Properties of H-diazepam binding to benzodiazepine receptors in rat cerebral cortex

The recently discovered specific binding sites for benzodiazepines in the central nervous system have been characterized further. Specific ³H-diazepam binding to synaptosomal preparations from rat cerebral cortex is saturable with the dissociation constant for diazepam K_D = 3.6 ± 0.1 nM. The maximal amount of specifically bound ³H-diazepam is 0.88 ± 0.05 pmole per mg protein. In the binding assay equilibrium between the specific binding sites and ³H-diazepam is reached within 15 min. Specific ³H-diazepam binding shows a linear dependency on protein up to 3.0 mg protein per assay and a sharp pH-optimum between pH 7.0 to pH 7.4 ³H-diazepam binding is temperature dependent with the highest amount bound at 4° C. Specific ³H-diazepam binding is altered by treating the synaptosomal preparations with proteolytic enzymes: trypsin and -chymotrypsin lead to the complete disappearance of specific ³H-diazepam binding; carboxypeptidase A or B decrease specific ³H-diazepam binding by 36 %, while leucineaminopeptidase was without effect.     

Temperature-dependent regulation of d-cis-[H]diltiazem binding to Ca channels by 1,4-dihydropyridine channel agonists and antagonists

The binding of the Ca²⁺-channel blocker d-cis-[³H]diltiazem to guinea pig skeletal muscle microsomes is temperature-dependent. At 2°C the K_D is 39 nM and B_max is 11 pmol/mg protein. The binding is fully reversible (K₋₁ = 0.02 min⁻¹). The binding sites discriminate between the diastereoisomers 1- and d-cis-diltiazem, recognize verapamil, gallopamil and tiapamil, and are sensitive to La³⁺-inhibition. At 30°C the K_D is 37 nM and the B_max is 2.9 pmol/mg protein. D-cis-diltiazem-labelling is regulated by the 1,4-dihydropyridine Ca²⁺-channel blockers and a novel Ca²⁺-channel activator in a temperature-dependent manner. At 30°C an enhancement of d-cis-diltiazem binding by the channel blockers is observed. This is attributed to a B_max increase. EC₅₀-values for enhancement and the maximal enhancement differ for the individual 1,4-dihydropyridines. At 2°C 1,4-dihydropyridines inhibit d-cis-[³H]diltiazem binding. This is attributed to a B_max decrease. We have directly labelled one of the drug receptor sites within the Ca²⁺-channel which can allosterically interact with the 1,4-dihydropyridine binding sites.     

Characterization of the cytoplasmic androgen receptor of rat seminal vesicle

Postmitochondrial supernatant (PMS) (1) has been prepared from the homogenate of rat seminal vesicles and the characteristics of the binding reaction of 5-dihydrotestosterone (DHT) to the cytoplasmic androgen receptor have been studied using a charcoal adsorption procedure.

At 0°C apparent equilibrium of binding is reached between 60 and 90 min of incubation but no exchange of bound (³H)DHT can be observed in the presence of a 100-fold excess of unlabelled DHT.

Saturation analysis shows a single class of independent binding sites for DHT with an apparent dissociation constant of 1 nM at 0°C and 2 nM at 25°C. Concentration of binding sites is in the range of 25–80 fmoles/mg protein.

When not occupied by DHT the receptor molecules are inactivated spontaneously following first order reaction kinetics. A rate constant of 0.27 hours⁻¹ at 0°C was determined for the inactivation reaction.

In the (³H)DHT-binding reaction testosterone and 19-nortestosterone are even more efficient competitors than unlabelled DHT, while hydrocortisone does not compete at all. On the other hand significant binding of (³H) testosterone could not be demonstrated.

The (³H)DHT-receptor complex is precipitated from the cytosol by 0 to 33% saturation of ammonium sulphate and sediments as a single, 3.1 S peak in sucrose gradients prepared in 0.4 M NaCl.     

