3H-spiroperidol binding sites in blood platelets

3H-spiroperidol, an antagonist of dopamine receptors in brain (striatum), was found to bind to human and rat platelet membrane preparations. The binding was rapid, reversible, saturable and specific. Unlabelled haloperidol displaced the specifically bound 3H-spiroperidol. Binding equilibrium was attained in 15 min at pH 7.4 and 37 degrees C. Scatchard analysis of 3H-spiroperidol binding revealed a single population of binding site with Kd of 7.6 nM in rat platelet membrane and Kd of 15 nM in human platelet membrane. Unlabelled 5-hydroxytryptamine produced no significant effect on 3H-spiroperidol binding to rat or human blood platelet membranes in the presence or absence of haloperidol. Some dopaminergic agents, known to inhibit spiroperidol binding in corpus striatum, also inhibited the same in rat and human blood platelet membranes under in vitro conditions. This study suggests the presence of specific 3H-spiroperidol binding sites in blood platelets.     

Heparin and heparan sulfate binding sites on B-16 melanoma cells

We have reported previously that the production of a tumor cell factor that stimulates synthesis of fibroblast collagenase is influenced by a fibroblast-deposited matrix component, possibly heparan sulfate-proteoglycan. In this study, binding sites for heparin and heparan sulfate on mouse B-16 melanoma cells have been demonstrated. Binding of 3H-heparin and 35S-heparan sulfate has been shown to occur to whole cells, isolated membranes, and to a component(s) of detergent extracts of the membranes. Scatchard analysis of binding of 3H-heparin yielded a Kd of 2-5 x 10(-8) M and a Bmax of 0.5 x 10(7) heparin molecules bound per cell. Binding of 35S-heparan sulfate was of at least an order of magnitude lower affinity than heparin, but the Bmax was similar to that for heparin. Competition studies showed that 35S-heparan sulfate binding was inhibited totally by heparin and heparan sulfate and partially by dermatan sulfate, but no inhibition was obtained with hyaluronate or chondroitin sulfate. Binding of 3H-heparin was inhibited totally by heparin but to different extents by preparations of heparan sulfate from different tissue sources. The heparin/heparan sulfate binding activity is a protein(s) because it is destroyed by treatment with trypsin. Binding of 3H-heparin to transblots of the detergent extract of the B-16 cell membranes indicated that at least part of the binding activity is a 14,000-dalton protein.     

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.     

Characterization of neuropeptide Y binding sites in rat cardiac ventricular membranes

Neuropeptide Y (NPY) binding sites in rat cardiac ventricular membranes have been characterized in detail. 125I-NPY bound to the membranes with high affinity. Binding was saturable, reversible and specific, and depended on time, pH and temperature. Analysis of the binding data obtained under optimal conditions, 2 hr, 18 degrees C and at pH 7.5, revealed the presence of low and high affinity binding sites. The high affinity binding sites had an apparent dissociation constant (Kd) of 0.38 nM and a binding capacity (Bmax) of 7.13 fmol/mg protein. The apparent Kd and Bmax for low affinity binding sites were 22.34 nM and 261.25 fmol/mg protein, respectively. Peptides unrelated to NPY did not compete with 125I-NPY for the binding sites even at 1 microM concentrations, whereas homologous peptides, peptide YY (PYY) and pancreatic polypeptide (PP), and NPY(13-36) inhibited 125I-NPY binding but with lower potency compared to NPY. 125I-NPY binding was sensitive to the nonhydrolyzable GTP analog, Gpp(NH)p, suggesting that the NPY receptor is coupled to the adenylate cyclase system. The ventricular membrane receptor characterized in this study may play an important role in mediating the physiological effects of NPY in the heart.     

Interaction of calmodulin with the particulate fraction of cardiac muscle

Tritiated calmodulin (T-CM) was bound to the EGTA-treated particulate fraction of cardiac muscle in a calcium-dependent manner with half-maximal binding occurring between 0.8 to 1.2 microM calcium. Binding exhibited high specificity at an optimum pH of 7.4-7.6. An excess of parvalbumin and other globular proteins did not displace T-CM. The Kd for the interaction was 2.5 +/- 0.83 microM. Binding was trypsin-sensitive, inhibited by high ionic strength and was heat inactivated at a midpoint of 48 - 50 degrees C. Competitive displacement of T-CM occurred with unlabeled troponin C and calmodulin over the same concentration range. The first-order rate constant of T-CM dissociation was 3.27 min-1. Calcium-dependent binding of T-CM was inhibited equally by both mepacrine and trifluoperazine with 50 percent inhibition occurring at 70 microM.     

