Effect of salicyclic acid on gluccorticoid receptor in cultured fibroblasts derived from rat carrageenin granuloma.

The binding activity of [3H]dexamethasone to the specific receptor was studied in the cytoplasmic fraction of a established fibroblast line derived from rat carrageenin granuloma in culture condition. Specific receptor to dexamethasone was demonstrated. Scatchard analysis revealed a single class of binding sites with a dissociation constant for [3H]dexamethasone of 3.64 - 10(-8) M and a concentration of binding sites of 0.825 pmol per mg cytosol protein. The number of cytoplasmic binding sites per cell was calculated at 1.15 - 10(5). Total binding activity to [3H]dexamethasone of the cytoplasmic fraction was enhanced when the cells were cultured in a medium containing salicylic acid was at 37 degrees C. The maximum enhancement was seen at the concentration of 10(-3)M and in 3h treatment of salicylic acid. This enhancement by salicylic acid was lost when cycloheximide was added to the culture medium at the same time. If salicyclic acid was added to the cell free system, it showed no effect on the binding activity. The other non-steroidal anti-inflammatory drugs; phenylbutazone and indomethacin,also enhanced the total binding activity to [3H]dexamethasone of the cytoplasmic fraction at the concentration of 2 - 10(-5) M and 2 - 10(-7) M, respectively.     

Specific binding sites on human phagocytic blood cells for Gly-Leu-Phe and Val-Glu-Pro-Ile-Pro-Tyr, immunostimulating peptides from human milk proteins.

Two immunostimulating peptides were isolated from human milk proteins by enzymatic digestion, the tripeptide GLF and the hexapeptide VEPIPY. These peptides increased the phagocytosis of human and murine macrophages and protected mice against Klebsiella pneumoniae infection. The present study showed that this activity may be correlated to the presence of specific binding sites on human blood phagocytic cells. The receptor molecules implicated were different for the two peptides. [3H]GLF specifically bound to PMNL and monocytes, whereas [3H]VEPIPY only bound to monocytes. The leukemic promyelocytic cell line HL-60 differentiated into granulocytes or into macrophages (depending on inducer used) coroborated these results. Specific binding of [3H]GLF on plasma membrane preparations of human PMNL (20 degrees C) was saturable and Scatchard analysis indicated two classes of binding sites: high-affinity sites of Kd 2.3 +/- 1.0 nM and Bm 60 +/- 9 fmol/mg protein and low-affinity sites of Kd 26.0 +/- 3.5 nM and Bm 208 +/- 45 fmol/mg protein. [3H]GLF binding was inhibited in a concentration-dependent manner by various analogous peptides, such as LLF, GLY, LLY and RGDGLF, but not by RGD, RGDS, VEPIPY and the chemotactic peptide f-Met-Leu-Phe (f-MLF). Only at high concentrations the direct analog MLF competed with labeled GLF. An important inhibitory effect was also observed with C1q component of the complement whereas C3 and BSA were uneffective. Specific binding of [3H]VEPIPY on monocyte membranes (20 degrees C) was saturable and Scatchard analysis was consistent with one class of binding sites of Kd 3.7 +/- 0.3 nM and Bm 150 +/- 6 fmol/mg protein.     

t-[35S]Butylbicyclophosphorothionate Binding Sites in Invertebrate Tissues

Specific high affinity binding of the cage convulsant t-[35S]butylbicyclophosphorothionate (TBPS) was observed in membrane homogenates of housefly heads and crayfish abdominal muscles. [35S]TBPS binding in these two invertebrate tissues was inhibited by biologically active cage convulsants, picrotoxin analogs, and barbiturates. The housefly binding sites were inhibited most potently by several insecticides. Approximately 50% of total binding was displaceable by excess (0.1 mM) nonradioactive TBPS, picrotoxinin, ethyl bicyclophosphate, or dieldrin. Optimal binding assay conditions for housefly homogenates included pH 7.5, 22 degrees C temperature, 0.3 M chloride concentration, and incubation for 60 min; for crayfish homogenates, 4 degrees C temperature and 150-min incubations were optimal. Scatchard plots of equilibrium binding indicated one site in both tissues (KD = 50 nM, Bmax = 250 fmol/mg protein in housefly; KD = 25 nM, Bmax = 100 fmol/mg protein in crayfish). Association kinetics in housefly were consistent with one rate constant (k+1 = 8 X 10(6) M-1 min-1), but dissociation was described better by two rate constants (k-1 = 0.28 min-1 and 0.042 min-1; calculated KD values of 80 nM and 12 nM). Displacement by cage convulsants showed Hill numbers near 0.5, also consistent with two populations of affinity, while displacement by other drugs showed Hill numbers near 1.0. [35S]TBPS binding in insects was most potently inhibited by the insecticides dieldrin (IC50 = 50 nM), aldrin, and lindane (200 nM), in a stereospecific manner, consistent with this binding site being the receptor for biological toxicity. [35S]TBPS binding was also inhibited by relatively high concentrations of some pyrethroid insecticides, such as deltamethrin and cypermethrin (1-2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of covalent binding of [14C]-2-chloro-4-acetotoluidide to microsomes of starling liver and kidney

