scholarly journals Insights into Hydrophobicity and the Chaperone-like Function of αA- and αB-crystallins

2005 ◽  
Vol 280 (23) ◽  
pp. 21726-21730 ◽  
Author(s):  
M. Satish Kumar ◽  
Mili Kapoor ◽  
Sharmistha Sinha ◽  
G. Bhanuprakash Reddy
Keyword(s):  
Binding Sites ◽  
Chaperone Activity ◽  
Titration Calorimetry ◽  
Affinity Binding ◽  
Hydrophobic Surfaces ◽  
Ans Binding ◽  
High Affinity Binding ◽  
Chaperone Function ◽  
High Affinity ◽  
Αb Crystallin

α-Crystallin, composed of two subunits, αA and αB, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions. The exposed hydrophobic surfaces of α-crystallins have been implicated in this process, but their exact role has not been elucidated. In this study, we quantify the hydrophobic surfaces of αA- and αB-crystallins by isothermal titration calorimetry using 8-anilino-1-napthalenesulfonic acid (ANS) as a hydrophobic probe and analyze its correlation to the chaperone potential of αA- and αB-crystallins under various conditions. Two ANS binding sites, one with low and another with high affinity, were clearly detected, with αB showing a higher number of sites than αA at 30 °C. In agreement with the higher number of hydrophobic sites, αB-crystallin demonstrated higher chaperone activity than αA at this temperature. Thermodynamic analysis of ANS binding to αA- and αB-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding. Interestingly, although the number of ANS binding sites was similar for αA and αB at 15 °C, αA was more potent than αB in preventing aggregation of the insulin B-chain. Although there was no change in the number of high affinity binding sites of αA and αB for ANS upon preheating, there was an increase in the number of low affinity sites of αA and αB. Preheated αA, in contrast to αB, exhibited remarkably enhanced chaperone activity. Our results indicate that although hydrophobicity appears to be a factor in determining the chaperone-like activity of α-crystallins, it does not quantitatively correlate with the chaperone function of α-crystallins.

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.

scholarly journals [3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

1992 ◽  
Vol 40 (6) ◽  
pp. 771-779 ◽  
Author(s):  
A A Maki ◽  
D G Baskin ◽  
W L Stahl
Keyword(s):  
Nervous System ◽  
Rat Brain ◽  
Cardiac Glycoside ◽  
Binding Sites ◽  
Quantitative Autoradiography ◽  
Affinity Binding ◽  
Ouabain Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Kd Values

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.

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