scholarly journals Equilibrium-dialysis studies of the interaction between cholic acid and 100000g-supernatant preparations from the rat liver

1976 ◽  
Vol 156 (2) ◽  
pp. 427-433 ◽  
Author(s):  
R C Strange ◽  
I A Nimmo ◽  
I W Percy-Robb
Keyword(s):  
Cholic Acid ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Proteolytic Enzymes ◽  
Equilibrium Dialysis ◽  
Complete Loss ◽  
Affinity Binding ◽  
Acid Transport ◽  
High Affinity ◽  
Rat Livers

1. The binding of cholic acid to 100000g supernatants from rat livers was investigated by equilibrium dialysis and gel-exculsion chromatography. 2. Supernatants were found to contain at least two classes of binding site for cholic acid. 3. These recptor molecules are probably proteins since incubation with proteolytic enzymes resulted in complete loss of cholic acid binding. 4. Supernatants were added to columns of Sephadex G-75, and two groups of fractions were shown to bind cholic acid. One of these contained low-affinity binding sites and the other contained both low- and high-affinity binding sites. 5. Feeding cholestyramine had no effect on cholic acid binding. 6. Increased cholic acid binding occurred after injection of phenobarbitone. There was an increase in the amount of the low-affinity component but no change in the high-affinity component. 7. The dissociation constants of the binding of cholic acid suggest that the binding proteins may be involved in bile acid transport.

scholarly journals Differences between binding of one-chain and two-chain tissue plasminogen activators to non-cross-linked and cross-linked fibrin clots

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 999-1006 ◽  
Author(s):  
SS Husain ◽  
AA Hasan ◽  
AZ Budzynski
Keyword(s):  
Dissociation Constant ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Aminocaproic Acid ◽  
Factor Xiiia ◽  
Single Type ◽  
Affinity Binding ◽  
High Affinity ◽  
Tissue Plasminogen ◽  
Fibrin Clots

Abstract Interaction of tissue plasminogen activator (t-PA) with fibrin plays a key role in regulation of plasminogen activation and clot dissolution. Previous investigations of t-PA-fibrin interaction, using incorporation of t-PA into polymerizing fibrin clots, have suggested that no significant differences exist in the binding of one-chain or two-chain t-PA to non-cross-linked or cross-linked fibrin. In the present study, binding of 125I-labeled and affinity-purified one-chain and two-chain forms of t-PA to preformed non-cross-linked or cross-linked, sonicated suspension of fibrin was investigated. Interaction of one-chain t-PA with cross-linked fibrin involved a single type of binding site with dissociation constant (kd) of 0.58 mumol/L and a stoichiometry (n) of 1.5. Interaction of one-chain t-PA with non-cross-linked fibrin, however, involved two classes of binding sites with dissociation constants of 0.32 and 1.5 mumol/L and corresponding number of binding sites equal to 0.57 and 2.0, respectively. In contrast to the binding of one-chain t-PA to cross-linked fibrin by a limited number of sites, two-chain t-PA appeared to involve a considerably greater number of sites (minimum six) whose dissociation constant was 3.2 mumol/L. Interaction of two-chain t-PA with non-cross-linked fibrin also showed the presence of many binding sites (minimum seven) with approximate dissociation constant of 6.4 mumol/L, as well as a few (n = 0.012) high- affinity sites with a kd of 0.011 mumol/L epsilon-Aminocaproic acid did not completely reverse the binding of either one-chain t-PA or two- chain t-PA to fibrin. The present findings suggest that the fibrin- binding properties of t-PA undergo considerable changes on proteolytic conversion from one-chain to two-chain t-PA, catalyzed under physiologic conditions by plasmin. The cleavage of one-chain t-PA to two-chain t-PA allows to bind to a large number of low-affinity binding sites on fibrin. Cross-linking of fibrin by factor XIIIa results in masking of high-affinity binding sites that are present in non-cross- linked fibrin. We propose that both plasmin and factor XIIIa play an important regulatory role in dissolution of blood clots by modulating t- PA-fibrin interaction.