In this study, we have characterized the covalent binding of [14C]-2-chloro-4-acetotoluidide (CAT) radioactivity to microsomes of starling liver and kidney. The maximal velocity (Vmax) of covalent binding and apparent Michaelis constant (Km) for both tissues were similar. The Vmax for liver and kidney were 52.8 and 68.9 pmol/min/mg protein, and the apparent Kms were 0.54 and 0.87 mM, respectively. The covalent binding of radioactivity to heat-denatured microsomes of liver and kidney was reduced by 62% and 15%, respectively. Incubation at 0 degrees C reduced the binding by 80% to liver and 70% to kidney microsomes. Absence of nicotinamide adenine dinucleotide phosphate (NADP) and molecular O2 reduced the binding to liver microsomes by 36 and 53%, as opposed to 28% increase and 26% decrease in binding to kidney microsomes, respectively. Inducers of cytochrome P450 monooxygenase (P450), phenobarbital, and 3-methylcholanthrene (3-MC), had opposite effects on the covalent binding of [14C]-CAT radioactivity to hepatic and renal microsomes. Phenobarbital increased the binding to hepatic microsomes by 100% and had no effect on binding to renal microsomes. 3-MC, on the other hand, increased the binding to kidney microsomes by threefold and had no effect on the binding to hepatic microsomes. SKF 525A, an inhibitor of P450, inhibited the binding to hepatic microsomes by 60% at 0.5 mM but failed to have any effect on binding to renal microsomes. alpha-Naphthoflavone, another inhibitor of P450, had no effect on the covalent binding of [14C]-CAT radioactivity to microsomes of either tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of dolichyl pyrophosphate N-acetylglucosaminyl intermediates in liver microsomes from hibernating ground squirrels (Citellus citellus L.)

Dolichyl pyrophosphate N-acetyl[14C]glucosamine was synthesized after incubation of liver microsomes from hibernating ground squirrels with UDP-N-acetyl[14C )glucosamine. The radioactivity of glycolipid formed by liver microsomes from hibernating ground squirrels was about 2-fold greater than by liver microsomes from active animals. Addition of exogenous dolichyl phosphate to the incubation mixture increased the formation of dolichyl pyrophosphate N-acetyl[14C]glucosamine by microsomes from both active and hibernating ground squirrels about 6 times. Liver microsomes from hibernating ground squirrels converted dolichyl pyrophosphate N-acetyl[14C]glucosamine into dolichyl pyrophosphate N,N'-diacetyl[14C]chitobiose in the presence of unlabelled UDP-N-acetylglucosamine. This conversion was maximal at 1.0 M concentration of unlabelled UDP-N-acetylglucosamine. The level of dolichyl phosphate assessed by the level of dolichyl pyrophosphate N-acetylglucosamine formation was nearly 2 times greater in liver microsomes from hibernating ground squirrels than from active animals.     

Specific binding of a calcium channel activator, [3H]BAY k 8644, to membranes from cardiac muscle and brain

BAY k 8644 is a member of a new class of drugs that directly activates Ca2+ channels. This 1,4-dihydropyridine was found to bind to both high and low affinity sites on rabbit ventricular microsomes and guinea pig brain synaptosomes. The dissociation constant obtained from Scatchard analysis with [3H]BAY k 8644 was 2 to 3 nM for the high affinity binding site, and the estimated maximal number of binding sites was 0.8 and 0.4 pmol/mg protein for heart and brain membranes, respectively, at 15 degrees C. Competition between nitrendipine and [3H]BAY k 8644 indicated a common high affinity binding site for Ca2+ channel activators and antagonists. The results suggest that the 1,4-dihydropyridine Ca2+ channel antagonists do not act as simple channel plugs.     

