high levels of Lp(a).

Enhancement of t-PA-mediated plasminogen activation by bacterial surface receptors

Summary A total of 63 strains representing 7 species of plasminogen binding bacteria, were examined for the ability to potentiate t-PA and urokinase (u-PA) mediated activation of human plasminogen to plasmin. Tested species included β-haemolytic streptococci groups A, C, and G, Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae and Proteus mirabilis. The effect on plasminogen activation was measured using a coupled enzymatic assay with a chromogenic substrate. The changes in absorbance were found to be linear functions of t2 in agreement with theoretical expectations. Most plasminogen binding strains were capable to dramatically enhance the rate of t-PA induced plasminogen activation. The most pronounced potentiation was observed with a Streptococcus equisimilis strain which increased the activation rate 130-fold. High potentiations was also detected for N. meningitidis and S. pneumoniae strains. P. mirabilis was an exception as some strains were capable to bind plasminogen without any influence on plasminogen activation. Also, u-PA mediated plasminogen activation was enhanced in the presence of bacteria. However the stimulation factor obtained were in most strains much less than those obtained with t-PA. For different strains within a species, the capability to enhance the rate of the t-PA mediated activation correlated with plasminogen binding (r=0.74–0.92). Both plasminogen binding and the stimulation of plasminogen activation were abolished in the presence of 6-aminohexanoic acid (6-AHA, ɛ-aminocaproic acid) suggesting that the lysine-binding sites in the plasminogen molecule are involved in both phenomena. The binding of plasminogen and facilitation of its activation provide bacteria with an active proteolytic shield of possible importance in tissue penetration and infection.

Conformation of Lys-plasminogen and the kringle 1-3 fragment of plasminogen analyzed by small-angle neutron scattering

Native human Glu-plasminogen (Glu1-Asn791) was previously shown to have a radius of gyration of 39 A and a shape best described by a prolate ellipsoid [Mangel, W. F., Lin, B., & Ramakrishnan, V. (1990) Science 248, 69-73]. Upon occupation of a weak lysine-binding site, the shape reversibly changes to that best described by a Debye random coil with a radius of gyration of 56 A. Conversion from the closed to the open form is not accompanied by any change in secondary structure, hence the closed conformation is formed by interaction between domains, the five kringles and the protease domain, and this is abolished upon conversion to the open form. Here we analyzed by small-angle neutron scattering the conformations of human Lys-plasminogen (Lys78-Asn791) and the fragment K1-3 that contains the first three kringles of plasminogen (Tyr80-Val338 or Tyr80-Val354). The shape of Lys-plasminogen was best described by a Debye random coil with a radius of gyration of 51 A, and occupation of its lysine-binding sites by 6-aminohexanoic acid did not dramatically alter its conformation. Thus Lys-plasminogen was in the open form, similar to that of Glu-plasminogen with its lysine-binding sites occupied. The fragment K1-3 in the absence or presence of 6-aminohexanoic acid had a shape best described equally either by an elongated prolate ellipsoid or by a Debye random coil, with a radius of gyration of 29 A. Our model for the two forms of plasminogen is that, in the closed form, domain interaction generates a compact, almost globular, structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a continuing accumulation of APSAC (Anisoylated plasminogen streptokinase activator complex) by human clots: Comparison with the binding of other plasminogen activators and Plasminogen

The uptake of [ 125I-plasminogen]-APSAC into crosslinked human plasma clots from autologous plasma was significantly greater than the uptake of equimolar iodinated lys77-plasminogen. The uptake of APSAC in the presence of 6-aminohexanoic acid (10 mM) was also significantly greater than the uptake of plasminogen in the presence of 6-aminohexanoic acid. The accumulation of APSAC continued for at least 2 h (in nonlysing conditions), was similar in platelet-rich, platelet-poor and whole-blood clots and was not affected by prior aging of the clots (up to 6 h). The uptakes of APSAC and streptokinase plasminogen were slightly affected by the plasma concentration of anti-SK IgG and, in general, the fibrin-binding of APSAC was similar to the binding of radiolabelled t-PA. Urokinase (high-molecular weight) did not demonstrate fibrin- binding, the uptake being explained by simple diffusion (quantified using albumin). The relative order of continuing accumulation APSAC = t-PA > lys-plasminogen > urokinase is similar to the order of initial binding (< 1 min incubation), and we conclude that the formation of an acylated, stabilised, activator complex of lys-plasminogen and streptokinase imparts additional fibrin-binding to lys-plasminogen.