Somatostatin binding to dissociated cells from rat cerebral cortex

A method has been developed for the study of somatostatin (SS) binding to dissociated cells from rat cerebral cortex. Binding of [¹²⁵I][Tyr¹¹]SS to cells obtained by mechanical dissociation of rat cerebral cortex was dependent on time and temperature, saturable, reversible and highly specific. Under conditions of equilibrium, i.e., 60 min at 25°C, native SS inhibited tracer binding in a dose-dependent manner. The Scatchard analysis of binding data was linear and yielded a dissociation constant of 0.60±0.08 nM with a maximal binding capacity of 160±16 fmol/mg protein. The binding of [¹²⁵I][Tyr¹¹]SS was specific as shown in experiments on tracer displacement by the native peptide, SS analogues, and unrelated peptides.     

Investigation of the transport of intact glutathione in human and rat type II pneumocytes

The aim of the study was to investigate whether there is transmembrane transport of intact glutathione ([³H]-GSH, 0.1 μCi) in rat and human type II pneumocytes (T2P), and if this transport might be dependent on the redox state of the extracellular fluid. The T2P were pretreated with acivicin (250 μM) to inhibit γ-glutamyl-transferase activity and with L-buthionine-[SR]-sulfoximine (1 mM) to inhibit intracellular GSH synthesis. After 48 h in culture, initial GSH influx rate was 0.70 ± 0.20 nmol/min/mg protein (37°C) and 0.35 ± 0.04 nmol/min/mg protein (4°C) during the first 5 min in rat T2P. In human T2P, the initial GSH influx rate was 0.36 ± 0.30 nmol/min/mg protein (37°C) and 0.32 ± 0.06 nmol/min/mg protein (4°C) during the first 10 min. Thereafter no further influx was found. The influx of 1 mM GSH in freshly isolated rat and human T2P in suspension was 2.3 ± 0.3 and 1.2 ± 0.3 nmol/mg protein after 15 min at 37°C, and 2.8 ± 0.2 and 1.0 ± 0.3 nmol/mg protein at 4°C, respectively. When GSH influx was studied at different concentrations between 0 and 40 mM, a linear increase without saturation or difference between 37°C and 4°C was found. Preexposure to ouabain had no effect on GSH influx. Efflux of GSH was stimulated and influx inhibited by preexposure of the cells to reduced thiols, while disulphides inhibited efflux and favoured inward uptake. Thus, in human and rat T2P a GSH-carrier exists which operates as an effluxer. At GSH concentrations in the physiological range no uptake is seen, but some uptake can be observed at GSH concentrations above normal physiological levels. The uptake appears to be energy-independent and non-saturable. Efflux of GSH is stimulated and influx inhibited by reduced thiols, while disulphides inhibit the efflux and favour inward uptake. GSH uptake in T2P thus may depend on concentration gradients and driving forces, such as the redox state of the extracellular fluid.     

Uptake of iron from transferrin by isolated hepatocytes

Isolated rat hepatocytes containing 0.56-1.79 micrograms iron/10(6) cells and with an intracellular ATP concentration of 3-4 mM, accumulate iron from transferrin linearly with time for at least 3 h. At 37 degrees C the rate of uptake amounts to 0.3-0.7 pmol/mg cell protein per min. The uptake reaches a saturation level of 21-40 pmol/mg cell protein per h at 2.2 microM iron. At 5 degrees C the uptake does not increase over the time of incubation. Uptake of iron, but not binding of transferrin is increased 4-5-fold at oxygen concentrations 10-20 microM. At oxygen concentrations beyond these limits iron uptake is decreased. Iron taken up at low oxygen concentrations can be chelated by bathophenanthroline and bathophenanthroline disulphonate , but only if the chelators are present during the uptake experiments. The results suggest that iron uptake from transferrin by hepatocytes in suspension involves reductive removal of iron.     

Temperature dependent binding of mebendazole to tubulin in benzimidazole-susceptible and -resistant strains of Trichostrongylus colubriformis and Caenorhabditis elegans.