Microsomal binding sites for antioestrogens in rat liver. Properties and detergent solubilization.

The properties of an antioestrogen binding site (AEBS), which has high affinity and specificity for nonsteroidal antioestrogens and structurally related compounds, have been studied in rat liver microsomes. When subcellular organelles were separated on Percoll density gradients the distribution of the AEBS paralleled that of NADPH-cytochrome c reductase, indicating that the AEBS is associated with the endoplasmic reticulum. Saturation analysis showed that [3H]tamoxifen was bound to a single class of saturable binding sites in liver microsomes with a KD of 0.9 +/- 0.1 nM at 0 degrees C. The equilibrium KD was not significantly different at 22 degrees C. The KD calculated from the association and dissociation rate constants for [3H]tamoxifen binding at 0 degrees C and 22 degrees C was compatible with the KD measured at equilibrium. Ligand specificity studies using tamoxifen analogues showed qualitatively similar structure-affinity relationships for the AEBS from both rat liver and the MCF 7 breast cancer cell line. In general structural modifications caused correspondingly greater changes in affinity for rat liver AEBS than for MCF 7 AEBS. The AEBS was solubilized from microsomal membranes with sodium cholate. This was the only detergent of nine tested that solubilized the site in high yield without loss of activity. Solubilization using cholate was more effective in the presence of 1 M-NaCl. In the solubilized state there was an apparent loss of [3H]tamoxifen binding activity which could be restored by dilution of the detergent. Gel filtration indicated an Mr of 440,000-490,000 for the AEBS-cholate complex. These studies demonstrate that rat liver contains high concentrations of a microsomal AEBS which has similar properties and specificity to the AEBS previously described in human breast cancer cells. This site can be solubilized by sodium cholate to supply material suitable for further purification.     

125I]EXP985: a highly potent and specific nonpeptide radioligand antagonist for the AT1 angiotensin receptor.

[125I]EXP985 is the first nonpeptide radioligand with high specific activity for the AT1 angiotensin receptor. The biochemical and pharmacological profiles of this ligand were determined using either ligand-receptor binding techniques in rat adrenal cortical microsomes or cellular Ca2+ mobilization in rat smooth muscle cells. Specific binding with 0.1 nM [125I]EXP985 increased slowly with time reaching an equilibrium at 60 min of incubation (22 degrees C). Scatchard analysis of the inhibition/binding data revealed a single class of binding sites having a Kd of 1.49 +/- 0.06 nM and a Bmax of 3.6 +/- 0.1 pmol/mg protein. These sites were saturable and the ligand-receptor complex dissociated with a t1/2 of 58 min. The binding was inhibited by Ang peptides with the following order of potency and IC50 (nM): Ang II (3.7) > Ang III (69) > Ang I (3650), and by the nonpeptide AT1 receptor antagonist, losartan, with an IC50 of 3.2 nM. PD123177, an AT2 selective antagonist, showed minimal inhibitory effect. Specific binding of [125I]EXP985 was found on rat aortic smooth cells. Ang II-induced Ca2+ mobilization in these cells was blocked by EXP985 in a noncompetitive manner. These data show that [125I]EXP985 (or its unlabeled) is a potent and highly specific radioligand or noncompetitive antagonist which represents a novel tool to further our understanding of the biochemistry of AT1 receptors.     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.     

Purification of a functional receptor for calcium-channel blockers from rabbit skeletal-muscle microsomes