Characterization of the high-affinity interaction between human plasminogen and pro-urokinase.

Activation of human Glu-plasminogen, Lys-plasminogen and low-Mr plasminogen (lacking lysine-binding sites) by pro-urokinase (pro-UK), obtained from a human lung adenocarcinoma cell line (Calu-3, ATCC), obeys Michaelis-Menten kinetics. Activation occurs with a comparable affinity (Km 0.40-0.77 microM), while the catalytic rate constant (kcat) is comparable for Glu-plasminogen (0.0022s-1) and low-Mr plasminogen (0.0034 s-1), but is somewhat higher for Lys-plasminogen (0.0106 s-1). The rate of activation of plasminogen by pro-UK is not significantly influenced by the presence of 6-aminohexanoic acid, purified fragments LBS I or LBS II or histidine-rich glycoprotein, indicating that the high affinity of pro-UK for plasminogen is not mediated via the high-affinity lysine-binding site of plasminogen located in kringles 1-3 (LBS I) nor via the low-affinity lysine-binding site comprised within kringle 4 (LBS II). The site(s) in plasminogen involved in the high-affinity interaction with pro-UK thus appear to be located within the low-Mr plasminogen moiety.

Fluorescence polarization and spectropolarimetric studies on the conformational changes induced by ω-aminoacids in two isozymes of glu-plasminogen (I and II)

Conformational changes of two isozymes of Glu-plasminogen(Glu-plg I and II) induced by ω-aminoacids were studied by using fluorescence polarization and spectropolarimetry. The rotational relaxation times ( P h ) of FITC labeled Glu-plg I and II decreased in the presence of 6-aminohexanoic acid (6AHA) or tranexamic acid (t-x), which may mean increase in Brownian motion of FITC labeled region (possibly N-terminal region) of Glu-plg I and II when 6AHA or t-x binds with lysine binding sites (LBS) of these plasminogens. Glu-plg II seems to have longer rotational relaxation time compared to that of Glu-plg I, which may mean smaller extent of Brownian motion of FITC labeled region of Glu-plg II in comparison to that of Glu-plg I. The far ultraviolet circular dichroism (CD) spectra indicate that there may be some difference in the polypeptide backbone between Glu-plg I and II, possibly more of β-structure and less of random coil structure in Glu-plg II in comparison to Glu-plg I The presence of 6AHA or t-x gave rise to larger change of the negative ellipticity at around 208 nm in Glu-plg I in comparison to its change in Glu-plg II, which may mean the larger extent of conformational change of Glu-plg I induced by 6AHA or t-x than that of Glu-plg II.

Properties of three different molecular forms of the alpha 2 plasmin inhibitor.

The α2plasmin inhibitor prepared by affinity chromatography on plasminogen-Sepharose 4B appears mainly as a molecular form with Mr67000 but also as forms with Mr65000 and 60000. The 67000-Mr and 60000. The 67000-Mrform spontaneously converts to the lower-molecular-mass forms. The rate of conversion is temperature-dependent. All three molecules react with specific immuno globulin against α2plasmin inhibitor. The plasminogen-binding capacity of the 67000-Mr form is lost upon its conversion into the 65000-Mrform, but it has retained its capacity to inhibit plasmin. Upon conversion to the 60000-Mr form the capacity to inhibit plasmin is also lost. The presumed catalytic effect of a copurified serine proteinase on these reactions could not be substantiated since the addition of a number of known serine proteinases and serine proteinase inhibitors did not influence the rate of conversion of the 67000-Mrform to the modified forms. The functional properties of the 65000-Mrnon-plasminogen-binding α2plasmin inhibitor form were compared to those of the 67000-Mrplasminogen-binding form. In a clot lysis assay the plasminogen-binding inhibitor was a more efficient inhibitor of fibrinolysis than the non-plasminogen-binding inhibitor. Plasminogen was displaced from fibrin more readily by the plasminogen-binding inhibitor than by the non-plasminogen-binding form, and activation of Glu-plasminogen by urokinase and tissue activator was more rapid with the plasminogen-binding inhibitor than with the non-plasminogen-binding form. Kinetic studies showed that the plasminogen-binding α2plasmin inhibitor reacted more readily with plasmin than the non-plasminogen-binding form and that the inactivation of plasmin by the former was a biphasic process in the absence of 6-aminohexanoic acid, but monophasic in its presence. Inactivation of plasmin by the non-plasminogen-binding α2plasmin inhibitor was a monophasic reaction both in the absence and the presence of 6-aminohexanoic acid and identical to the reaction with the plasminogen-binding form in the presence of 6-aminohexanoic acid.