The binding of [³H]mebendazole ([³H]MBZ) to tubulin in benzimidazole-susceptible (BZ-S) and benzimidazole-resistant (BZ-R) strains of Trichostrongylus colubriformis and Caenorhabditis elegans was examined in order to investigate the biochemical changes to tubulin that result in BZ resistance in parasitic and free-living nematodes. In both species the extent of [³H]MBZ binding to tubulin was significantly reduced in the BZ-R strain compared with the BZ-S strain. The decrease in [³H]MBZ binding in the BZ-R strain of each species was the result of a significant reduction in the amount of charcoal stable [³H]MBZ-tubulin complexes and was not related to a change in the association constant of the [³H]MBZ-tubulin interaction. [³H]MBZ binding to tubulin was temperature dependent, reaching maximum levels at 37°C in BZ-S T. colubriformis and 10°C in BZ-R T. colubriformis. Both the BZ-S and BZ-R strains of C. elegans displayed maximum [³H]MBZ binding at 4°C. Resistance ratios derived from the amount of [³H]MBZ binding in the BZ-S and BZ-R strains and in vitro development assays demonstrated that the temperature dependence and extent of drug binding was indicative of BZ resistance status and was species specific in the BZ-S isolates. These results indicate that biochemical differences exist in the binding of benzimidazole carbamates to tubulin in nematode species, and suggest that the susceptibility of the parasitic nematodes to the benzimidazole anthelmintics is the result of a unique high affinity and/or high capacity interaction ofbenzimidazole carbamates with tubulin.     

Effect of hypoxia and glucose deprivation on ATP level, adenylate energy charge and [Ca]0-dependent and independent release of [H]dopamine in rat striatal slices

Release of [³H]dopamine ([³H]DA) from rat striatal slices kept under hypoxic or/and glucose-free conditions was measured using a microvolume perfusion method. The corresponding changes in nucleotide content were determined by reverse-phase high-performance liquid chromatography (RPHPLC). The resting release of [³H]DA was not affected by hypoxia, but under glucose-free conditions massive [Ca²⁺]₀-independent release of [³H]DA was observed. Hypoxia reduced the energy charge (E.C.) and the total purine content from 19.36 ± 4.15 to 6.98 ± 1.83 mol/mg protein. Glucose deprivation by itself, or in combination with hypoxia, markedly reduced the levels of adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The E.C. under glucose-free conditions was significantly reduced from 0.73 ± 0.04 to 0.44 ± 0.20. When the tissue was exposed to hypoxic and glucose-free conditions for 18 min the level of ATP was reduced to 3.15 ± 0.11 mol/mg protein. However, when the exposure time was 30 min the ATP level was further reduced to 1.11 ± 0.37 nmol/mg protein. The resting release was enhanced in a [Ca²⁺]₀-independent manner, but there was no release in response to stimulation, and tetrodotoxin did not affect the enhanced resting release, indicating that the release was not associated with axonal activity. Similarly, 50 μM ouabain, inhibitor of Na⁺/K⁺-activated ATPase, enhanced the release of [³H]DA at rest in a [Ca²⁺]₀-independent manner. It seems very likely that the reduced ATP level under glucose-free conditions leads to an inhibition of the activity of Na⁺/K⁺-ATPase that results in reversal of the uptake processes and in [Ca²⁺]₀-independent [³H]DA release from the axon terminals.     

Effect of reaction temperature and reaction time on the preparation of low-molecular-weight chitosan using phosphoric acid

Low-molecular-weight chitosan were prepared using 85% phosphoric acid at different reaction temperatures and reaction time. At room temperature, the viscosity average-molecular weights (M_v) of chitosan decreased to 7.1×10⁴ from 21.4×10⁴ after 35 days treatment. The degradation rate decreased with increasing hydrolysis time. The yields of chitosan also continuously decreased from 68.4 to 40.2% after 35 days. At 40, 60 and 80 °C, the molecular weight decreased to 3.70×10⁴, 3.50×10⁴ and 2.00×10⁴ on 8 h hydrolysis, respectively. The yields of chitosan remain at a high level compared with that at room temperature and were 86.5, 71.4 and 61.3% at 40, 60 and 80 °C treatment, respectively. The different reaction time gave chitosan with different molecular weights. At 60 °C, the molecular weight of products decreased to 7.40×10⁴ from 21.4×10⁴ within 4 h, then decreased slowly to 1.90×10⁴ in 15 h. It was also found that the water-solubility of chitosan increased as the molecular weight decreased. Results show the changes in yields and molecular weight of chitooligomers were strongly dependent on the reaction temperature and reaction time.     

