Palmitate binding to and efflux kinetics from human erythrocyte ghost

At 0 degrees C, pH 7.3, palmitate (PA) binds to human erythrocyte ghosts suspended in 0.2% bovine serum albumin (BSA) solution with molar ratios of PA to BSA, v, between 0.2 and 1.3. The binding depends on the water phase PA concentration, measured in equilibrium experiments, using BSA-filled ghosts as semipermeable bags. The saturable binding has a capacity of 19.4 +/- 7.5 nmol g-1 packed ghosts (7.2 x 10(9) cells) and Kd = 13.5 +/- 5 nM. PA exchange efflux kinetics to 0.2% BSA is recorded from ghosts without and with 0.2% BSA with a resolution time of about 1 s. Data are analyzed in terms of compartmental models. Using BSA-free ghosts the kinetics is essentially monoexponential. The rate constant is 0.0287 +/- 0.0022 s-1. Using ghosts with BSA, the kinetics is biexponential with widely different rate constants. Extrapolated zero-time values reflect, according to additional investigations, 'instantaneous' release of PA from the outer surface of the ghosts. Analyses of the biexponential curve up to about 55% tracer efflux assign unequivocally values to three model parameters. (1) k1, the dissociation rate constant of the PA-BSA complex is (1.47 +/- 0.03) x 10(-3) s-1 and (2.56 +/- 0.08) x 10(-3) s-1 and (4.08 +/- 0.13) x 10(-3) s-1 at v = 0.2, 0.6 and 1.4, respectively. (2) k3*, the overall rate constant of PA transport from the inside of the ghost membrane to the medium is 0.0269 +/- 0.0020 s-1 independent of v. (3) Qkin, the ratio of PA on the inside of the membrane to PA on BSA within the ghosts is v dependent and smaller than a corresponding ratio Qeq measured in equilibrium by a value corresponding to PA on the outer surface. This fraction is released with a rate constant, k5, which is of the order of 1 s-1. The data suggest a maximum PA transport capacity, Jmax, of 2 pmol min-1 cm-2, 0 degrees C, pH 7.3.     

Exchange efflux of [3H]palmitate from human red cell ghosts to bovine serum albumin in buffer. Effects of medium volume and concentration of bovine serum albumin.

[3H]Palmitate, PA, exchange efflux kinetics is recorded from human erythrocyte ghosts to buffer with bovine serum albumin, BSA, at 0 degrees C. The effects have been investigated of three medium/ghost volume ratios: 36, 80 and 500, of six BSA concentrations, [BSA]: 0.01, 0.02, 0.05, 0.2, 1 and 2% (1.5, 3.0, 7.5, 30, 150 and 300 microM) and of various v, molar ratios of palmitate to BSA, between 0.15 and 0.94. Data are analyzed in terms of a virtually closed three-compartment model. In theory, the tracer efflux is biexponential and the rate coefficients differ at least 20 fold [1]. The efflux rate at 2% BSA is monoexponential beyond our resolution time of about 1 s, but nearly biexponential at or below 0.2% BSA with a well-defined smallest-rate coefficient beta. beta depends strongly on [BSA] but is remarkably v independent. The medium/ghost volume ratio has no effect on beta when [BSA] > or = 0.2%, although beta measured at 2% BSA is almost 2-fold higher than at 0.2%. This suggests the presence of an unstirred layer, USL. According to our model, the observations are understood quantitatively on basis of our previously published dissociation rate constants of the PA-BSA complex, as well as PA equilibrium bindings to ghost membranes (Bojesen, I.N. and Bojesen, E. (1991) Biochim. Biophys. Acta 1069, 297-307). Essentially, beta is theoretically a function of two terms, one comprising the membrane transport parameters and the other the medium-dependent variables. Most important is the clearance with respect to monomer concentration adjacent to the membrane. The clearance is calculated on basis of quasi-stationary diffusion in USL. The data are compatible with a planar USL of 6 microns depth and with the same area as a ghost but not with a spherical USL.     

