Nongenomic inhibition of oxytocin binding by progesterone in the ovine uterus

Progesterone (P4) has been reported to inhibit oxytocin (OT) binding to its receptor in isolated murine endometrial membranes. The purpose of the present research was to 1). examine the in vivo and in vitro effect of P4 on the binding of OT to its receptor in the ovine endometrium and 2). determine whether the endometrial plasma membranes have high-affinity binding sites for P4. Ovariectomized ewes were pretreated with a sequence of estradiol-17beta (2 days) and P4 (5 days) before being treated with estradiol-17beta plus either vehicle (corn oil), P4, or P4 + mifepristone (RU 486) for 3 consecutive days. Treatment of ewes with 10 mg P4/day for 3 days suppressed binding of OT (P < 0.01) compared with that of controls, whereas concomitant treatment with the progestin antagonist RU 486 (10 mg/day) blocked the effect of P4. Similarly, incubation of endometrial plasma membranes with P4 (5 ng/ml) inhibited binding of OT (P < 0.05), whereas this effect of P4 was blocked by the presence of RU 486 (10 ng/ml). By radioreceptor assay, the endometrial plasma membranes were found to contain a high-affinity binding site for P4 and the progestin agonist promegestone (Kd 1.2 x 10-9 and 1.74 x 10-10M, respectively). Incubation of endometrial plasma membranes with P4 (5 ng/ml) significantly increased the concentration of progestin binding sites. Binding of labeled promegestone (R 5020) was competitively inhibited by excess unlabeled R 5020, P4, RU 486, and OT but not by estradiol-17beta, cortisol, testosterone, and arginine vasopressin. These data suggest a direct suppressive action of P4 on the binding of OT to OT receptors in the ovine endometrial plasma membrane.     

Characterization of the parathyrin receptor in renal plasma membranes by labelled hormone and labelled antibody binding techniques.

The parathyrin receptor in renal cortex has been investigated by studying the binding of 125I-labelled parathyrin, or of unlabelled parathyrin detected with 125I-labelled antibodies, to a partially purified plasma membrane fraction. The kinetics of hormone uptake demonstrated a biphasic response in both systems at 22 degrees C but this phenomenon was not detectable at 37 degrees C. Specific displacement of lactoperoxidase labelled 125I-labelled parathyrin occurred with 8 ng unlabelled bovine parathyrin. The apparent affinity constant was 2.3-10(8) M(-1) and the apparent binding capacity of the membranes 1.25 pmol/mg protein. Using the labelled antibody technique the receptor showed maximal binding at pH 7.0-7.5. As little as 80 pg bovine parathyrin produced a significant increase in binding of labelled anti-bovine parathyrin antibody and saturation of binding sites was demonstrated at 2.5 pmol/mg protein. Oxidized hormone showed undetectable binding. Treatment of membranes with phospholipases A or D, or Trypsin greatly reduced subsequent hormone binding. Prior incubation of membranes with 1-34 synthetic parathyrin decreased the binding of intact hormone whereas gastrin, insulin and glucagon had no effect. Growth hormone and calcitonin slightly increased parathyrin binding.     

Calcitonin modifies ligand binding to muscarinic receptor in CNS membranes

Calcitonin (CT) is a peptide produced by the thyroid gland, whose best described role is to prevent bone reabsorption, though it also participates in other biological functions through both central and peripheral mechanisms. CT is able to inhibit brain Na(+), K(+)-ATPase activity (Rodríguez de Lores Arnaiz, López Ordieres, Peptides 1997;18:613-5) and a relationship between such enzyme activity and cholinergic function has been suggested. Accordingly, we tested CT effect on [(3)H]-quinuclidinyl benzilate ([(3)H]-QNB) binding to rat CNS membranes to determine whether the peptide is able to modify the cholinergic muscarinic receptor as well. It was found that 1x10(-7)-1x10(-5) M CT decreased 20-70% ligand binding to hippocampal, cerebellar, cortical and striatal membranes. Scatchard analysis of saturation curves showed that 5x10(-6) M CT significantly modified binding kinetic constants, thus it increased roughly 220% K(d) values and decreased 20-36% B(max) values in cerebral cortical and cerebellar membranes. Since the peptide decreases affinity ligand binding and reduces the number of binding sites, CT may well be acting as a cholinergic modulator through a decrease in muscarinic receptor functionality.     