Oxytocin receptors in the oviduct during the oestrous cycle of the ewe

1990 ◽  
Vol 124 (3) ◽  
pp. 353-359 ◽  
Author(s):  
V. J. Ayad ◽  
S. A. McGoff ◽  
D. C. Wathes
Keyword(s):  
Binding Sites ◽  
Luteal Phase ◽  
Dissociation Constants ◽  
Relative Concentration ◽  
Oestrous Cycle ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Lower Affinity ◽  
High Affinity ◽  
Oxytocin Receptors

ABSTRACT The presence of oxytocin receptors in ovine oviduct has been investigated. High-affinity binding sites for [3H]oxytocin were detected in crude membrane fractions prepared from the oviducts of ewes killed during the oestrous period. The dissociation constant calculated for these sites in competition studies was 1·7 nmol/l. Similar dissociation constants were calculated for [Arg8]-vasopressin and the oxytocin-specific agonists [Gly7]-oxytocin and [Thr4, Gly7]-oxytocin, indicating that these sites represent oxytocin receptors. At least one additional site of lower affinity and undetermined identity was present. The relative concentration of oxytocin-binding sites in preparations of oviduct membranes were estimated in ewes killed at different stages of the oestrous cycle using a single concentration of [3H]oxytocin. Binding was low during the luteal phase of the cycle but increased to a maximum at oestrus (77·7 fmol/mg protein). Binding fell after ovulation, reaching what appeared to be basal concentrations by the early luteal stage of the cycle. Binding to oviductal membranes from prepubertal, anoestrous and pregnant ewes was also low, but in anoestrous animals which had been treated with progesterone and oestrogen it was similar to values measured in ewes at oestrus. These results are consistent with the existence of oviductal oxytocin receptors which are regulated by ovarian steroids. We conclude that oxytocin receptors are present in the oviduct of the ewe around the time of ovulation. The significance of oxytocin to events taking place in the oviduct at this time remains to be determined. Journal of Endocrinology (1990) 124, 353–359

High affinity GDP binding sites on brown adipose tissue mitochondria of genetically obese ‘fa/fa’ rats

1985 ◽  
Vol 5 (2) ◽  
pp. 159-166 ◽  
Author(s):  
R. R. French ◽  
S. J. Holt ◽  
D. A. York
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Binding Sites ◽  
Equilibrium Dialysis ◽  
Affinity Binding ◽  
High Affinity ◽  
Brown Adipose ◽  
Obese Rats

The number of high affinity [3H]GDP binding sites in brown adipose tissue mitochondria is normal in obese (f a / f a) rats in contrast to the reduced number of low affinity GDP binding sites. Adrenalectomy corrected the loss of low affinity binding sites in fa/fa rats but had no effect on the number of high affinity sites in either lean or obese rats. Equilibrium dialysis was used to show the presence of both high and low affinity binding sites on the purified 32 kdalton protein.

scholarly journals Differences between binding of one-chain and two-chain tissue plasminogen activators to non-cross-linked and cross-linked fibrin clots

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 999-1006
Author(s):  
SS Husain ◽  
AA Hasan ◽  
AZ Budzynski
Keyword(s):  
Dissociation Constant ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Aminocaproic Acid ◽  
Factor Xiiia ◽  
Single Type ◽  
Affinity Binding ◽  
High Affinity ◽  
Tissue Plasminogen ◽  
Fibrin Clots