The dihydropyridine receptor was purified from rabbit skeletal muscle microsomes in the presence of [3H]nitrendipine plus diltiazem or [3H](+)PN 200-110 to an apparent density of 1.5-2 nmol binding sites/mg protein. Sodium dodecyl sulfate gel electrophoresis in the absence of reducing agents yielded three peptide bands of 142, 56 and 30 kDa in a relative ratio of 11:1:1.3, whereas in the presence of 40 mM dithiothreitol bands of 142, 122, 56, 31, 26 and 22 kDa were obtained in a relative ratio of 5.5:2.2:1:0.9:14:0.09. This gel pattern was observed regardless of whether the receptor was purified as a complex with nitrendipine plus diltiazem or with (+)PN 200-110. cAMP-dependent protein kinase phosphorylated preferentially the 142-kDa band up to a stoichiometry of 0.82 +/- 0.07 (15) mol phosphate/mol peptide. The 56-kDa band was phosphorylated only in substoichiometric amounts. [3H]PN 200-110 bound at 4 degrees C to one site with apparent Kd and Bmax values of 9.3 +/- 1.7 nM and 2.2 +/- 0.3 (3) nmol/mg protein, respectively. The binding was stereospecific and was not observed in the presence of 1 mM EGTA. Desmethoxyverapamil interfered with the binding of [3H]PN 200-110 in an apparent allosteric manner. (-)Desmethoxyverapamil inhibited the binding of [3H]PN 200-110 at 37 degrees C and stimulated it at 18 degrees C. In agreement with these results, (-)desmethoxyverapamil increased the dissociation rate of [3H]PN 200-110 from 0.29 min-1 to 0.38 min-1 at 37 degrees C and decreased it threefold from 0.046 min-1 to 0.017 min-1 at 18 degrees C. The (+)isomer of desmethoxyverapamil inhibited PN 200-110 binding at all temperatures tested. d-cis-Diltiazem stimulated the binding of [3H]PN 200-110 at 37 degrees C with an apparent EC50 of 1.4 microM and decreased the dissociation rate from 0.29 min-1 to 0.11 min-1. The stimulatory effect of d-cis-diltiazem was temperature-dependent and was seen only at temperatures above 18 degrees C. These results suggest that the purified dihydropyridine receptor retains the basic properties of the membrane-bound receptor and contains separate sites for at least dihydropyridines and phenylalkylamines.     

Calcium-induced membrane metabolic alterations modify the sex steroids binding into dog brain synaptosomal plasma membranes

The binding of 45Ca2+ into synaptosomal plasma membranes (SPM) of dog brain follows a sigmoid path. In graphical analysis of this binding the mean Hill coefficient (h) was 1.64 +/- 0.09 (r2 = 0.96 +/- 0.02). Binding of Ca2+ into SPM was saturable, with an apparent binding constant of 1.2 +/- 0.1 microM. At saturation, such calcium specific binding sites corresponded to 11.2 +/- 0.9 nmol/mg SPM protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of calcium into SPM of at least one class of high affinity specific binding sites. [14C]estradiol, [14C]estrone and [14C]progesterone, when incubated with SPM up to a concentration of 10 microM for 2 hr at 37 degrees C, bind into SPM at nmolar concentrations. Ca2+ ions up to 5 mM considerably increase steroids binding into SPM. This effect of calcium was concentration-dependent, reached saturation at approx 4-5 mM. Once calcium has promoted steroids binding, the subsequent addition of 25 mM EGTA failed to displace bound steroids. Molecular interactions between calcium and SPM was assessed by measuring the steady-state fluorescence polarization (P) of 1,6-diphenyl-1,3,5-hexatriene (DPH), and by estimating the production of malondialdehyde (MDA) during 2 hr incubation of Ca2+ (5 mM) with SPM at 37 degrees C. The effect of Ca2+ on the SPM structure was to increase both the rigidity of the membrane and the MDA production. Chelation of Ca2+ (5 mM) with EGTA (25 mM) did not reverse the increase in the rigidity owing to metabolic alterations of SPM lipids (e.g. production of MDA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical Bases for the Loss of Basipetal IAA Transport with Advancing Physiological Age in Etiolated Helianthus Hypocotyls: Changes in IAA Movement, Net IAA Uptake, and Phytotropin Binding

Basipetal transport of [¹⁴C]IAA in hypocotyl segments isolated from various regions of etiolated Helianthus annuus L. cv NK 265 seedlings declines with increasing physiological age. This decline was the result of a reduction in both transport capacity and apparent velocity. Net IAA uptake was greater and the abilities of auxin transport inhibitors to stimulate net IAA uptake were reduced in older tissues. Net IAA accumulation by microsomal vesicles exhibited a similar behavior with respect to age. Specific binding of [³H]N-1-naphthylphthalamic acid (NPA) to microsomes prepared from young and older hypocotyl regions was saturable and consistent with a single class of binding sites. The apparent affinity constants for NPA binding in microsomes prepared from young versus older tissues were 6.4 and 10.8 nanomolar, respectively, and the binding site densities for young versus old tissues were 7.44 and 3.29 picomoles/milligram protein, respectively. Specific binding of [³H]NPA in microsomes prepared from both tissues displayed similar sensitivities toward unlabeled flurenol and exhibited only slight differences in sensitivity toward 2,3,5-triiodobenzoic acid. These results demonstrate that the progressive loss of basipetal IAA transport capacity in etiolated Helianthus hypocotyls with advancing age is associated with substantial alterations in the phytotropin-sensitive, IAA efflux system and they suggest that these changes are, at least partially, responsible for the observed reduction of polar IAA transport with advancing tissue age.     