Different arachidonate and palmitate binding capacities of the human red cell membrane

Human red cell membrane bindings of arachidonate and palmitate at pH 7.3 are investigated at temperatures between 0 and 38°C by equilibrating ghosts with the long-chain fatty acids bound to bovine serum albumin in molar ratios (v) within the physiological range (<1.7). Linearized relations of ghost uptakes and fatty acid monomer concentrations in buffer provide estimates of the binding capacities and corresponding equilibrium dissociation constants (K _dm ). The temperature-independent arachidonate binding capacity, 5.5 ± 0.5 nmol g^–1 packed ghosts, is approximately fivefold smaller than that of palmitate, 26.6 ± 2.0 nmol g^–1. While K _dm of arachidonate binding 5.1 ± 0.5 nm is temperature independent, K _dm of palmitate increases with temperature from 3.7 nm at 0°C to 12.7 nm at 38°C.The large difference in binding capacities suggests the presence of at least two different fatty acid binding domains in human red cell membranes.     

Binding of anandamide to bovine serum albumin

The endocannabinoid anandamide is of lipid nature and may thus bind to albumin in the vascular system, as do fatty acids. The knowledge of the free water-phase concentration of anandamide is essential for the investigations of its transfer from the binding protein to cellular membranes, because a water-phase shuttle of monomers mediates such transfers. We have used our method based upon the use of albumin-filled red cell ghosts as a dispersed biological "reference binder" to measure the water-phase concentrations of anandamide. These concentrations were measured in buffer (pH 7.3) in equilibrium with anandamide bound to BSA inside resealed human red cell membranes at low molar ratios below one. Data were obtained at 0 degrees C, 10 degrees C, 23 degrees C, and 37 degrees C. The equilibrium dissociation constant (Kd) increases with temperature from 6.87 +/- 0.53 nM at 0 degrees C to 54.92 +/- 1.91 nM at 37 degrees C. Regression analyses of the data suggest that BSA has one high-affinity binding site for anandamide at all four temperatures. The free energy of anandamide binding (DeltaG0) is calculated to -43.05 kJ mol-1 with a large enthalpy (DeltaH0) contribution of -42.09 kJ mol-1. Anandamide has vasodilator activity, and the binding to albumin may mediate its transport in aqueous compartments.     

Membrane transport of anandamide through resealed human red blood cell membranes

The use of resealed red blood cell membranes (ghosts) allows the study of the transport of a compound in a nonmetabolizing system with a biological membrane. Transmembrane movements of anandamide (N-arachidonoylethanolamine, arachidonoylethanolamide) have been studied by exchange efflux experiments at 0 degrees C and pH 7.3 with albumin-free and albumin-filled human red blood cell ghosts. The efflux kinetics is biexponential and is analyzed in terms of compartment models. The distribution of anandamide on the membrane inner to outer leaflet pools is determined to be 0.275 +/- 0.023, and the rate constant of unidirectional flux from inside to outside is 0.361 +/- 0.023 s(-1). The rate constant of unidirectional flux from the membrane to BSA in the medium ([BSA]o) increases with the square root of [BSA]o in accordance with the theory of an unstirred layer around ghosts. Anandamide passed through the red blood cell membrane very rapidly, within seconds. At a molar ratio of anandamide to BSA of <1, membrane binding of anandamide increases with increasing temperatures between 0 degrees C and 37 degrees C, and the equilibrium dissociation constants are in the nanomolar range. The nature of membrane binding and the mechanism of membrane translocation are discussed.     

Effects of preparative procedures on the volume and content of resealed red cell ghosts

