Bioactive potential of : a study on morphology, phytochemicals, and antioxidant activity.

The present study aimed to investigate the morphological features, phytochemicals, phenolic content, and antioxidant activity in different parts of . The morphological features depicted that the plant is 7.9 ± 1.

Characterization of the protein Z-dependent protease inhibitor interactive-sites of protein Z.

Background: Protein Z (PZ) has been reported to promote the inactivation of factor Xa (FXa) by PZ-dependent protease inhibitor (ZPI) by about three orders of magnitude. Previously, we prepared a chimeric PZ in which its C-terminal pseudo-catalytic domain was grafted on FX light-chain (Gla and EGF-like domains) (PZ/FX-LC). Characterization of PZ/FX-LC revealed that the ZPI interactive-site is primarily located within PZ pseudo-catalytic domain.

Polyphosphate amplifies proinflammatory responses of nuclear proteins through interaction with receptor for advanced glycation end products and P2Y1 purinergic receptor.

The extracellular nuclear proteins, histone H4 (H4) and high mobility group box 1 (HMGB1), released by injured cells during the activation of inflammation and coagulation pathways provoke potent inflammatory responses through interaction with pathogen-related pattern recognition receptors (ie, Toll-like receptors [TLRs] and receptor for advanced glycation end products [RAGE]) present on vascular and innate immune cells. Inorganic polyphosphate (polyP) has emerged as a key modulator of coagulation and inflammation. Here, we demonstrate that polyP binds to both H4 and HMGB1 with high affinity, thereby dramatically potentiating their proinflammatory properties in cellular and in vivo models.

Contribution of the NH2-terminal EGF-domain of factor IXa to the specificity of intrinsic tenase.

Factor IXa (FIXa) is a vitamin K-dependent coagulation serine protease which binds to factor VIIIa (FVIIIa) on negatively charged phospholipid vesicles (PCPS) to catalyse the activation of factor X (FX) to factor Xa (FXa) in the intrinsic pathway. Fluorescence resonance energy transfer (FRET) studies have indicated that the Gla-domain-dependent interaction of FIXa and FX with PCPS in the presence of FVIIIa positions the active-site of the protease at an appropriate height above the membrane surface to optimise the catalytic reaction. In this study, we investigated the contribution of the NH2-terminal EGF-domain (EGF1) of FIXa to the recognition specificity of intrinsic tenase by constructing an EGF1 deletion mutant of FIXa (FIXa-desEGF1) and characterising the properties of the mutant in kinetic, direct binding and FRET assays.

Role of the residues of the 39-loop in determining the substrate and inhibitor specificity of factor IXa.

The activation of antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reactions by 300- to 500-fold. Relative to FXa, AT inhibits FIXa with approximately 40-fold slower rate constant. Structural data suggest that differences in the residues of the 39-loop (residues 31-41) may partly be responsible for the differential reactivity of the two proteases with AT.

Inhibitory properties of the P1 Tyr variant of antithrombin.

Antithrombin (AT) and protein Z-dependent protease inhibitor (ZPI) are among two physiological serpin inhibitors in plasma that are involved in the regulation of the clotting cascade. Unlike AT, which can inhibit the proteolytic activity of all coagulation proteases, ZPI has narrower protease specificity, inhibiting only factors Xa (fXa) and XIa. Unlike an Arg at the P1 site of the AT reactive center loop (RCL), this residue is a Tyr in ZPI.

Role of P2 glycine in determining the specificity of antithrombin reaction with coagulation proteases.

Structural data suggests that bulky hydrophobic residues at the S2-S4 sub-sites of factor Xa (fXa) restrict the preference of this pocket for small and non-polar residues like Gly at the P2 position of substrates and inhibitors. However, kinetic studies monitoring the cleavage specificity of 10-residue peptides by fXa have identified Phe as the most preferred P2 residue and Gln-Phe-Arg-Ser-Leu-Ser as the most preferred P3-P3' residues for recognition by fXa. To determine whether this mechanism of specificity is also true for fXa reaction with antithrombin (AT), we prepared two AT mutants having either a Phe at the P2 or Gln-Phe-Arg-Ser-Leu-Ser at the P3-P3' positions of the reactive center loop.

Mutagenesis studies toward understanding allostery in thrombin.

The binding of thrombomodulin (TM) to exosite-1 and the binding of Na(+) to 225-loop allosterically modulate the catalytic activity and substrate specificity of thrombin. To determine whether the conformation of these two cofactor-binding loops are energetically linked to each other and to the active site, we rationally designed two thrombin mutants in which either the 70-80 loop of exosite-1 or the 225-loop of the Na(+)-binding site was stabilized by an engineered disulfide bond. This was possible by replacing two residues, Arg-67 and Ile-82, in the first mutant and two residues, Glu-217 and Lys-224, in the second mutant with Cys residues.

Membrane-dependent interaction of factor Xa and prothrombin with factor Va in the prothrombinase complex.

Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) had been deleted. The fVa-mediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS).

One- and two-metal ion catalysis: global single-turnover kinetic analysis of the PvuII endonuclease mechanism.

Ester hydrolysis is one of the most ubiquitous reactions in biochemistry. Many of these reactions rely on metal ions for various mechanistic steps. A large number of metal-dependent nucleases have been crystallized with two metal ions in their active sites.