The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils. I. Evidence for two binding sites with different affinities

F Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe binding to rabbit peritoneal neutrophils and purified membranes were measured at 4 degrees C silicone oil centrifugation assays, and the results were analyzed by the LIGAND computer program, which permits analysis of ligand binding to multiple classes of binding sites. LIGAND analysis of peptide binding to intact neutrophil indicated that both f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe detected two population of binding sites. The apparent Kd values for f Met-Leu-[3H]Phe binding were 1.6 +/- 1.0 X 10(-9) M and 2.2 +/- 0.9 X 10(-8) M, respectively, and 3.1 +/- 0.2 X 10(-9) M and 1.2 +/- 0.6 X 10(-7) M for f Nle-Leu-[3H]Phe. Furthermore, the higher affinity sites detected on whole cells comprised approximately 15 to 30% of the total sites. Two populations of binding sites were also detected on purified neutrophil plasma membranes by both radiolabeled chemotactic peptides. LIGAND analysis of peptide binding to purified membranes yielded apparent Kd values of 5.0 +/- 2.5 X 10(-10) M and 4.8 +/- 0.6 X 10(-8) M for f Met-Leu-[3H]Phe binding, and 4.7 +/- 4.2 X 10(-10) M and 3.0 +/- 1.3 X 10(-8) M for f Nle-Leu-[3H]Phe. The percentage of higher affinity sites detected by f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe on purified membranes were 1 to 5% of the total sites detected. These data are consistent either with the existence of two independent binding sites for formylpeptides on rabbit neutrophils or receptor negative cooperativity.     

A specific gastrin receptor site in the rat stomach.

A specific receptor for gastrin I has been demonstrated in the rat stomach fundus. Specific binding of 125I-labelled gastrin I was localised to particles sedimenting between 250--20 000 X g. Saturation of binding sites occurred with a gastrin concentration of 10(-11) M in an assay system containing 0.6--1.7 mg/ml of homogenate protein. Gastrin binding was shown to be reversible, temperature- and pH-dependent, and susceptible to tryptic digestion. Electron microscopic and enzymatic studies showed the binding fraction to contain predominantly mitochondria. Preincubation of the homogenate with 10(-8) M cholecystokinin or secretin inhibited gastrin binding to a greater extent than an equimolar concentration of pentagastrin. Cimetidine, a histamine receptor antagonist, did not affect binding of gastrin to the receptor.     

Mechanism of membrane binding by the bovine seminal plasma protein,PDC-109: a surface plasmon resonance study

PDC-109, the major protein of bovine seminal plasma, binds to sperm plasma membranes upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. The binding process is mediated primarily by the specific interaction of PDC-109 with choline-containing phospholipids. In the present study the kinetics and mechanism of the interaction of PDC-109 with phospholipid membranes were investigated by the surface plasmon resonance technique. Binding of PDC-109 to different phospholipid membranes containing 20% cholesterol (wt/wt) indicated that binding occurs by a single-step mechanism. The association rate constant (k(1)) for the binding of PDC-109 to dimyristoylphosphatidylcholine (DMPC) membranes containing cholesterol was estimated to be 5.7 x 10(5) M(-1) s(-1) at 20 degrees C, while the values of k(1) estimated at the same temperature for the binding to membranes of negatively charged phospholipids such as dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidic acid (DMPA) containing 20% cholesterol (wt/wt) were at least three orders of magnitude lower. The dissociation rate constant (k(-1)) for the DMPC/PDC-109 system was found to be 2.7 x 10(-2) s(-1) whereas the k(-1) values obtained with DMPG and DMPA was about three to four times higher. From the kinetic data, the association constant for the binding of PDC-109 to DMPC was estimated as 2.1 x 10(7) M(-1). The association constants for different phospholipids investigated decrease in the order: DMPC > DMPG > DMPA > DMPE. Thus the higher affinity of PDC-109 for choline phospholipids is reflected in a faster association rate constant and a slower dissociation rate constant for DMPC as compared to the other phospholipids. Binding of PDC-109 to dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylethanolamine, which are also zwitterionic, was found to be very weak, clearly indicating that the charge on the lipid headgroup is not the determining factor for the binding. Analysis of the activation parameters indicates that the interaction of PDC-109 with DMPC membranes is favored by a strong entropic contribution, whereas negative entropic contribution is primarily responsible for the rather weak interaction of this protein with DMPA and DMPG.     

