scholarly journals The interaction of bovine factor IX, its activation intermediate, factor IX α, and its activation products, factor IXaα and factor IXaβ, with acidic phospholipid vesicles of various compositions

1986 ◽  
Vol 236 (3) ◽  
pp. 861-869 ◽  
Author(s):  
J M Beals ◽  
F J Castellino
Keyword(s):  
Binding Sites ◽  
Dissociation Constants ◽  
Factor Ix ◽  
Phospholipid Vesicles ◽  
Acidic Phospholipid ◽  
Factor Ixa ◽  
Activation Products ◽  
Alpha Factor ◽  
Number Of Binding Sites ◽  
Bovine Factor

The interactions of bovine factor IX, its activation intermediate, Factor IX alpha, and its activation products, Factor IXa alpha and Factor IXa beta, with phospholipid vesicles, of mean radius of approx. 30 nm, containing various amounts of phosphatidylserine (PS) and phosphatidylcholine (PC), have been examined. For Factor IX, Factor IX alpha, Factor IXa alpha and Factor IXa beta, the dissociation constants, at saturating levels of Ca2+, are independent of the PS concentration in the vesicle after levels of 20-30% (w/w) have been reached, and attain minimum values of approx. 1.7, 1.7, 0.7 and 1.0 microM, respectively, with vesicles containing 50% PS. The amount of protein bound per vesicle particle is independent of the PS content, above 20% PS, for Factor IX and Factor IXa beta, with values of approx. 995-1197 and 1128-1566 molecules/vesicle, respectively. With Factor IX alpha, a dependence on the amount of protein bound with the content of PS is seen, which ranges from 338 to 619 molecules/vesicle with membranes containing 30-50% PS. For Factor IXa alpha, no regularity is noted and a range of 583-1083 molecules of protein/vesicle is observed with the systems employed. Examination of the radii of the proteins on the vesicle demonstrates that Factors IX alpha and IXa alpha occupy considerably more of the surface than do Factors IX and IXa beta, suggesting that a reason for the decreased number of binding sites for the former two proteins on the vesicle may be related to their greater surface spatial requirements.

Adrenergic receptors on cerebral microvessels: pericyte contribution

1989 ◽  
Vol 256 (1) ◽  
pp. R224-R230 ◽  
Author(s):  
R. M. Elfont ◽  
P. R. Sundaresan ◽  
C. D. Sladek
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Radioligand Binding ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Binding Studies ◽  
Cerebral Microvessels ◽  
Beta Adrenoceptor ◽  
Number Of Binding Sites

R224-R230, 1989.--[125I]iodocyanopindolol ([125I]ICYP) and [3H]rauwolscine were used to quantitate, respectively, the beta- and alpha 2-adrenergic receptors in freshly isolated bovine cerebral microvessels and in pericyte cultures derived from these microvessels. Morphological and immunocytochemical criteria distinguished the pericytes from endothelial cells. Competitive binding studies established the specificity of the radioligand binding. The maximal number of binding sites (Bmax) for [125I]ICYP in the pericytes constituted only 8% of that in the microvessels (3.5 +/- 1.3 vs. 44.4 +/- 6.6 fmol/mg protein). In contrast, the Bmax for [3H]rauwolscine in the pericytes was 50% of that in the microvessels (55.4 +/- 11.8 vs. 111.1 +/- 9.5 fmol/mg protein). The dissociation constants for both [125I]ICYP and [3H]rauwolscine were similar in the two preparations. No alpha 1-adrenergic receptors, as defined by the specific binding of [3H]prazosin, were identified either in the pericytes or microvessels. Overall, our results suggest that pericytes contribute minimally to the total beta-adrenoceptor number of cerebral microvessels, and thus the beta-adrenoceptors must be located predominantly on endothelial cells. However, the contribution of pericytes to the total alpha 2-adrenoceptor number of the microvessels may be substantial.

scholarly journals Role of gamma-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 398-405 ◽  
Author(s):  
R Rawala-Sheikh ◽  
SS Ahmad ◽  
DM Monroe ◽  
HR Roberts ◽  
PN Walsh
Keyword(s):  
Binding Sites ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Factor Ix ◽  
Factor X ◽  
Apparent Dissociation Constant ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Carboxyglutamic Acid ◽  
Factor X Activation

To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/- 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX.