Motilin receptors in rabbit stomach and small intestine

Motilin receptors in rabbit antral and duodenal smooth muscle tissue were characterized by direct binding technique using 125I-labeled porcine motilin as a tracer ligand. Binding at 30 degrees C was maximal at 90 min, was saturable and partially reversible. Displacement studies with natural porcine motilin, synthetic leucine-motilin or norleucine-motilin indicated a dissociation constant (Kd) of 1.1 +/- 0.3 nM and a maximal binding capacity (Bmax) of 42 +/- 10 fmol/mg protein. Binding was unaffected by glucagon, pancreatic polypeptide and somatostatin, but substance P interfered via an unknown mechanism. By density gradient centrifugation motilin receptors were shown to be present in plasma membranes. Binding could only be demonstrated in preparations from antrum and upper duodenum. These observations provide evidence for a localized target region for motilin in the gastrointestinal tract, and for a direct interaction of motilin with gastrointestinal smooth muscle tissue.     

Cyclic AMP-dependent protein kinase phosphorylation of cardiac (Na+ + K+)-ATPases. Effect on calcium binding.

1. Calcium binding to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from beef and pig heart preparations of varying degrees of purity was measured. 2. Binding was inhibited by Mg2+, Na+ and K+. Inhibition by Na+ and K+ appeared to be due to an ionic strength effect. 3. Four classes of binding sites were identified with Kd values for calcium of about 0.03, 1, 15 and 200 micrometer. 4. Cyclic AMP-dependent phosphorylation of the enzyme by protein kinase (ATP: protamine O-phosphotransferase, EC 2.7.1.70) had no effect on (Na+ + K+)-ATPase activity. 5. Phosphorylation also had no effect on either Kd or Bmax for calcium binding at any of the four sites whether measured in the presence of absence of NaCl or KCl. 6. It is concluded that previous reports of an effect of phosphorylation on calcium binding to a (Na+ + K+)-ATPase preparation may have been due to the presence of membrane material not directly associated with (Na+ + K+)-ATPase.     

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.     

On the binding of dolichol by bovine liver supernatant

Experimental evidence is presented that a bovine liver pH 5.1 supernatant possesses binding capacity towards dolichol. Optimal binding is found at physiological pH and at 5 degrees C. At higher temperature the binding is drastically reduced. After binding, the labelled ligand cannot be chased by unlabelled dolichol. Scatchard analysis indicates a single class of binding sites (Bmax = 3.6 pmol/mg protein) with an apparent Kd of 1.8 X 10(-11) M. Only dolichol and dolichyl derivatives drastically reduce the binding phenomenon. The involvement of a protein-like structure is inferred from ammonium sulphate precipitation and proteolysis experiments. Exclusion chromatography and gel electrophoresis under nondenaturating conditions indicate a high molecular weight of the binding complex. Upon SDS electrophoresis, bound [3H]dolichol comigrates with a single protein band (Mr approximately equal to 25,000).     

Characterization of central melatonin receptors using 125I-melatonin

The binding of 125I-melatonin, a potent analog of melatonin, to rat brain synaptosomal preparations was investigated. 125I-melatonin bound with high affinity (Kd = 38 nM) to a single class of sites (Bmax = 81 fmol/mg protein). Kinetic studies indicated that binding was time-dependent and reversible. Specific 125I-melatonin binding was inhibited by melatonin, and was unaffected by other structurally related compounds including serotonin. Binding of 125I-melatonin was greatly reduced if the synaptosomal preparations were pretreated by heat or trypsin but was unaffected by freeze-thawing. These results suggest that 125I-melatonin may serve as a valuable probe for studying melatonin receptors.     

Binding of hyaluronate to the surface of cultured cells

The binding of hyaluronate to SV-3T3 cells was measured by incubating a suspension of cells (released from the substratum with EDTA) with 3H-labeled hyaluronate and then applying the suspension to glass fiber filters which retained the cells and the bound hyaluronate. The extent of binding was a function of both the concentration of labeled hyaluronate and the cell number. Most of the binding took place within the first 2 min of the incubation and was not influenced by the presence or absence of divalent cations. The binding of labeled hyaluronate to SV-3T3 cells could be prevented by the addition of an excess of unlabeled hyaluronate. High molecular weight preparations of hyaluronate were more effective in preventing binding than low molecular weight preparations. The binding of [3H]hyaluronate was inhibited by high concentrations of oligosaccharide fragments of hyaluronate consisting of six sugars or more, and by chondroitin. The sulfated glycosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, heparin, and heparan sulfate) had little or no effect on the binding. The labeled hyaluronate bound to the cells could be totally removed by incubating the cells with testicular hyaluronidase, streptomyces hyaluronidase, or trypsin, indicating that the hyaluronate-binding sites are located on the cell surface.     