Change of coupling system of receptor-adenylate cyclase induced by epinephrine and GTP in plasma membranes of rat liver.

1. The binding of [3H]epinephrine to plasma membranes was affected (temporary release of bound epinephrine and characteristic retardation of epinephrine binding) not only by GTP but also by dGTP and guanylylimidodiphosphate, whereas the binding of [3H]dihydroalprenolol was not affected by GTP. GTP affected the binding of [3H]epinephrine in the presence of alpha-antagonists, but not in the presence of beta-antagonists, suggesting that the GTP effects are specific to beta-agonists and beta-receptors. 2. The half-maximal release of bound [3H]epinephrine was found at 8.8 . 10(-6) M GTP in the absence of ATP, whereas it was found at 1.6 . 10(-6) M GTP in the presence of 0.3 mM ATP in coincidence with the half-maximal activation of adenylate cyclase by GTP in the presence of 0.3 mM ATP (as measured at 30 s of incubation). 3. In the presence of 4 . 10(-5) M GTP, adenylate cyclase activity as measured at 30 s of incubation (State I) tended to increase with epinephrine concentration, showing no saturation tendency even at 1 . 10(-4) M epinephrine. The activity of State II, which is established at 4 min of incubation, was much lower than that of State I but was found to reach a plateau as the epinephrine concentration increased, showing half-maximal activation at an epinephrine concentration between 2 . 10(-6) and 2 . 10(-7) M. 4. Apparent kinetic parameters (Km and V) for State I as assayed at 30 s of incubation suggested that GTP alone may increase V slightly, whereas epinephrine plus GTP may increase the V to a further extent and simultaneously decrease the Km. 5. Adenylate cyclase of plasma membranes pretreated with epinephrine plus GTP was stimulated by GTP alone similarly to untreated membranes, but it was no longer responsive to the synergistic activation by epinephrine plus GTP. Accordingly, the binding of [3H]epinephrine to the pretreated plasma membranes was no longer affected by GTP. 6. The results of the present study seem to support the idea that the most active and coherently coupling state (State I) of the beta-receptor-adenylate cyclase system generated in the presence of epinephrine plus GTP is very labile and degenerates before reaching equilibrium. In turn, State II, in which the coherently coupling mechanism is largely impaired, seems to be established in due time. The characteristic biphasic kinetics of [3H]epinephrine binding in the presence of GTP seem to be related to the above change occurring in the beta-receptor-adenylate cyclase system.     

Properties of follicle-stimulating-hormone receptor in cell membranes of bovine testis.