Interaction of tissue plasminogen activator (t-PA) with fibrin plays a key role in regulation of plasminogen activation and clot dissolution. Previous investigations of t-PA-fibrin interaction, using incorporation of t-PA into polymerizing fibrin clots, have suggested that no significant differences exist in the binding of one-chain or two-chain t-PA to non-cross-linked or cross-linked fibrin. In the present study, binding of 125I-labeled and affinity-purified one-chain and two-chain forms of t-PA to preformed non-cross-linked or cross-linked, sonicated suspension of fibrin was investigated. Interaction of one-chain t-PA with cross-linked fibrin involved a single type of binding site with dissociation constant (kd) of 0.58 mumol/L and a stoichiometry (n) of 1.5. Interaction of one-chain t-PA with non-cross-linked fibrin, however, involved two classes of binding sites with dissociation constants of 0.32 and 1.5 mumol/L and corresponding number of binding sites equal to 0.57 and 2.0, respectively. In contrast to the binding of one-chain t-PA to cross-linked fibrin by a limited number of sites, two-chain t-PA appeared to involve a considerably greater number of sites (minimum six) whose dissociation constant was 3.2 mumol/L. Interaction of two-chain t-PA with non-cross-linked fibrin also showed the presence of many binding sites (minimum seven) with approximate dissociation constant of 6.4 mumol/L, as well as a few (n = 0.012) high- affinity sites with a kd of 0.011 mumol/L epsilon-Aminocaproic acid did not completely reverse the binding of either one-chain t-PA or two- chain t-PA to fibrin. The present findings suggest that the fibrin- binding properties of t-PA undergo considerable changes on proteolytic conversion from one-chain to two-chain t-PA, catalyzed under physiologic conditions by plasmin. The cleavage of one-chain t-PA to two-chain t-PA allows to bind to a large number of low-affinity binding sites on fibrin. Cross-linking of fibrin by factor XIIIa results in masking of high-affinity binding sites that are present in non-cross- linked fibrin. We propose that both plasmin and factor XIIIa play an important regulatory role in dissolution of blood clots by modulating t- PA-fibrin interaction.

Chronic treatment with bovine growth hormone upregulates high-affinity hepatic somatotropic receptors in sheep

1991 ◽  
Vol 124 (3) ◽  
pp. 307-313 ◽  
Author(s):  
Helga Sauerwein ◽  
Bernhard H. Breier ◽  
John J. Bass ◽  
Peter D. Gluckman
Keyword(s):  
Growth Hormone ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Hormone Treatment ◽  
Bovine Growth Hormone ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Gh Therapy ◽  
High Affinity

Abstract. We evaluated the effect of chronic bovine growth hormone treatment on the hepatic somatotropic receptor. Growing lambs were treated with bGH at 0, 0.05, 0.15, 0.25 or 0.55 mg · kg−1 · day−1 daily (N=5/group) for 56 days. The binding of ovine GH to hepatic membranes washed with 4 mol/l MgCl2 and prepared in the presence of aprotinin was examined. The specific binding of oGH was increased (p<0.01) from 7.1±1.2% in saline-treated controls to 17.4±1.5% in the 0.55 mg · kg−1 · day−1 group. Scatchard analysis showed curvilinear plots that best fitted a two-site model in 22/25 livers. The two sites had estimated dissociation constants (Kd) of 3 to 13 nmol/l for the low-affinity site and a Kd ranging from 0.17 to 0.31 nmol/l for the high-affinity site. Treatment with bGH had no consistent effect on the affinity of either binding site. However, bGH therapy was associated with a dose-dependent increase (p<0.01) in the number of high-affinity somatotropic receptors. There was no effect of bGH therapy on the concentration of low-affinity binding sites. The concentration of high-affinity receptors correlated with weight gain (r=0.54, p<0.01), fat content (r=−0.54, p<0.01), protein content (r=0.40, p<0.05), and plasma IGF-I (r=0.57, p<0.005). The concentrations of low-affinity binding sites showed no such correlations. These observations demonstrate that an important effect of chronic GH therapy in animals with an intact somatotropic axis is to increase significantly the concentration of high-affinity somatotropic receptors. It is suggested that enhancement of the number of high-affinity somatotropic receptors is central to the determination of the efficacy of GH therapy.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

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