Characterization of amylin binding sites in a human hepatoblastoma cell line

Amylin binding sites in a human hepatoblastoma cell line (HepG2) have been characterized in detail. ¹²⁵I-Amylin (rat) bound to HepG2 cells with high affinity. Binding was reversible and selective, and dependent on time and temperature. Scatchard analysis revealed the presence of high (K_d = 0.11 ± 0.04 nM) and low (K_d = 1.3 ± 0.4 μM) affinity binding sites for ¹²⁵I-amylin in HepG2 cells. The dissociation experiments also showed that ¹²⁵I-amylin dissociated from high- and low-affinity sites. The association data, however, indicated the presence of only one binding site. Rat amylin was more potent than human amylin and rat calcitonin gene-related peptide (CGRP) in displacing ¹²⁵I-amylin bound to HepG2 cells. Nonhomologous peptides did not displace ¹²⁵I-amylin. Rat amylin was, however, less potent than rat CGRP in displacing ¹²⁵I[Tyr⁰]CGRP from HepG2 cells. Pretreatment of HepG2 cells with rat amylin (10 nM) reduced the specific binding of ¹²⁵I-amylin by 75%, whereas rat CGRP (10 nM) pretreatment had no effect on amylin binding. Calcitonin gene-related peptide, as well as rat and human amylin, stimulated the adenylate cyclase activity of HepG2 cell membrane preparation in a dose-dependent manner, with an order of potency of CGRP > rat amylin > human amylin. A CGRP antagonist, CGRP(8–37), significantly attenuated the stimulatory effect of both amylin and CGRP on adenylate cyclase activity. These investigations show that distinct receptors of amylin and CGRP are present in HepG2 cells and that amylin stimulates adenylate cyclase activity through CGRP receptors. This system could now be exploited for studying amylin receptors and amylin-mediated signal transduction.     

The uptake of the GABA agonist, [H]isoguvacine, by the isolated retina of the rabbit

Previous autoradiographic studies aimed at showing neurones using GABA as their neurotransmitter have been hampered by the fact that the substance is a ubiquitous metabolite and therefore accumulated by a large variety of cells, including glia. Consequently, GABA uptake markers without this widespread uptake are desirable, and one, [³H]isoguvacine, has shown promising results in autoradiographic experiments. Its uptake has now been further studied with quantitative radiochemical techniques.

The uptake of the drug was slow compared to GABA uptake and reached a tissue/medium ratio of about 3 after 120 min. The uptake could be inhibited by GABA, beta-alanine or ouabain, and by incubating the retinas at 0°C. The uptake kinetics were complex but suggested a high affinity uptake system (K_m about 10⁻⁸ M) and perhaps one or several systems with lower affinities.

The results indicate that [³H]isoguvacine and [³H]GABA are accumulated and retained by the same neurones, which most likely use GABA as their neurotransmitter.     

Effects of detergents on binding of 5-hydroxytryptamine3 receptor antagonist [H]GR65630 to rat cortical membranes

In the absence of detergent, specific binding of [³H]GR65630, a 5-hydroxytryptamine₃ (5-HT₃) antagonist, determined in the presence of 5-HT₃ receptor antagonist ICS205-930, was at most 30% of the total binding. To decrease the level of nonspecific binding, the effects of detergents on [³H]GR65630 binding to rat cortical membranes were investigated. The use of a detergent (0.1% Lubrol PX or Triton X-100) decreased nonspecific binding, increasing the proportion of specific binding to 70% of total binding. In the presence of 0.1% Triton X-100, binding of [³H]GR65630 was rapid, reversible and saturable at 25°C. The rank order of 5-HT₃ receptor active drugs in inhibiting [³H]GR65630 binding was quipazine > ICS205-930 > 2-methyl-5-HT = 5-HT > metoclopramide, which confirmed that [³H]GR65630 efficiently labeled 5-HT₃ receptors in the presence of Triton X-100. Triton X-100 improved 5-HT₃ receptor binding with rat brain membranes.     