Covalent binding of an intermediate(s) in prostaglandin biosynthesis to guinea pig lung microsomal protein

We have investigated the possible covalent binding of intermediates in prostaglandin (PG) biosynthesis to tissue macromolecules. Following incubation of arachidonic acid -1-[14]C (AA) with guinea pig lung microsomes, radioactivity was associated with the microsomal protein which was not dissociated from the protein by exhaustive solvent extraction. Furthermore, filtration of the protein complex through a Sephadex G-25 column failed to dissociate the radioactivity from the protein. This probably indicates covalent binding of AA metabolite(s) to protein. [3]H-PGE₂, [3]H-PGF_2α, and [3]H-thromboxane B₂ (TXB₂) did not show this high affinity binding to microsomal protein. The covalent binding of AA metabolites was greatly reduced in denatured microsomes and was inhibited by the addition of glutathione (GSH) or indomethacin to the incubation mixtures. Chromatographic analysis of the water layers obtained from microsomal incubations with either [3]H-AA or [3]H-GSH suggested the presence of one or more glutathione conjugates derived from AA. These studies indicate that most likely an intermediate formed during PG synthesis from AA covalently binds to tissue macromolecules. This covalent binding may be of physiological and pathological significance.     

Binding sites for inositol trisphosphate in the bovine adrenal cortex

Binding sites for inositol trisphosphate (IP3) have been identified in bovine adrenal cortex, employing [32P]IP3 prepared from human erythrocytes radiolabeled with [32P]ATP. IP3 was bound to adrenal microsomes with high affinity (Kd = 5 nM) and low capacity (186 fmol/mg protein). During kinetic studies, half-maximal binding was reached in less than one min at 4 degrees C, and dissociation was even more rapid with t1/2 of about 10 sec. [32P]IP2 showed no binding to the microsomal sites, which represent putative receptors at which IP3 acts to elevate intracellular calcium concentration during the actions of peptide hormones such as angiotensin II.     

Evidence for muscarinic cholinergic receptors in dog portal vein: binding of [3H]quinuclidinyl benzilate

Muscarinic cholinergic receptor sites in dog portal veins were analyzed directly using [3H]quinuclidinyl benzilate (QNB) as a ligand. Specific [3H]QNB binding to crude membrane preparations from the isolated veins was saturable, reversible and of high affinity (KD = 15.5 +/- 2.8 pM) with a Bmax of 110 +/- 14.7 fmol/mg protein. Scatchard and Hill plot analyses of the data indicated one class of binding sites. From kinetic analysis of the data, association and dissociation rate constants of 1.91 X 10(9) M-1 min-1 and 0.016 min-1, respectively, were calculated. The dissociation constant calculated from the equation KD = K-1/K+1 was 8.3 pM, such being in good agreement with the Scatchard estimate of KD (15.5 pM). Specific binding of [3H]QNB was displaced by muscarinic agents. Nicotinic cholinergic agents, alpha-bungarotoxin, nicotine and hexamethonium, were ineffective in displacing [3H]QNB binding at 10 microM. Our findings provide direct evidence for the existence of muscarinic cholinergic receptors in dog portal veins.     

Adenosine binding sites of rat pheochromocytoma PC 12 cell membranes: partial characterization and solubilization

Rat pheochromocytoma PC 12 cell membranes were shown to possess A2-like adenosine binding sites as assessed by using 5'-N-ethylcarboxamide[3H]adenosine [( 3H]NECA). Specific [3H]NECA binding to PC 12 cell membrane at 0 degrees C was saturable and showed a monophasic saturation profile. In contrast, [3H]NECA binding to PC 12 cell membrane at 30 degrees C exhibited a biphasic profile suggesting the presence of two specific binding site. The rank order of potency for inhibition of [3H]NECA binding at 0 degrees C was NECA greater than 2-chloroadenosine greater than 2',5'-dideoxyadenosine greater than isobutylmethylxanthine much greater than phenylisopropyladenosine. These adenosine binding sites were solubilized with sodium cholate and the solubilized portion retained the same ligand binding characteristics as those of the membrane-bound form. Gel filtration experiments indicated an apparent Stokes radius of 6.7 nm for these adenosine binding sites/detergent complexes.     