Saccharomyces cerevisiae mutants unresponsive to alpha-factor pheromone: alpha-factor binding and extragenic suppression

1987 ◽  
Vol 7 (4) ◽  
pp. 1311-1319
Author(s):  
D D Jenness ◽  
B S Goldman ◽  
L H Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
The Other ◽  
Restrictive Temperature ◽  
Mrna Accumulation ◽  
Alpha Factor ◽  
Number Of Binding Sites ◽  
One Step ◽  
Extragenic Suppression ◽  
Six Genes

Mutations in six genes that eliminate responsiveness of Saccharomyces cerevisiae a cells to alpha-factor were examined by assaying the binding of radioactively labeled alpha-factor to determine whether their lack of responsiveness was due to the absence of alpha-factor receptors. The ste2 mutants, known to be defective in the structural gene for the receptor, were found to lack receptors when grown at the restrictive temperature; these mutations probably affect the assembly of active receptors. Mutations in STE12 known to block STE2 mRNA accumulation also resulted in an absence of receptors. Mutations in STE4, 5, 7, and 11 partially reduced the number of binding sites, but this reduction was not sufficient to explain the loss of responsiveness; the products of these genes appear to affect postreceptor steps of the response pathway. As a second method of distinguishing the roles of the various STE genes, we examined the sterile mutants for suppression. Mating of the ste2-3 mutant was apparently limited by its sensitivity to alpha-factor, as its sterility was suppressed by mutation sst2-1, which leads to enhanced alpha-factor sensitivity. Sterility resulting from each of four ste4 mutations was suppressed partially by mutation sst2-1 or by mutation bar1-1 when one of three other mutations (ros1-1, ros2-1, or ros3-1) was also present. Sterility of the ste5-3 mutant was suppressed by mutation ros1-1 but not by sst2-1. The ste7, 11, and 12 mutations were not suppressed by ros1 or sst2. Our working model is that STE genes control the response to alpha-factor at two distinct steps. Defects at one step (requiring the STE2 gene are suppressed (directly or indirectly) by mutation sst2-1, whereas defects at the other step (requiring the STE5 gene) are suppressed by the ros1-1 mutation. The ste4 mutants are defective for both steps. Mutation ros1-1 was found to be allelic to cdc39-1. Map positions for genes STE2, STE12, ROS3, and FUR1 were determined.

Antibody to C1 Domain of Factor VIII Alters Interaction of Factor Xase Complex with Factor X.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1738-1738
Author(s):  
Gary E. Gilbert ◽  
Anu Bhimavarapu ◽  
Patricia Price ◽  
Marc Jacquemin
Keyword(s):  
Factor Viii ◽  
Factor Ix ◽  
Phospholipid Bilayer ◽  
Phospholipid Vesicles ◽  
Factor X ◽  
Apparent Affinity ◽  
Factor Ixa ◽  
Factor Viiia ◽  
C1 Domain ◽  
Gla Domain