Studies on the chemical nature of lysine-binding sites and on their localization in human plasminogen

The isolated ‘kringle’ structures 1 and 4 of human plasminogen lost lysine affinity upon photo-oxidation of histidine, but mostly retained it in the presence of 6-aminohexanoic acid. Lysine affinity was lost and could be partially restored after blocking of histidine with diethylpyrocarbonate and deblocking, or after esterification of COOH-groups and saponification. Only His-31 and most likely Asp-54 qualify as participants in a lysine binding site when the primary structures of the ‘kringles’ are considered.

Effects of α2-Plasmin Inhibitor on Fibrin Clot Lysis. Its Comparison with α2-Macroglobulin

SummaryThe major plasmin inhibitors namely α2-plasmin inhibitor and α2-macroglobulin were compared for their effects on lysis of fibrin clot.Plasmin fibrinolytic activity was immediately inhibited by α2-plasmin inhibitor, whereas α2-macroglobulin inhibited plasmin progressively. Urokinase(plasminogen activator)-induced clot lysis was inhibited efficiently by α2-plasmin inhibitor present in the clot. Inhibition of urokinase-induced clot lysis by α2-macroglobulin was weak and the molar concentration necessary for α2-macroglobulin to achieve the same degree of inhibition as that achieved with α2-plasmin inhibitor was about 10 times higher than that of α2-plasmin inhibitor.Binding of Lys-plasminogen to fibrin was inhibited by α2-plasmin inhibitor but not by α2-macroglobulin. Molar concentrations of α2-plasmin inhibitor which were effective in inhibiting the binding were 30 times less than that of 6-aminohexanoic acid. α2-Plasmin inhibitor was found to interact with Lys-plasminogen to form a weakly-bound complex which is dissociable in the presence of 6-aminohexanoic acid, suggesting that inhibition of binding of Lys-plasminogen to fibrin by α2-plasmin inhibitor may be due to interaction of α2-plasmin inhibitor with a specific site of the plasminogen molecule and that the site may be 6-aminohexanoic acid-binding site.It is suggested that α2-plasmin inhibitor is more reactive and efficient inhibitor of fibrinolysis than α2-macroglobulin.

Differences in the binding to fibrin of native plasminogen and plasminogen modified by proteolytic degradation influence of ω-aminocarboxylic acids

Pretreatment of native plasminogen with plasmin or activators resulted in a pronounced increase in the binding of plasminogen to fibrin. The pretreated plasminogen was considered to be identical to the proteolytically degraded proenzyme with NH 2-terminal lysine, valine or methionine, which is formed as an intermediate stage during activation of plasminogen. Bound plasminogen could be extracted by 6-aminohexanoic acid indicating a reversible binding between plasminogen and fibrin. Adsorption of pretreated plasminogen decreased when increasing concentrations of 6-aminohexanoic acid or trans-4-aminomethylcyclohexane-1-carboxylic acid (t-AMCHA) were present during fibrin formation. The concentration of amino acid producing a decrease in the binding of pretreated plasminogen to 0.5 of the amount bound in the absence of amino acid was 8.0 · 10 −5 M with 6-aminohexanoic acid and 1.7 · 10 −5 M with t-AMCHA. The decrease in binding is most likely related to an effect of the amino acids on plasminogen, since agarose gel electrophoresis of pretreated plasminogen in the presence of 6-aminohexanoic acid or t-AMCHA showed a cathodic shift in mobility at the same range of concentrations of amino acid, which produced the decrease in binding of plasminogen to fibrin. Evidence is provided that the decrease in binding of proteolytically degraded plasminogen may result in an inhibition of fibrinolysis caused by activators.