Functional properties and active-site topographies of factor X Gla- and prothrombin Gla-domain chimeras of activated protein C.

Substitution of the Gla-domain of activated protein C (APC) with the Gla-domain of prothrombin (APC-PTGla) improves the anticoagulant activity of APC independent of protein S. Previous FRET studies showed that this substitution alters the active-site topography of this mutant, rendering it identical to the active site of the APC-protein S complex. In this study, we characterized the functional properties and the active-site topography of another APC chimera containing the Gla-domain of factor X (APC-FXGla).

Protein Z-dependent protease inhibitor binds to the C-terminal domain of protein Z.

Protein Z (PZ) is a multidomain vitamin K-dependent plasma protein that functions as a cofactor to promote the inactivation of factor Xa (fXa) by PZ-dependent protease inhibitor (ZPI) by three orders of magnitude. To understand the mechanism by which PZ improves the reactivity of fXa with ZPI, we expressed wild-type PZ, PZ lacking the gamma-carboxyglutamic acid domain (GD-PZ), and a chimeric PZ mutant in which both Gla and EGF-like domains of the molecule were substituted with identical domains of fXa. The ZPI binding and the cofactor function of the PZ derivatives were characterized in both binding and kinetic assays.

Factor Va alters the conformation of the Na+-binding loop of factor Xa in the prothrombinase complex.

Structural and mutagenesis data have indicated that the 220-loop of thrombin is stabilized by a salt-bridge between Glu-217 and Lys-224, thereby facilitating the octahedral coordination of Na (+) with contributions from two carbonyl O atoms of Arg-221a and Lys-224. All three residues are also conserved in fXa and the X-ray crystal structure of fXa indicates that both Glu-217 and Lys-224 are within hydrogen-bonding distance from one another. To investigate the role of these three residues in the catalytic function of fXa and their contribution to interaction with Na (+), we substituted them with Ala and characterized their properties in both amidolytic and proteolytic activity assays.

Autolysis loop restricts the specificity of activated protein C: analysis by FRET and functional assays.

We previously demonstrated that the substitution of the autolysis loop (residues 143-154 in chymotrypsin numbering) of APC with the corresponding loop of trypsin (APC-Tryp 143-154) has no influence on the proteolytic activity of the protease toward fVa, however, this substitution increases the reactivity of APC with plasma inhibitors so that the mutant exhibits no anticoagulant activity in plasma. To further investigate the role of the autolysis loop in APC and determine whether this loop is a target for modulation by protein S, we evaluated the activity of APC-Tryp 143-154 toward fVa and several plasma inhibitors both in the absence and presence of protein S. Furthermore, we evaluated the active-site topography of APC-Tryp 143-154 by determining the average distance of the closest approach (L) between a fluorescein dye tethered to a tripeptide inhibitor, attached to the active-site of APC-Tryp 143-154, and octadecylrhodamine dyes incorporated into PCPS vesicles both in the absence and presence of protein S.

FRET studies with factor X mutants provide insight into the topography of the membrane-bound factor X/Xa.

FRET (fluorescence resonance energy transfer) studies have shown that the vitamin K-dependent coagulation proteases bind to membrane surfaces perpendicularly, positioning their active sites above the membrane surfaces. To investigate whether EGF (epidermal growth factor) domains of these proteases play a spacer function in this model of the membrane interaction, we used FRET to measure the distance between the donor fluorescein dye in the active sites of Fl-FPR (fluorescein-D-Phe-Pro-Arg-chloromethane)-inhibited fXa (activated Factor Xa) and its N-terminal EGF deletion mutant (fXa-desEGF1), and the acceptor OR (octadecylrhodamine) dye incorporated into phospholipid vesicles composed of 80% phosphatidylcholine and 20% phosphatidylserine. The average distance of closest approach (L) between fluorescein in the active site and OR at the vesicle surface was determined to be 56+/-1 A (1 A=0.

A unique molten globule state occurs during unfolding of cytochrome c by LiClO4 near physiological pH and temperature: structural and thermodynamic characterization.

We have carried out denaturation studies of bovine cytochrome c (cyt c) by LiClO4 at pH 6.0 and 25 degrees C by observing changes in difference molar absorbance at 400 nm (Deltaepsilon400), mean residue ellipticities at 222 nm ([theta]222) and difference mean residue ellipticity at 409 nm (Delta[theta]409). The denaturation is a three-step process when measured by Deltaepsilon400 and Delta[theta]409, and it is a two-step process when monitored by [theta]222.

Equilibrium studies of the effect of difference in sequence homology on the mechanism of denaturation of bovine and horse cytochromes-c.

We have carried out equilibrium studies of the effect of the amino acid residue difference in the primary structure of bovine cytochrome-c (b-cyt-c) and horse cyt-c (h-cyt-c) on the mechanism of their folding <--> unfolding processes at pH 6.0 and 25 degrees C. It has been observed that guanidinium chloride (GdmCl)-induced denaturation of b-cyt-c follows a two-state mechanism and that of h-cyt-c is not a two-state process.

Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants.

The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation <--> X (intermediate) conformation and the second denaturation phase, X conformation <--> D (denatured) conformation are reversible.