Are there one or two high-affinity binding sites for GDP in brown adipose tissue mitochondria?

1986 ◽  
Vol 14 (6) ◽  
pp. 1201-1202
Author(s):  
RACHEL E. MILNER ◽  
SHELAGH WILSON ◽  
JONATHAN R. S. ARCH ◽  
PAUL TRAYHURN
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Binding Sites ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Brown Adipose

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.

Acute influences on the two GDP-binding sites in brown-adipose-tissue mitochondria

1984 ◽  
Vol 4 (6) ◽  
pp. 523-533 ◽  
Author(s):  
K. R. Bryant ◽  
N. J. Rothwell ◽  
M. J. Stock
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Conformational Changes ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Mitochondrial Protein ◽  
Scatchard Analysis ◽  
Slight Reduction ◽  
High Affinity ◽  
Brown Adipose

Scatchard analysis of3H-guanosine diphosphate (GDP) binding to rat brown-adipose-tissue mitochondria demonstrated that binding to the high- and low-affinity sites (Kd = 0.05 and 2.0 μM) was abolished by denaturation at 100°C but non-specific binding remained constant (0.2% of free-GDP). Prior incubation of mitochondria at 37°C reduced binding to the high-affinity site, but this could be reversed by incubating samples at 0°C. Addition of palmitic acid (5–40 nmole/mg of mitochondrial protein) did not affect GDP-binding, but similar concentrations of palmitoyl CoA caused a slight reduction in the number of high-affinity sites and a significant decrease in the number of lower-affinity sites. Acute treatments known to stimulate thermogenesis in vivo (a single meal, cold exposure, or noradrenaline injection 40–80 min before sacrifice) all increased binding to both binding sites, and tended to raise the dissociation constants, whereas injection of 2-deoxy-D-glucose, which depresses metabolic rate in the rat, decreased dissociation constants of both sites and the maximum number of high-affinity sites. These data indicate that both GDP-binding sites respond rapidly to acute thermogenic stimuli, possibly due to conformational changes in the mitochondrial inner membrane, and that palmitoyl CoA may influence mitochondrial proton conductance via an association with purine nucleotide binding sites.

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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