A simple method for preparing plasma membranes from bovine testes is described. Bovine testicular receptor has a high affinity and specificity for 125I-labelled human FSH (follicle-stimulating hormone). The specific binding of 125I-labelled human FSH to the plasma membranes is a saturable process with respect to the amounts of receptor protein and FSH added. The association and dissociation of 125I-labelled human FSH are time- and temperature-dependent, and the binding of labelled human FSH to bovine testicular receptor is strong and not readily reversible. Scatchard [Ann. N.Y. Acad. Sci. (1949) 51, 660-672] analysis indicates a dissociation constant, Kd, of 9.8 X10(-11)M, and 5.9 X 10(-14)mol of binding sites/mg of membrane protein. The testicular membrane receptor is heat-labile. Preheating at 40 degrees C for 15 min destroyed 30% of the binding activity. Specific binding is pH-dependent, with an optimum between pH 7.0 and 7.5. Brief exposure to extremes of pH caused irreversible damage to the receptors. The ionic strength of the incubation medium markedly affects the association of 125I-labelled human FSH with its testicular receptor. Various cations at concentrations of 0.1M inhibit almost completely the binding of 125I-labelled human FSH. Nuclectides and steroid hormones at concentrations of 1mM and 5mu/ml respectively have no effect on the binding of FSH to its receptor. Incubation of membranes with and chymotrypsin resulted in an almost complete loss of binding activity, suggesting that protein moieties are essential for the binding of 125I-labelled human FSH. Binding of 125I-labelled human FSH to bovine testicular receptor does not result in destruction or degradation of the hormone.     

Neurotensin receptors in canine intestinal smooth muscle: preparation of plasma membranes and characterization of (Tyr3-125I)-labelled neurotensin binding

To study the binding of (Tyr3-125I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%-33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5'-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (Kd1 = 0.118 nM, Kd2 = 3.18 nM, Bmax1 = 9.73 fmol/mg and Bmax2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the Kd value equal to 0.107 nM. Carboxy terminal amino acid residues 8-13 were found to be essential for the binding activity and replacement of Tyr11 by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl2, CaCl2 and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.     

Properties of the interaction between bovine thyrotropin and bovine thyroid plasma membranes.

Studies have been conducted to characterize further the interaction between 125I-labeled bovine thyrotropin (TSH) and bovine thyroid plasma membranes. Sequential subcellular fractionation of thyroid homogenates yielded preparations of progressively greater specific binding activity, highest activity being found in fractions previously shown to contain predominately plasma membranes (Amir, S. M., Carraway, T.F., Kohn, L.D., and Winand, R.J. (1973) J. Biol. Chem. 248, 4092-4100). Although binding of 125I-TSH by plasma membranes was greatest at pH 6.0, studies were conducted at pH 7.45 as well as pH 6.0, and results obtained differed quantitatively, but not qualitatively. Binding was maximal at 0 degrees, 15 degrees, and 22 degrees and steady state values remained unchanged for at least 22 hours. At 37 degrees, binding was decreased by 40% at 1 hour; the loss was even greater (65%) at 50 degrees. A similar loss of binding was evident when membranes were preincubated without TSH at 37 degrees or higher and were then incubated with 125I-TSH at 0 degrees. Lineweaver-Burk analysis indicated that preincubation resulted in loss of receptor sites without change in affinity of residual receptors. Addition of Ca2+ (1 to 10 mM) to the preincubation medium prevented the effect of preincubation at 37 degrees by preserving the number of receptor sites without altering their affinity. Under similar conditions, Na+ and K+ were without protective effect. Membranes bound 45Ca2+ in a specific and saturable manner. Scatchard plots indicated a dissociatiion constant (Kd) of 9 X 10(-5) M and a capacity (n) of 54 nmol/mg of membrane protein. 45Ca2+ was also displaced from membranes by Mg2+ and Mn2+. Ca2+ had a biphasic effect on binding; low concentrations (1 to 10 muM) added to the incubation mixture stimulated binding, while higher concentrations (0.1 mM) caused inhibition. Mg2+ and Mn2+, at comparable concentrations, were also inhibitory, Na+ and K+ less so. In the case of Ca2+, both the stimulatory and inhibitory concentrations were lower than those required to achieve saturation of Ca2+-binding sites. Proteolytic enzymes (trypsin, alpha-chymotrypsin, and pronase) sharply reduced binding of 125I-TSH, owing to a decrease in receptor sites. Phospholipases A and C enhanced binding of TSH, while neuraminidase and beta-galactosidase were without measurable effect.     