Abstract The role of the C1 domain in function of factor VIII has not been clearly defined. In contrast, functional interactions have been identified for the three A domains and the C2 domain. We hypothesized that the C1 domain of factor VIII participates in both phospholipid binding and interaction with factor X and/or factor IXa. We evaluated inhibition of the factor Xase complex by LE2E9, a human inhibitor IgG4k mAb against C1. We utilized altered catalytic activity of the factor Xase complex in a defined assay to report the inhibition by LE2E9. Inhibition by LE2E9 was also evaluated when soluble phosphatidylserine replaced vesicles to support the factor Xase complex and when Gla-domainless factor X was the substrate. The deglycosylated form of LE2E9 was also evaluated to better define the mechanism through which LE2E9 exerts its effect. We found that LE2E9 bound to factor VIIIa with an apparent KD of 0.5 nM. The apparent affinity of factor VIIIa for sonicated phospholipid vesicles of phosphatidylserine:phosphatidylethanolamine:phosphatidylcholine 4:20:76 increased 3-fold in the presence of LE2E9. The apparent affinity of factor VIIIa for factor IXa was not significantly changed. The KM of the factor VIIIa-factor IXa complex was 20 ± 2 nM with LE2E9 vs. 40 ± 2 nM without. LE2E9 decreased the Vmax by 77 ± 6% indicating that the affinity of factor X for the factor Xase complex is increased while the rate of cleavage is decreased. When Gla-domainless factor X was used as the substrate for the factor Xase complex, LE2E9 did not inhibit activity indicating that inhibition occurs via an interaction that involves the factor X Gla domain. When the factor VIIIa-factor IX complex was supported by dihexanoyl phosphatidylserine rather than phospholipid vesicles the inhibition of Vmax was 47% indicating that the inhibitory effect does not require a phospholipid bilayer. Deglycosylated LE2E9 did not significantly change the KM but decreased the Vmax by 22% while both antibodies bound to factor VIII with the same affinity. These results suggest that LE2E9 inhibition relates largely to interaction of a carbohydrate moiety with factor VIII or factor X rather than binding the core C1 epitope. We conclude that LE2E9 decreases the KM, and the Vmax for the factor VIIIa-factor IXa complex, but only when the factor X Gla domain is present. These results suggest that in the factor Xase complex the C1 domain of factor VIII is intimately associated with the Gla domain of factor X and that interaction between these domains enhances the kcat for the factor VIIIa-factor IXa complex.

scholarly journals The binding of calcium to the activation products of bovine factor IX.

1979 ◽  
Vol 254 (14) ◽  
pp. 6333-6336 ◽  
Author(s):  
G W Amphlett ◽  
R Byrne ◽  
F J Castellino
Keyword(s):  
Factor Ix ◽  
Activation Products ◽  
Bovine Factor

Identification of two mitochondrial GDP-binding sites in rat brown adipose tissue

1983 ◽  
Vol 3 (7) ◽  
pp. 589-598 ◽  
Author(s):  
K. R. Bryant ◽  
N. J. Rothwell ◽  
M. J. Stock ◽  
D. Stribling
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Mitochondrial Protein ◽  
Scatchard Analysis ◽  
Nucleotide Binding Sites ◽  
Guanosine Diphosphate ◽  
Brown Adipose ◽  
Number Of Binding Sites

Scatchard analysis of specific guanosine-diphosphate-([3H]GDP-) binding to rat brown-adipose-tissue mitochondria revealed two distinct binding sites with apparent dissociation constants (Kd) of approximately 0.05 and 2.0 μM. Binding to both sites was insensitive to atractyloside. Reducing the pH of the binding medium from 7.1 to 6.6 caused marked reductions in the Kd of both sites, but at pH 7.6, the dissociation constants were increased about 3-fold. Acute treatment of rats with noradrenaline, 1 h before sacrifice, increased the maximum number of binding sites (Bmax, pmol/rng mitochondrial protein) of both sites and also increased the dissociation constants. The Bmax of the lower-affinity site was elevated in rats exposed to 5°C or fed a palatable cafeteria diet for 10 d, compared to control animals, with the greater changes occurring in the cold-adapted group. The high-affinity site was unaltered by cold adaptation or cafeteria feeding. These results indicate the presence of two distinct nucleotide-binding sites in brown-fat mitochondria, both of which may be involved in thermogenesis.