Identification of specific binding sites for leukotriene C4 in human fetal lung

Specific leukotriene C4 (LTC4)1 binding sites were identified in membrane preparations from human fetal lung. Specific binding of [3H]-LTC4 represented 95 percent of total binding, reached steady-state within 10 minutes and was rapidly reversible upon addition of excess unlabeled LTC4. Binding assays were performed at 4 degrees C under conditions which prevented metabolism of [3H]-LTC4 (80 mM serine-borate, 10 mM cysteine, 10 mM glycine). Under these conditions, greater than 95 percent of the membrane bound radioactivity, as analyzed by high performance liquid chromatography, co-eluted with the LTC4 standard. Computer-assisted analyses of saturation binding data showed a single class of binding sites with a dissociation constant (Kd) of 26 + 6 nM and a density (Bmax) of 84 + 18 pmol/mg protein. Pharmacological specificity was demonstrated by competition studies in which specific binding of [3H]-LTC4 was displaced by LTC4 and its structural analogs with inhibition constants (Ki) of 10 to 30 nM, whereas LTD4, diastereoisomers of LTD1, LTE4 and the end organ antagonist FPL 55712 were 150 to 700 fold less potent competitors than LTC4. These results provide evidence for specific, reversible, saturable, high affinity binding sites for [3H]-LTC4 in human fetal lung membranes.     

Purification and characterization of fatty acid-binding protein from rat kidney

We detected the presence of a fatty acid-binding protein (FABP) in rat kidney cytosols. This protein was eluted and purified 9.3-fold by sequential gel filtration and anion-exchange chromatography. Homogeneity was shown by a single band on polyacrylamide gel with a molecular weight of about 15,500. It had an optimum binding pH of 7.4. The binding of palmitate to the protein was saturable. Examination of fatty acid binding revealed the presence of a single class of fatty acid-binding sites. The apparent dissociation constant was 1.0 microM and the maximal binding capacity was 48 nmol/mg of protein. This protein showed similar binding characteristics for palmitate, oleate, and arachidonate. Rabbit antibody to this cytosolic FABP gave a single precipitin line with the antigen and selectively inhibited [14C]palmitate binding to the protein.     

Characterization and visualization of neurotensin binding to receptor sites in human brain

The binding of monoiodo [125I-Tyr3]-neurotensin to human brain was characterized and visualized using radioreceptorassay and autoradiographic techniques. Specific binding to homogenates of human substantia nigra at 25 degrees C was maximal at 20 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two populations of binding sites with Kd values of 0.26 nM and 4.3 nM. Corresponding binding capacities were 26 and 89 fmol/mg of protein. Neurotensin analogs inhibited the binding of iodinated neurotensin with relative potencies that demonstrated the crucial role of the C-terminal hexapeptide portion of neurotensin for binding to its receptors. Autoradiography of human substantia nigra sections incubated with iodinated neurotensin revealed high levels of specific binding in the nucleus paranigralis and substantia nigra, pars compacta, and low levels in the substantia nigra, pars reticulata.     

Binding of β1-adrenoreceptor antagonist [3H]CGP 26505 in rat cerebral cortex and sinus atrial nodeantagonist [3H]CGP 26505 in rat cerebral cortex and sinus atrial node

Specific β₁-adrenoreceptors antagonist [³H]CGP 26505 binding was characterized in rat cerebral cortex and heart sinus atrial node. In both tissues [³H]CGP 26505 binding was maximal at 25°C, it was specific, saturable and protein concentration dependent. Scatchard analysis of saturation isotherms of specific [³H]CGP 26505 binding in cerebral cortex showed that [³H]CGP 26505 binds a single class of high affinity sites with a dissociation constant (K_D) of 1±0.3 nM and a maximal number of binding sites (B_max) of 40±2 fmol/mg of protein. In sinus atrial node, [³H]-CGP 26505 binds a single class of high affinity sites (K_D=1.9±0.4 nM, B_max=28±2 fmol/mg of protein).