HPLC-purified 2-[125I]iodomelatonin labels multiple binding sites in hamster brain

Binding of 2-[125I]iodomelatonin in hamster brain synaptosomal membranes at 0 degrees C is rapid, saturable, reversible and sensitive to heat and trypsin treatment. Computer resolution of curvilinear Scatchard plots yielded high- and low-affinity components as follows: Kd1 = 0.32 +/- 0.14 nM, Bmax1 = 5.6 +/- 1.7 fmol/mg protein and Kd2 = 10.5 +/- 3.2 nM, Bmax2 = 123 +/- 33 fmol/mg protein (n = 3). Competition experiments indicated that 2-iodomelatonin and prazosin are the most potent inhibitors of high-affinity binding. Unlike prazosin, several alpha-adrenergic agents and various neurotransmitters were ineffective. These findings suggest that prazosin may be a potent antagonist at a unique, non-alpha-adrenergic, high-affinity binding site for melatonin.     

Modulation of 3H-prostaglandin E2 binding sites in the rabbit gastric mucosa

The binding of 3H-prostaglandin E2 (PGE2) to rabbit gastric mucosa was investigated. Binding depended on incubation time, temperature and pH, and was saturable and reversible. Scatchard plot analysis revealed a single class of binding sites with a dissociation constant (Kd) of 5.33 +/- 0.21 nM and a maximum number of binding sites (Bmax) of 138.1 +/- 3.4 fmol/mg protein. PGE1 and 16,16-dimethyl PGE2 potently competed with 3H-PGE2 for the binding sites of gastric mucosa, whereas PGA2, PGF2 alpha, 6-keto PGF1 alpha and thromboxane B2 were less potent. The gastric mucosa prepared from the rabbits given indomethacin (5 mg/kg s.c. three times) showed a lower Kd (2.47 +/- 0.19 nM) for 3H-PGE2 than that from untreated one. Treatment with a PGE1 analog, misoprostol (320 micrograms/kg s.c. three times) lowered the Bmax to 74.1 +/- 2.4 fmol/mg protein without any significant effect on the Kd value. It is concluded that rabbit gastric mucosa has specific binding sites for 3H-PGE2 which may be modulated by the levels of PGs in vivo.     

Interaction of single-chain urokinase-type plasminogen activator with human endothelial cells

The interaction of urokinase-type plasminogen activators with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) express receptors for single-chain urokinase (scu-PA) on the cell surface and examined the effect of such binding on plasminogen activator activity. Binding of 125I-labeled scu-PA to HUVEC, performed at 4 degrees C, was saturable, reversible, and specific (k+1 4 +/- 1 X 10(6) min-1 M-1, k-1 6.2 +/- 1.4 X 10(-3) min-1, Kd 2.8 +/- 0.1 nM; Bmax 2.2 +/- 0.1 X 10(5) sites/cell; mean +/- S.E.). Binding of radiolabeled scu-PA was inhibited by both natural and recombinant wild-type scu-PA, high molecular weight two-chain u-PA (tcu-PA), catalytic site-inactivated tcu-PA, an amino-terminal fragment of u-PA (amino acids 1-143), and a smaller peptide (amino acids 4-42) corresponding primarily to the epidermal growth factor-like domain. Binding was not inhibited by low molecular weight urokinase or by a recombinant scu-PA missing amino acids 9-45. Cell-bound scu-PA migrated at its native molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of plasminogen, scu-PA bound to endothelial cells generated greater plasmin activity than did scu-PA in the absence of cells. In contrast, when tcu-PA was added directly to HUVEC, sodium dodecyl sulfate-stable complexes formed with cell or matrix-associated plasminogen activator inhibitors with a loss of plasminogen activator activity. These studies suggest that endothelial cells in culture express high affinity binding sites for the epidermal growth factor domain of scu-PA. Interaction of scu-PA with these receptors may permit plasminogen activator activity to be expressed at discrete sites on the endothelial cell membrane.     