Solid-phase assay for insulin-like growth factor (IGF) binding to IGF-binding protein-3: application to the study of the effects of antibodies and heparin

1997 ◽  
Vol 153 (1) ◽  
pp. 87-97 ◽  
Author(s):  
J A Koedam ◽  
C M Hoogerbrugge ◽  
S C van Buul-Offers ◽  
J L Van den Brande
Keyword(s):  
Binding Sites ◽  
Dissociation Constants ◽  
Solid Phase ◽  
Binding Protein ◽  
Alkylating Agents ◽  
Igf Binding Proteins ◽  
Igf I ◽  
Igf Binding ◽  
Number Of Binding Sites ◽  
Igfbp 3

Abstract The availability and activity of insulin-like growth factors (IGF-I and IGF-II) are largely determined by a group of IGF-binding proteins (IGFBPs). We have developed a new assay to characterize the interaction between the IGFs and IGFBP-3. In this assay, recombinant IGFBP-3 (5 ng) was immobilized on plastic microtitre wells, after which radiolabelled IGF-I or -II was allowed to bind. The assay is highly specific, since neither IGF bound to control wells blocked with albumin. By constructing both saturation and competition binding curves, equivalence of binding between the radiolabelled and native IGF ligands could be demonstrated. From these curves, reliable specific activities of the tracers were calculated. Scatchard plots of both types of data produced identical results for dissociation constants and number of binding sites. The affinity of IGF-II was twice as high as the affinity of IGF-I (dissociation constants of 44 and 102 pm respectively). The assay was used to show that polyclonal anti-IGFBP-3 antibodies could block binding of IGF. Alkylating agents and NaCl were without effect, but chaotropic salts such as CaCl2 and NaSCN decreased IGF binding to IGFBP-3. IGFBP-1 and IGFBP-3, but not an N-terminal fragment of IGFBP-3, could effectively block binding of both IGF-I and IGF-II to the solid-phase IGFBP-3. Increasing concentrations of heparin had little or no effect on IGF-I binding, but strongly inhibited IGF-II binding. This was shown to be a consequence of a decrease in both the affinity and the number of binding sites. Possibly, the interaction of IGFBP-3 with heparin or heparin-like structures in vivo can lead to the selective release of IGF-II from this binding protein. Our results with heparin also suggest that the binding sites on IGFBP-3 for IGF-I and IGF-II are not completely identical. This assay can be applied to the study of various aspects of the interaction between the IGFs and IGFBP-3, such as the effects of interfering substances and structure–function relationships of both moieties of the complex. Journal of Endocrinology (1997) 153, 87–97

Activation of factor IX zymogen results in exposure of a binding site for low-density lipoprotein receptor–related protein

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3459-3465 ◽  
Author(s):  
Jaap G. Neels ◽  
Birgit M. M. van den Berg ◽  
Koen Mertens ◽  
Hans ter Maat ◽  
Hans Pannekoek ◽  
...  
Keyword(s):  
Dissociation Constants ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Factor Ix ◽  
Lipoprotein Receptor ◽  
Factor Ixa ◽  
Density Lipoprotein Receptor ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation ◽  
Lipoprotein Receptor Related Protein

The interaction between the endocytic receptor low density lipoprotein receptor–related protein (LRP) and either coagulation factor IX or its active derivative factor IXa was studied. Purified factor IX was unable to associate with LRP when analyzed by surface plasmon resonance. By contrast, factor XIa–mediated conversion of factor IX into factor IXa resulted in reversible dose- and calcium-dependent binding to LRP. Active-site blocking of factor IXa did not affect binding to LRP, whereas LRP binding was efficiently inhibited in the presence of heparin or antibodies against factor IX or LRP. The factor IXa–LRP interaction could be described by a 2-site binding model with equilibrium dissociation constants of 27 nmol/L and 69 nmol/L. Consistent with this model, it was observed that factor IXa binds to 2 different recombinant receptor fragments of LRP (denoted cluster II and cluster IV) with equilibrium dissociation constants of 227 nmol/L and 53 nmol/L, respectively. The amount of factor IXa degraded by LRP-deficient cells was 35% lower than by LRP-expressing cells, demonstrating that LRP contributes to the transport of factor IXa to the intracellular degradation pathway. Because ligand binding to LRP is often preceded by binding to proteoglycans, the contribution of proteoglycans to the catabolism of factor IXa was addressed by employing proteoglycan-deficient cells. Degradation of factor IXa by proteoglycan-deficient cells proceeded at a 83% lower rate than wild-type cells. In conclusion, the data presented here indicate that both LRP and proteoglycans have the potential to contribute to the catabolism of factor IXa.