The effects of variations in preparative procedures on the volume and content of resealed red cell ghosts have been investigated. Following hypotonic lysis at 0 degrees C, and after a variable delay time (td), concentrated buffer was added to restore isotonicity; resealing was then induced by incubation at 37 degrees C for one hour. Using this procedure, both the resealed ghost volume and the residual hemoglobin (Hb) content decreased for increasing td. If ghosts were maintained at 0 degree C (i.e., no 37 degrees C incubation), they remained nearly spherical until isotonicity was restored. Their volume then fell abruptly, but subsequently increased toward an intermediate level. The fall in volume was greater and the final level achieved was smaller for longer delay times. At 0 degree C, return to isotonicity also halted the otherwise gradual loss of residual Hb from unsealed ghosts. In addition, ghosts with internal osmolality of 40 to 300 mosmol/kg were prepared by adding different amounts of concentrated buffer before resealing for one hour at 37 degrees C. Under these conditions, the final ghost volume was inversely related to the resealing osmolality (i.e., lower osmolality yielded a larger volume). Ghost volume also increased, along with Hb content, if the quantity or concentration of the red cell suspension added to the lysing medium was increased. We conclude that resealed ghost volume is influenced by the ratio of lysate to resealing medium osmolality and by the colloid osmotic pressure of the residual ghost Hb. These data indicate methods by which ghosts with desired characteristics can be prepared, and have potential application for studies of ghost mechanical and biophysical behavior.     

Equilibrium-binding studies of pig laryngeal cartilage proteoglycans with hyaluronate oligosaccharide fractions.

The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 x 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 x 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) a method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol..wt. 670 000), an average Kd of 12 x 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.     

The preparation and properties of folate-binding protein from cow''s milk.

An improved affinity-chromatographic method for the preparation of folate-binding protein from cow's milk is described. Under dissociating conditions the protein appeared homogeneous in the ultracentrifuge, with a molecular weight of 35 000 +/- 1500, but it was heterogeneous on electrophoresis and ion-exchange chromatography and evidently consisted of several glycoproteins with similar molecular weights that all bound folic acid. Overall, the protein contained a high proportion of half-cystine (18 residues/molecule) and 10.3% of carbohydrate. At saturation it bound approx. 1 mol of folate/mol of protein at pH 7.2. Equilibrium-dialysis measurements of the binding of folic acid and 5-methyltetrahydrofolate to the purified protein gave non-linear Scatchard plots, the shapes of which depended on pH. The results were interpreted in terms of ligand binding to a polymerizing system in which the affinity of ligand for monomer was greater than its affinity for polymer. When the protein concentration was similar to that in cow's milk, dissociation constants (Kd) for folate and 5-methyltetrahydrofolate were 3 nM and 5 nM respectively at pH 7.2 and 37 degrees C, whereas Kd for the binding of folate to monomer was about 50 pM. The properties of the binding protein are discussed in relation to its possible role in folate absorption in the gut.     

Binding of diclofenac sodium with bovine serum albumin at different temperatures, pH and ionic strengths

The study was designed to examine the binding of diclofenac sodium with bovine serum albumin (BSA) at different temperatures (20 degrees, 30 degrees and 40 degrees C), pH (6.4, 7.4 and 8.4) and ionic strengths (micro = 0.1, 0.2 and 0.3) by means of equilibrium dialysis method. The concentration of diclofenac sodium was maintained at wider range from 15 to 900 micromole/l and BSA concentration was maintained at 61.5 micromole/l. The data obtained were interpreted by nonlinear regression method using Graphpad prism software. The analysis showed that the interaction between diclofenac sodium with BSA results in two-site saturable binding. A decrease in association constant was observed with increasing temperature. The average standard free energy change (deltaGdegrees) value was -7.07 (site I) and -4.2 (site II) Kcal/mol. The standard enthalpy change (deltaHdegrees) and the standard entropy change (deltaSdegrees) were -7.8 Kcal/mole, -2.35 cal/mole (site I) and -7.4 Kcal/mole, -10.5 cal/mole (site II), respectively. The negative enthalpy change suggested the binding between diclofenac sodium and the binding sites of BSA were spontaneous and exothermic. The negative value of deltaHdegrees and deltaSdegrees indicated hydrogen bonding and van der Waal's force was the major mechanism for diclofenac sodium and BSA interaction. Increase in pH and ionic strength also caused decrease in association constant of diclofenac sodium and BSA binding.     