Correlation between trypsin binding to a specific receptor and prostacyclin production in cultured vascular endothelial cells

The correlation between the binding and processing of trypsin and its effect on prostacyclin (PGI2) production in cultured adult bovine aortic endothelial (ABAE) cells was studied. ABAE cells demonstrated an ability to produce PGI2 in a dose-response manner to trypsin at the range of 0.1-2.0 micrograms/ml with a saturation at a concentration of 1 microgram/ml. Likewise, 125I-trypsin binding to the cultured cells increased in a dose-response way and reached saturation at a concentration of about 1 microgram/ml; 125I-trypsin was bound to a specific high-affinity cell-surface receptor with a dissociation constant (Kd) of 1.5 X 10(-8) M and each of the confluent ABAE cells has about 1.2 X 10(5) such receptors sites. The cell-surface receptor for trypsin displayed specific characteristics and an excess amount of unlabeled trypsin successfully abolished 125I-trypsin binding while thrombin in excess failed to compete for 125I-trypsin binding. Only a small fraction of the cell-surface-bound 125I-trypsin was internalized and subsequently degraded by ABAE cells as compared to the process of 125I-trypsin internalization by human skin fibroblasts (HSF). This study demonstrated that the stimulatory effect of trypsin on prostacyclin production and release by ABAE cells might be mediated by a specific cell-surface receptor for trypsin on these cells distinct from the thrombin receptor.     

Characterization of glucagon receptors in Golgi fractions of rat liver: evidence for receptors that are uncoupled from adenylyl cyclase

Glucagon receptors have been identified and characterized in intermediate (Gi) and heavy (Gh) Golgi fractions from rat liver. At saturation, plasma membranes bound 3500 fmol of hormone/mg of membrane protein, while Gi and Gh bound 24 and 60 fmol of 125I-glucagon/mg of protein, respectively. Half-maximal saturation of binding to plasma membranes, Gi, and Gh occurred at approximately 4, 10, and 20 nM 125I-glucagon, respectively. Trichloroacetic acid precipitation of intact, but not degraded, glucagon was used to correct binding isotherms for hormone degradation. After such correction, half-maximal saturation of binding to plasma membranes, Gi, and Gh was observed in the presence of approximately 2, 7, and 14 nM hormone, respectively. After 90 min of dissociation in the absence of guanosine 5'-triphosphate (GTP), 86% of 125I-glucagon remained bound to plasma membranes, whereas only 42% remained bound to Golgi membranes. GTP significantly increased the fraction of 125I-glucagon released from plasma membranes but only slightly augmented the dissociation of hormone from Golgi fractions. 125I-Glucagon/receptor complexes solubilized from plasma membranes fractionated by gel filtration as high molecular weight (Kav = 0.16), GTP-sensitive complexes and lower molecular weight (Kav = 0.46), GTP-insensitive complexes. 125I-Glucagon complexes solubilized from Golgi membranes fractionated almost exclusively as the lower molecular weight species. The lower affinity of Golgi than plasma membrane receptors for hormone, the ability of glucagon to stimulate plasma membrane, but not Golgi membrane, adenylyl cyclase, and the near absence of high molecular weight, GTP-sensitive complexes in solubilized Golgi membranes demonstrate that plasma membrane contamination of Golgi fractions cannot account for the 125I-glucagon binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the structural domain of ApoAI recognized by high density lipoprotein receptors.

There is good evidence that high density lipoprotein (HDL) interacts with high affinity sites present on hepatocytes. The precise nature of the ligand recognized by putative HDL receptors remains controversial, although there is a consensus that apolipoprotein AI (apoAI) is involved. This suggestion would be strengthened if a biologically active site demonstrating a high affinity for the receptor could be isolated. Cyanogen bromide fragments (CF) of apoAI (CF1-CF4) were complexed with phospholipid, and their ability to associate with the receptor was compared in various binding studies. Careful analysis of the concentration-dependent association of 125I-labeled dimyristoyl phosphatidylcholine (DMPC) recombinants to rat liver plasma membranes revealed high and low affinity binding components. As all DMPC recombinants displayed the low affinity binding component, it was postulated that this interaction was independent of the protein present in the particle and may well represent a lipid-lipid or lipid-protein association with the membranes. Only 125I-labeled CF4.DMPC displayed a high affinity binding component with similar Kd and Bmax (8 x 10(-9) M, 1.6 x 10(-12) mol/mg plasma membrane protein) to that of 125I-labeled AI.DMPC (7 x 10(-9), 1.4 x 10(-12) mol/mg plasma membrane protein). Similarly, egg yolk phosphatidylcholine complexes containing CF4 (CF4.egg PC) showed higher affinity binding than CF1-egg yolk phosphatidylcholine complexes confirming the results obtained with DMPC complexes. Furthermore, ligand blotting studies showed that only 125I-labeled CF4.DMPC associated specifically with HB1 and HB2, two HDL binding proteins recently identified in rat liver plasma membranes. We conclude that a region within the carboxyl-terminus of apoAI is responsible for the interaction with putative HDL receptors present in rat liver plasma membranes.     