Binding and endocytosis of heparin by human endothelial cells in culture

Binding of heparin and low molecular weight heparin fragments (CY 222, Mr range 1500-8000) to human vascular endothelial cells was studied. Primary culture of human umbilical vein endothelial cells and either 125I or 3H-labeled heparin or [125I]CY 222 were used. Slow, saturable and specific binding was found. No other tested glycosaminoglycan, excepting a highly sulfated heparan fraction, was able to compete for heparin binding. Two groups of binding sites for [3H]heparin could be distinguished: one with high affinity (Kd = 0.12 microM) and another with lower affinity (Kd = 1.37 microM) and a relative large capacity of binding (1.16 X 10(7) molecules/cell) was calculated. The Kd for unlabeled heparin, as calculated from competition experiments, was 0.23 microM. Much lower affinity was calculated for unlabeled low molecular weight heparin fragments CY 222 (Kd = 4.3 microM) from competition experiments with [125I]CY 222. The binding reversibility was only partial for unfractionated heparin. Even by chasing with unlabeled compound, a fraction of 25-30% was not dissociable from endothelial cells. This fraction was much lower if incubation was carried out at 4 degrees C. The addition of basic proteins (histones) to the incubation medium greatly enhanced the undissociable binding at 37 degrees C, but not at 4 degrees C. The undissociable fraction of heparin was not available to degradation by purified microbial heparinase. These results suggest that a fraction of bound heparin is internalized by the vascular endothelium.     

Studies on the binding of a 32K rat epididymal protein to rat epididymal spermatozoa

A glycoprotein of molecular weight 32K has been isolated and purified from the rat caudal epididymal fluid by gel filtration, ion-exchange and affinity chromatography. The highly purified protein was labeled with radioactive iodine and the binding of the 125I-labeled 32K rat epididymal protein (REP) to washed rat caudal epididymal sperm was studied under various conditions. Scatchard plots of the binding data revealed two binding kinetics. One bound with high affinity (KD = 2.6 X 10(-10) ) but low capacity. The other bound with lower affinity (KD = 2.2 X 10(-9)M) but high capacity. The rate of binding of the labeled protein to sperm was dependent on the temperature of the incubation medium. At the scrotal temperature of 33 degrees C, maximal binding was obtained after 40 min. However, at 22 degrees C equilibrium state was reached after 90 min and at 0 degrees C, the equilibrium rate was not reached even after 120 min of incubation. Binding showed dependence on extracellular pH (optimal pH at 4) and ionic strength of the incubation medium. High ionic strength was found to inhibit binding of the 125I-labeled 32K REP to rat caudal epididymal sperm. Specific binding was abolished by 100-fold molar excess unlabeled 32K REP or by native rat caudal epididymal fluid proteins, but not by albumin or ovalbumin. This indicates high specificity of binding. This study has provided direct evidence for the interaction of an epididymal protein with epididymal spermatozoa.     

Protein binding and stability of norepinephrine in human blood plasma. Involvement of prealbumin, alpha 1-acid glycoprotein and albumin

The binding of norepinephrine (NE) to plasma proteins of fresh human blood obtained from healthy volunteers was studied by ultrafiltration at different NE concentrations and incubation times at 37 degrees C. At 1.7 nM L-[3H]-NE binding was approximately 25%. The binding was rapid and was not influenced by the incubation time. [3H]-NE could be dissociated from its binding sites by acid precipitation and, after HPLC, showed to be unchanged NE. No difference in NE binding was found between plasma collected in EGTA-GSH or heparin solution. There was no degradation of NE when incubated in plasma at 37 degrees C for 10 h, even without the addition of antioxidants. Therefore, in the present study, binding represented interaction of unchanged NE with plasma proteins. The whole plasma binding was saturable over the range of 0.66 nM to 0.59 mM of NE. Scatchard plot of specific binding revealed high-affinity sites with a Kd of 5.4 nM and a Bmax of 3.9 fmoles.mg-1 protein, and low-affinity sites with a Kd of 2.7 microM and a Bmax of 3.3 pmoles.mg-1 protein. Electrophoretic characterization of NE-binding proteins showed that about 60% of bound NE was associated to albumin, and 20% to prealbumin. NE binding to pure human plasma proteins was also studied using ultrafiltration. Scatchard analyses revealed a single class of very high-affinity binding sites for prealbumin (Kd 4.9 nM), a single class of binding sites for alpha 1-acid glycoprotein (Kd 54 microM) and two classes of binding sites for albumin with high (Kd 1.7 microM) and low (Kd 0.8 mM) affinities respectively. The main results obtained in this study - a) reversibility of NE binding, b) stability of free and bound NE in plasma, c) involvement of the prealbumin as a specific binding protein - point out to a specific transport for NE in human blood plasma.     