Anion transport in red blood cells and arginine-specific reagents: The location of [14C]Phenylglyoxal binding sites in the anion transport protein in the membrane of human red cells

The reaction of phenylglyoxal, a reagent specific for arginine residues, with erythrocyte membrane at pH 7.4 results in complete inhibition of sulfate equilibrium exchange across human red cells. The inactivation was found to be concentration and time depenent. The binding sites of this reagent in the anion transport protein (band 3) under these conditions were determined by using [¹⁴C]phenylglyoxal. The rate of incorporation of the radioactivity into band 3 gave a good correlation with the rate of inactivation. Under conditions where the transport is completely inhibited about 6 mol [¹⁴C]phenylglyoxal are incorporated into 1 mol band 3. Treating the [¹⁴C]phenylglyoxalated ghosts at different degrees of inactivation with extracellular chymotrypsin showed that about two-thirds of these binding sites are located on the 60 kDa fragment.     

Temperature-dependent abnormalities of the erythrocyte membrane in porcine malignant hyperthermia

The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24 degrees C in both MH and normal erythrocyte ghosts. However, below 24 degrees C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33 degrees C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.     

Direct determination of unbound lipophilic ligands in aqueous solutions

Due to their hydrophobic nature, lipophilic compounds are always bound to proteins when transported in the organism. The transfer of such compounds between their binding proteins and cells as well as intracellular trafficking is mediated by a very low water-phase concentration of monomers. The use of protein filled resealed red cell membranes (erythrocyte ghosts) as semipermeable bags enables us to determine directly such water-phase concentrations in a biological system where the lipophilic compound is in equilibrium with the compound bound to its binding protein. Equilibrium dissociation constants (K_d’s) and number of binding sites are determined by regression analyses of data. We describe the method with the hydrophobic anion arachidonate and the neutral N-arachidonoylethanolamide as examples. Published: October 19, 2004.     

Human red-blood-cell Ca2+-antagonist binding sites. Evidence for an unusual receptor coupled to the nucleoside transporter

The human red blood cell ghost Ca2+-antagonist binding sites were characterized with (+/-)-[3H]nimodipine. The labelled 1,4-dihydropyridine bound in a non-cooperative, reversible manner with a Kd of 52 nM at 25 degrees C to 9.65 pmol sites/mg ghost protein. The stereochemistry of the binding domain was evaluated with the optically pure enantiomers of chiral 1,4-dihydropyridines. In contrast to the 1,4-dihydropyridine-selective receptors on Ca2+ channels in electrically excitable tissues, the (+) enantiomer of nimodipine and the (-) enantiomer of the benzoxadiazol 1,4-dihydropyridine (PN 200-110) were bound with higher affinity than the respective optical antipodes. The human red blood cell ghost [3H]nimodipine-labelled sites also interacted with the inorganic Ca2+-antagonist La3+ (increase in the number of binding sites), and were allosterically regulated by the optical enantiomers of the phenylalkylamine-type Ca2+-antagonists (e.g. verapamil, desmethoxyverapamil, methoxyverapamil). The benzothiazepines d- or l-cis-diltiazem were without effect. Nucleosides (adenosine approximately equal to inosine greater than cytidine) were inhibitory at the nimodipine-labelled site, as were the nucleoside uptake inhibitors dipyridamole, hexobendine, dilazep, nitrobenzylthioinosine and nitrobenzylthioguanosine. The binding sites have essential sulfhydryl groups, show trypsin sensitivity, but are relatively heat stable. When nitrobenzylthioinosine was employed as a covalent probe to inactivate the red blood cell ghost nucleoside carrier, [3H]nimodipine binding was irreversibly lost. (+)-Nimodipine greater than (-)-nimodipine inhibited [14C]adenosine transport into human red blood cells. A good correlation between IC50 values for inhibition of [3H]nimodipine binding and IC50 values for inhibition of [14C]adenosine uptake was found for 18 compounds. Sheep red blood cells (which lack the nucleoside transporter) had no detectable [3H]nimodipine binding sites. It is concluded that the Ca2+-antagonist receptor sites of the human erythrocyte are coupled to the nucleoside transporter.     