scholarly journals Role of gamma-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 398-405 ◽  
Author(s):  
R Rawala-Sheikh ◽  
SS Ahmad ◽  
DM Monroe ◽  
HR Roberts ◽  
PN Walsh
Keyword(s):  
Binding Sites ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Factor Ix ◽  
Factor X ◽  
Apparent Dissociation Constant ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Carboxyglutamic Acid ◽  
Factor X Activation

Abstract To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/- 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX.

scholarly journals An ordered sequential mechanism for Factor IX and Factor IXa binding to platelet receptors in the assembly of the Factor X-activating complex

2005 ◽  
Vol 390 (1) ◽  
pp. 157-167 ◽  
Author(s):  
Xia Yang ◽  
Peter N. Walsh
Keyword(s):  
Fold Increase ◽  
Factor Ix ◽  
Phospholipid Vesicles ◽  
Phospholipid Membranes ◽  
Factor X ◽  
Wild Type ◽  
Binding Assays ◽  
Epidermal Growth Factor Domain ◽  
Factor Ixa ◽  
Activated Platelets

To define the contributions of the Ω-loop of the Gla (γ-carboxyglutamic acid) domain and the EGF2 (second epidermal growth factor) domain of FIXa (Factor IXa) in the assembly of the FX-activating complex on activated platelets and phospholipid membranes, three recombinant FIXa chimeras were prepared with corresponding residues from the homologous coagulation protein, FVII: (i) Gly4–Gln11 (FIXa7Ωloop), (ii) Cys88–Cys124 (FIXa7EGF2), and (iii) both Gly4–Gln11 and Cys88–Cys124 (FIXa7Ωloop7EGF2). All three chimeras were similar to wild-type FIXa, as assessed by SDS/PAGE, active-site titration, content of Gla residues, activation rates by FXIa and rates of FXa generation in solution. Titrations of FX or FVIIIa on SFLLRN peptide-activated platelets and on phospholipid vesicles in the presence of FVIIIa revealed normal substrate and cofactor binding to all chimeras. In kinetic assays in the presence of phospholipid vesicles and FVIIIa, compared with wild-type FIXa Kd, app∼4 nM, the FIX7Ωloop chimera showed a 1.6-fold increase in Kd, app, the FIX7EGF2 chimera had a 7.4-fold increase in Kd, app, and the FIX7Ωloop7EGF2 chimera showed a 21-fold increase in Kd, app. In kinetic assays and equilibrium platelet-binding assays with activated platelets and FVIIIa, compared with wild-type FIXa (Vmax∼5 nM min−1; Kd, app∼0.5 nM; Bmax∼550 sites/platelet; Kd∼0.5 nM), the FIX7Ωloop chimera displayed 2-fold decreases in Vmax and Bmax and 2-fold increases in Kd, app and Kd. The FIX7EGF2 chimera displayed 2-fold decreases in Vmax and Bmax and 10-fold increases in Kd, app and Kd. The FIX7Ωloop7EGF2 chimera showed non-saturable curves and severely impaired rates of FXa generation, and non-saturable, non-specific, low-level binding to activated platelets. Thus both the Gla domain Ω-loop (Gly4–Gln11) and the EGF2 domain (Cys88–Cys124) are required to mediate the normal assembly of the FX-activating complex on activated platelets and on phospholipid membranes.

Sign in / Sign up

Export Citation Format

Share Document