Ca2+ binding sites in plasma membranes of rat liver and hepatoma cells, and effect of concanavalin A on the Ca2+ binding sites and cellular uptake of Ca2+

1. Plasma membranes isolated from rat livers and ascites hepatoma cells (AH-130, AH-7974) were assayed for specific Ca2+ binding sites using 45Ca2+ and a Millipore filtration technique. The presence of higher (Kd = 1.4--1.5 . 10(-5) M) and lower (Kd = 0.9--1.0 . 10(-4) M) affinity sites in both liver and hepatoma membranes was observed. The hepatoma plasma membranes however, showed 1.4--2.1-fold as many Ca2+ binding sites (higher and lower affinity sites) as the liver plasma membranes on the basis of protein. 2. Concanavalin A stimulated the specific Ca2+ binding to liver and hepatoma plasma membranes, showing a maximal stimulation (3--5-fold) at 100 microgram/ml. Succinyl concanavalin A was less effective, whereas wheat germ agglutinin and ricinus lectin were ineffective. 3. Concanavalin A stimulated the Ca2+ uptake by AH-7974 cells. The concanavalin A-mediated stimulation of Ca2+ uptake showed lectin-concentrations and Ca2+-concentration dependencies similar to those in the concanavalin A-mediated stimulation of Ca2+ binding.     

The expressed human hepatic receptor for low-density lipoproteins differs from the fibroblast low-density lipoprotein receptor

The role of the cellular receptor for the low-density lipoproteins (LDL) in cholesterol transport was initially defined through the study of nonhepatic cells in vitro. Since the liver is central in plasma lipoprotein metabolism, an investigation of hepatic lipoprotein receptors is important for understanding normal lipoprotein transport. Utilizing human hepatic and fibroblast membranes, the characteristics of receptors for LDL from hepatic and nonhepatic tissues were directly compared. Human hepatic membranes reversibly bound LDL within 5 min. Although both fibroblast and hepatic membranes saturably bound LDL at 37 degrees C, the fibroblast LDL receptor affinity (Kd = 2.5 X 10(-8) M) and number (5.5 X 10(12) sites/mg membrane protein) were greater than the hepatic receptor affinity (Kd = 10.8 X 10(-8) M) and number (0.5 X 10(12) sites/mg membrane protein). In contrast to the fibroblast LDL receptor which was unable to bind LDL in the presence of EDTA, the hepatic LDL receptor binding of LDL was only partially blocked by EDTA. The binding of LDL to its hepatic receptor is highly temperature-dependent, and studies utilizing both radiolabeled LDL and colloidal gold-labeled LDL indicate that little, if any, binding of LDL hepatic membranes occur at 0-4 degrees C. The hepatic membrane receptor(s) (Mr approximately equal to 270 000 and 330 000) differ from that of the fibroblast LDL receptor (Mr approximately equal to 130 000) and these proteins are present in hepatic membranes from a patient lacking the fibroblast LDL receptor. These data indicate that an expressed hepatic LDL receptor has unique properties different from those of the fibroblast LDL receptor and that the expressed protein(s) is genetically distinct from the fibroblast receptor.     