Solubilization and characterization of kainate receptors from goldfish brain

The binding of [3H]kainate to goldfish brain membrane fragments was investigated. Scatchard analysis revealed a single class of binding sites in Tris-HCl buffer with a Kd of 352 nM and a Bmax of 3.1 pmol/mg wet weight. In Ringer's saline, [3H]kainate bound with a Bmax of 1.8 pmol/mg wet weight and a Kd of 214 nM. Binding in Ringer's saline, but not Tris-HCl buffer, displayed positive cooperativity with a Hill coefficient of 1.15. The [3H]kainate binding sites were solubilized in Ringer's saline using the nonionic detergent n-octyl-beta-D-glucopyranoside. Approximately 30-50% of the total number of membrane-bound binding sites were recovered on solubilization. The Kd of [3H]kainate for solubilized binding sites was approximately 200 nM. The rank order of potency for glutamatergic ligands at inhibiting [3H]kainate binding was identical and the competitive ligands had similar Ki values in both membranes and solubilized extracts. In membrane preparations, [3H]kainate displayed a two component off-rate with koff values of 0.97 min-1 and 0.07 min-1; in solubilized extracts, however, only a single off-rate (koff = 0.52 min-1) was observed. The hydrodynamic properties of n-octyl-beta-D-glucopyranoside solubilized [3H]kainate binding sites was investigated by sucrose density centrifugation. A single well defined peak was detected which yielded a sedimentation coefficient of 8.3 S. The results presented in this report suggest that goldfish brain may provide an ideal system in which to study kainate receptor biochemistry.     

High-density lipoprotein binding to guinea-pig hepatic membranes. Comparison of guinea-pig and human ligands

Human and guinea-pig apo-E-free HDL were incubated with isolated guinea-pig hepatic membranes at 4 and 37 degrees C to determine the effects of temperature and heterologous systems on the equilibrium parameters of HDL binding. Receptor mediated HDL binding was highest at 37 degrees C for both lipoproteins. At 4 degrees C, a higher affinity constant (Kd) was observed when guinea-pig HDL was the ligand relative to human HDL; in contrast, both HDL preparations had similar Kd values at 37 degrees C. Calculated binding and receptor number (Bmax) were higher at both temperatures when guinea-pig HDL was the ligand. These results demonstrate a significant species difference in HDL binding to hepatic membrane which is partially temperature-dependent.     

Characterization of platelet-activating factor binding sites on uterine membranes from pregnant rabbits

One of the earliest signs of endometrial preparation for blastocyst implantation is a localized increase in capillary permeability, an event that is essentially inflammatory in character and thought to be a prerequisite for subsequent decidual tissue formation. Platelet-activating factor (PAF), chemically identified as 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine, is a very potent vasoactive compound that recently has been implicated in the implantation process. In the present study, PAF binding sites are characterized in the rabbit uterus. A specific, reversible, saturable, and thermally labile binding of [3H]PAF to uterine membranes has been demonstrated, exhibiting multiple binding sites. The equilibrium dissociation constant (Kd) of the higher affinity binding site (type 1) was 3.6 +/- 0.4 nM (mean +/- SD) with a binding capacity (Bmax) of 3.4 +/- 1.6 pmol/mg protein. The second (lower affinity) binding site (type 2) had an apparent Kd of 114.6 +/- 13.5 nM and a Bmax of 164.3 +/- 17.6 pmol/mg membrane protein, under the conditions of maximal [3H]PAF binding, 25 degrees C, 150 min. Incubations at 4 degrees C for up to 3 h yielded only 30% of the Bmax observed at 25 degrees C. In crude and purified endometrial membrane preparations in which the PAF binding was predominantly located, the affinity of the binding for PAF was significantly higher than for the whole uterus, giving Kds of 1.5 +/- 0.8 and 0.8 +/- 0.5 nM; these latter values were not significantly different. However, the Bmax values of 3.9 +/- 0.9 pmol/mg protein and 376.8 +/- 163.3 fmol/mg protein for the two endometrial preparations, respectively, did differ significantly. Kinetic analysis at 25 degrees C resulted in a calculated Kd of 3.28 +/- 1.14 nM, which did not differ from the value for for the whole uterus at the same temperature, but was greater than for the endometrial preparations. Using 4 nM [3H]PAF to selectively label only the type 1 binding sites, the relative potencies of PAF and its antagonists in displacing [3H]PAF were lyso-PAF greater than CV3988 greater than PAF greater than U66985 greater than A02405 greater than BN52021 greater than U66982. The antagonists SRI 63,441 and L652,731 were ineffective in displacing [3H]PAF at up to 5000-fold molar excess of [3H]PAF. [3H]Lyso-PAF binding at 4 nM was displaceable by PAF. All cations tested, i.e. Ca2+, Mg2+, K+, Na+, and Li+, inhibited [3H]PAF binding. Serine hydrolase inhibitors, diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), inhibited binding, but bacitracin, leupeptin, and antipain stabilized it.(ABSTRACT TRUNCATED AT 400 WORDS)