Concanavalin A binding to oligosaccharide chain leads to alterations in properties of band 3

Anion transport activity and thermotropic behavior of Band 3 are found to be altered after binding of concanavalin (Con A) to human erythrocyte ghosts and isolated Band 3. At lower Con A concentration, the rate coefficients of anion transport enhance with increasing Con A concentration, while noticeable changes of the largest calorimetric endotherm of human erythrocyte membranes termed the C transition (Band 3) can not be observed. With 50 micrograms/ml of Con A, the rate coefficient of Con A-modified ghosts increases 34.4% in comparison with that of normal ghosts. Binding of Con A in lower concentration to ghosts bring about increase of fluidity of lipid which maybe contribute to increase anion transport via Band 3. At higher Con A concentration, the C transition tend to lower temperature with increase in Con A concentration, the C transition is shifted from 69.25 degrees C to 66.25 degrees C with 2.5 mg/ml Con A. It is suggested that the Con A-modified Band 3 possess a looser structure than normal one.     

19F-NMR spin-spin relaxation (T2) method for characterizing volatile anesthetic binding to proteins. Analysis of isoflurane binding to serum albumin.

This paper characterizes the low-affinity ligand binding interactions of a fluorinated volatile anesthetic, isoflurane (CHF2OCHClCF3), with bovine serum albumin (BSA) using 19F-NMR transverse relaxation (T2). 19F-NMR spectra of isoflurane in aqueous BSA reveal a single isoflurane trifluoromethyl resonance, indicative of rapid exchange of isoflurane between protein-bound and aqueous (free) environments. The exchange is slow enough, however, that the chemical shift difference between bound and free isoflurane (delta omega = 0.545 ppm) contributes to the observed isoflurane T2. The contribution of delta omega to T2 can be minimized by shortening the interval between 180 degrees refocusing pulses in the Carr-Purcell-Meiboom-Gill pulse sequence used to monitor T2. Analysis of the dependence of T2 on interpulse interval additionally allows determination of the T2 (6.2 ms) and the average lifetime (tau b = 187 microseconds) of bound isoflurane molecules. By use of a short interpulse interval (less than 100 microseconds), T2 measurements can readily be used to analyze equilibrium binding of isoflurane to BSA. This analysis revealed a discrete saturable binding component with a KD = 1.4 mM that was eliminated either by coincubation with oleic acid (6 mol/mol of BSA) or by conversion of BSA to its "expanded" form by titration to pH 2.5. The binding was independently characterized using a gas chromatographic partition analysis (KD = 1.4 mM, Bmax = 3-4 sites). In summary, this paper describes a method whereby T2 measurements can be used to characterize equilibrium binding of low-affinity ligands to proteins without the confounding contributions of chemical shift.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metal binding and metal-induced conformational change of frog bone gamma-carboxyglutamic acid-containing protein

Ca2+ or Cd2+ binding and the conformational change induced by the metal binding in two frog bone Gla-proteins (BGP, termed BGP-1 and BGP-2) were studied by equilibrium dialysis and CD measurement. By CD measurement in the far-ultraviolet region, the alpha-helix content of both apoBGPs was found to be 8%. Binding of both Ca2+ and Cd2+ was accompanied with a change in the CD spectrum, and the alpha-helix content increased to 15 and 25% for BGP-1 and BGP-2, respectively. CD measurement in the near-ultraviolet region indicated that the environment of aromatic amino acid residues in the protein molecule was changed by metal binding. Equilibrium dialysis experiments indicated that each of these two protein binds specifically 2 mol of Ca2+, and nonspecifically an additional 3-4 mol of Ca2+ in 0.02 M Tris-HCl/0.15 M NaCl (pH 7.4), at 4 degrees C. According to the two separate binding sites model, BGP-1 has 1 high-affinity Ca2+ binding site (Kd1 = 0.17 mM) and 1 low-affinity site (Kd2 = 0.29 mM), and BGP-2 contains 1 high-affinity site (Kd1 = 0.14 mM) and 1 low-affinity site (Kd2 = 0.67 mM). In addition, 2 Cd2+ bound to a high-affinity binding site on BGP-1 with Kd1 of 10.4 microM, and 1 Cd2+ bound to a low-affinity binding site with Kd2 of 41.5 microM. On the other hand, BGP-2 had three classes of binding sites and 1 Cd2+ bound to each binding site with Kd1 = 3.6 microM, Kd2 = 16.3 microM, Kd3 = 51.7 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium binding to the epidermal growth factor homology region of bovine protein C