Ammonium sulfate modifies adenylate cyclase and the chemotactic receptor of Dictyostelium discoideum. Evidence for a G protein effect

(NH4)2SO4 was found to activate adenylate cyclase in Dictyostelium discoideum membranes. The effect of (NH4)2SO4 on the enzyme was observed after pretreatment of membranes but could not be observed if the salt was added to the assay mixture. Activation was seen when membranes were pretreated with 0.16 M (NH4)2SO4 and was maximal at 0.6-1.0 M. The maximal activation of the enzyme was observed within 3 min of pretreatment and was not readily reversible. The effect was specific for the NH+4 ion since pretreatment of membranes with other NH+4 salts could activate the enzyme, whereas pretreatment with NaCl or KCl could not. Pretreatment of plasma membranes with (NH4)2SO4 eliminated the sensitivity of the enzyme to the inhibitory effect of guanine nucleotides. (NH4)2SO4 pretreatment also significantly attenuated the inhibition by guanine nucleotides of cAMP binding to its plasma membrane receptor. The effect of (NH4)2SO4 on GTP inhibition of cAMP binding to its receptor was even more dramatic when the salt was present in the binding assay. (NH4)2SO4 also increased the ADP-ribosylation by cholera toxin of a 39,000-Da membrane protein. The data support the hypothesis that (NH4)2SO4-induced changes in adenylate cyclase and the cAMP receptor are due to an alteration of a putative G protein.     

Binding properties of high-density lipoprotein subfractions and low-density lipoproteins to rabbit hepatocytes

Primary cultures of rabbit hepatocytes which were preincubated for 20 h in a medium containing lipoprotein-deficient serum subsequently bound, internalized and degraded 125I-labeled high-density lipoproteins2 (HDL2). The rate of degradation of HDL2 was constant in incubations from 3 to 25 h. As the concentration of HDL2 in the incubation medium was increased, binding reached saturation. At 37 degrees C, half-maximal binding (Km) was achieved at a concentration of 7.3 micrograms of HDL2 protein/ml (4.06 X 10(-8)M) and the maximum amount bound was 476 ng of HDL2 protein/mg of cell protein. At 4 degrees C, HDL2 had a Km of 18.6 micrograms protein/ml (1.03 X 10(-7)M). Unlabeled low-density lipoproteins (LDL) inhibited only at low concentrations of 125I-labeled HDL2. Quantification of 125I-labeled HDL2 binding to a specific receptor (based on incubation of cells at 4 degrees C with and without a 50-fold excess of unlabeled HDL) yielded a dissociation constant of 1.45 X 10(-7)M. Excess HDL2 inhibited the binding of both 125I-labeled HDL2 and 125I-labeled HDL3, but excess HDL3 did not affect the binding of 125I-labeled HDL3. Preincubation of hepatocytes in the presence of HDL resulted in only a 40% reduction in specific HDL2 receptors, whereas preincubation with LDL largely suppressed LDL receptors. HDL2 and LDL from control and hypercholesterolemic rabbits inhibited the degradation of 125I-labeled HDL2, but HDL3 did not. Treatment of HDL2 and LDL with cyclohexanedione eliminated their capacity to inhibit 125I-labeled HDL2 degradation, suggesting that apolipoprotein E plays a critical role in triggering the degradative process. The effect of incubation with HDL on subsequent 125I-labeled LDL binding was time-dependent: a 20 h preincubation with HDL reduced the amount of 125I-labeled LDL binding by 40%; there was a similar effect on LDL bound in 6 h but not on LDL bound in 3 h. The binding of 125I-labeled LDL to isolated liver cellular membranes demonstrated saturation kinetics at 4 degrees C and was inhibited by EDTA or excess LDL. The binding of 125I-labeled HDL2 was much lower than that of 125I-labeled LDL and was less inhibited by unlabeled lipoproteins. The binding of 125I-labeled HDL3 was not inhibited by any unlabeled lipoproteins. EDTA did not affect the binding of either HDL2 or HDL3 to isolated liver membranes. Hepatocytes incubated with [2-14C]acetate in the absence of lipoproteins incorporated more label into cellular cholesterol, nonsaponifiable lipids and total cellular lipid than hepatocytes incubated with [2-14C]acetate in the presence of any lipoprotein fraction. However, the level of 14C-labeled lipids released into the medium was higher in the presence of medium lipoproteins, indicating that the effect of those lipoproteins was on the rate of release of cellular lipids rather than on the rate of synthesis.     