A high affinity calcium binding site that is independent of the gamma-carboxyglutamic acid-rich amino-terminal region, has been demonstrated in bovine protein C, as well as in the other vitamin K-dependent proteins (except prothrombin) involved in blood coagulation. gamma-Carboxyglutamic acid-independent calcium binding in protein C is required for its rapid activation by the thrombin-thrombomodulin complex. We have now isolated a Ca2+-binding fragment from a tryptic digest of bovine protein C. The isolated fragment contains the two domains that are homologous to the epidermal growth factor precursor from the light chain of protein C, and a small disulfide bound peptide derived from the heavy chain. The isolated fragment bound 1 mol of Ca2+/mol of protein with a dissociation constant (Kd) of approximately 1 x 10(-4) M. This is similar to the Kd previously determined for binding of a single Ca2+ ion to protein C lacking the gamma-carboxyglutamic acid region. Immunochemical evidence indicated that Ca2+ binding induced a conformational change both in protein C lacking the gamma-carboxyglutamic acid region and in the isolated fragment.     

Temperature dependence of anion transport inhibitor binding to human red cell membranes

The binding characteristics of the inhibitor of anion transport in human red cells, 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS), to the anion transport protein of red cell ghost membranes in buffer containing 150 mM NaCl have been measured over the temperature range 0-30 degrees C by equilibrium and stopped-flow fluorescence methods. The equilibrium dissociation constant Keq, increased with temperature. No evidence of a 'break' in the ln(Keq) vs. 1/T plot was found. The standard dissociation enthalpy and entropy changes calculated from the temperature dependence are 9.1 +/- 0.9 kcal/mol and 3.2 +/- 0.3 e.u., respectively. Stopped-flow kinetic studies resolve the overall binding into two steps: a bimolecular association of DBDS with the anion transport protein, followed by a unimolecular rearrangement of the DBDS-protein complex. The rate constants for the individual steps in the binding mechanism can be determined from an analysis of the concentration dependence of the binding time course. Arrhenius plots of the rate constants showed no evidence of a break. Activation energies for the individual steps in the binding mechanism are 11.6 +/- 0.9 kcal/mol (bimolecular, forward step), 17 +/- 2 kcal/mol (bimolecular, reverse step), 6.4 +/- 2.3 kcal/mol (unimolecular, forward step), and 10.6 +/- 1.9 kcal/mol (unimolecular, reverse step). Our results indicate that there is an appreciable enthalpic energy barrier for the bimolecular association of DBDS with the transport protein, and appreciable enthalpic and entropic barriers for the unimolecular rearrangement of the DBDS-protein complex.     

A thrombin receptor in resident rat peritoneal macrophages.

Resident rat peritoneal macrophages possess 6 x 10(2) high-affinity binding sites per cell for bovine thrombin with a Kd of 11 pM, and 7.5 x 10(4) low-affinity sites with a Kd of 5.8 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labeled bovine thrombin is achieved after 1 min at 37 degrees C, and after 12 min at 4 degrees C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 0.27 and 0.06 min-1 at 4 degrees C. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radio-activity migrates as intact thrombin upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3 treatment, and the receptor does not mediate a quantitatively important degradation of the ligand. The binding is not dependent on the catalytic site of thrombin, since irreversibly inactivated thrombin also binds to the receptor. 125I-labeled thrombin covalently cross-linked to its receptor migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr 160,000, corresponding to an approximate receptor size of Mr 120,000.     

Red cell membrane alterations in human chronic fluoride toxicity.

Red cells from humans exposed chronically to toxic levels of fluoride through drinking water showed significant increase in lipid peroxidation and membranous cholesterol and phospholipids. Additionally, electrophoretic patterns of ghost membrane proteins revealed the presence of a new band in the range of congruent to 66 Kd and increase in the high molecular weight protein and predominance of bands with a molecular weight of congruent to 93 Kd and congruent to 20 Kd. The activities of total, Na(+)-K(+)-, Mg(2+)- and Ca(2+)-ATPases were significantly decreased in the red cell ghosts of fluorotic patients.