Fibrinogen binding to human platelet plasma membranes. Identification of two steps requiring divalent cations

Fibrinogen binding to platelet plasma membranes, which is a prerequisite for platelet aggregation, was determined by incubating 125I-labeled fibrinogen with isolated membranes and measuring the amount of radioactivity sedimenting with the membranes through 15% sucrose. Fibrinogen binding was optimal at 10(-3) M Ca2+. Scatchard analyses of the fibrinogen binding showed that the membrane capacity for fibrinogen was 1.6 X 10(-12) mol/mg of membrane protein, with a dissociation constant (Kd) = 1.2 X 10(-8) M. When Ca2+ levels were manipulated by the addition of varying amounts of EGTA at a fixed Mg2+ concentration of 3 X 10(-3) M, specific binding of fibrinogen to platelet membranes occurred only at Ca2+ concentrations greater than or equal to 10(-6) M. Membranes isolated from platelets of an individual with Glanzmann's thrombasthenia bound only 12% as much fibrinogen as control platelets. The data in the present study suggest that there are two divalent cation binding sites that must be occupied for fibrinogen to bind: one site is specific for calcium and is saturated at 10(-6) M Ca2+; the other site is less specific and is saturated at a 10(-3) M concentration of either Ca2+ or Mg2+. Fibrinogen binding to intact platelets and, consequently, platelet aggregation only required 10(-3) M extracellular divalent cation and was not specific for Ca2+. These data indicate that the cytoplasm is a potential source for the requirement of 10(-6) M Ca2+, and that changes in the intracellular concentration of Ca2+ may cause the expression of fibrinogen receptors during ADP-induced platelet activation.     

A specific gastrin receptor on plasma membranes of antral smooth muscle

Plasma membranes with a 17 fold enrichment in 5'-nucleotidase over homogenate were prepared from antral smooth muscle. A specific gastrin receptor on the plasma membranes has been demonstrated. By Scatchard analysis receptor has a Kaff of 2x10(9)M(-1) and a binding capacity of 5x10(-14) moles/mg of membrane protein.     

Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity

Coated pits will assemble onto purified plasma membranes that are attached to a poly-L-lysine coated substratum (Moore, M. S., Mahaffey, D. T., Brodsky, F. M., and Anderson, R. G. W. (1987) Science 236, 558-563; Mahaffey, D. T., Moore, M. S., Brodsky, F. M., and Anderson, R. G. W. (1989) J. Cell Biol. 108, 1615-1624). To better understand the assembly reaction, we have purified both clathrin triskelion and AP-2 subunits from bovine brain and assayed for their ability to bind to the cytoplasmic surface of attached membranes. Two types of membranes were analyzed: those washed with a high pH buffer that selectively removes triskelions and those washed with a high salt buffer that removes both the AP-2 and the triskelion subunits. We found that purified AP-2 subunits bind with high affinity (Kd approximately 3 x 10(-8) M) to salt stripped membranes. Binding is saturable and abolished by treating membranes with less than 20 micrograms/ml of elastase. When membranes were treated with elastase before the salt wash and then salt washed and assayed for AP-2 binding, normal binding was seen, which indicates that the presence of clathrin-coated pits protects the binding site from the protease. Membranes that had rebound AP-2 did not bind purified triskelions, even though high pH buffer-washed membranes that bear endogenous AP-2 bound triskelions with high affinity (Kd approximately 3 x 10(-9) M) and supported lattice assembly. We conclude that coated pit assembly is initiated by the binding of AP-2 to an integral membrane protein but that the AP-2 complex must be activated by an unknown process before the coated pit lattice will assemble.