The effects of thermal acclimation upon ion transport in erythrocytes

The different components of ⁸⁶Rb⁺ influx (a marker for K⁺ influx) were measured in erythrocytes of 10°C and 30°C-acclimated carp. Passive influx was similar in both acclimation groups and was stimulated by increased temperature. The active and facilitated components of ⁸⁶Rb⁺ influx plateaued above 15°C in 10°C-acclimated carp and above 25°C in 30°C-acclimated carp. The furosemide-sensitive component, an ill-defined facilitated mechanism, was particularly affected by high temperatures. The influx rates of each acclimation group at 50°C were almost identical but at higher temperatures, the influx rates for 30°C-acclimated carp were substantially greater than for the 10°C-acclimated fish.     

C NMR relaxation studies of 1:2 LiCl-ethylaluminum dichloride solutions

A new room-temperature molten salt, 1:2 LiCl-ethylaluminum dichloride (LiCl-EtAlCl₂, f.p. about 178 K), is examined using ¹³C relaxation methods at 7.05 T (−25 to + 80 °C). The methylene carbon undergoes scalar relaxation of the ‘second kind’ as it is coupled to a faster relaxing (quadrupolar) nucleus. LiCl-EtAlCl₂ undergoes a significant liquid-state phase change between 5 and 15 °C as evidenced by observed changes in the relaxation properties of the methylene and methyl carbons and J(¹³C−²⁷Al). The J(¹³C−²⁷Al) coupling constants are 75 (− 10 to + 5 °C) and 11 Hz (15–65 °C), indicating a change in structure between 5 and 15 °C. Chemical shift anisotropies of 56 and 48 ppm are obtained for the methylene and methyl carbons in the EtAlCl₂ dimer part of the 1:2 LiCl-EtAlCl₂ solution.     

Interaction of sialosyl cholesterol with the cell surface of rat astrocytes and its biological activities

The interaction between sialosyl cholesterol (- or neuraminyl cholesterol, - or β-SC) and the plasma membrane of astrocytes was investigated by the use of ¹⁴C-labeled - or β-SC. Both - and β-SC were dose-dependently and time-dependently bound to rat astrocytes. The Scatchard plot analyses showed that rat astrocytes bound apparently 9.69 × 10⁹ molecules of both -SC/cell (apparent K_d = 2.29 × 10⁻⁵ M) and β-SC/cell (apparent K_d = 5.39 × 10⁻⁵ M) at 37°C. Both the binding of -SC to astrocytes and the subsequent inhibition of DNA synthesis were decreased at the low temperature (4°C), and also suppressed by serum proteins including albumin. One molecule of bovine serum albumin (BSA) bound 2.3 molecules of -SC with the slightly lower K_d-value (8.03 × 10⁻⁶ M) than that for the binding site on astrocytes. BSA not only suppressed the -SC-binding to astrocytes but also increased its release from the cells to the culture media. Gangliosides such as GM1 and GM3 unaffected the -SC-binding, promoted the small release of -SC from the cell surface, and inhibited the morphological changes of astrocytes induced by -SC. The mechanism of -SC-binding to cultured astrocytes with reference to the effects of serum or gangliosides is discussed.     

Characterization of growth hormone-releasing hormone (GHRH) binding to cloned porcine GHRH receptor

To study structure-activity relationships of growth hormone-releasing hormone (GHRH), a competitive binding assay was developed using cloned porcine adenopituitary GHRH receptors expressed in human kidney 293 cells. Specific binding of [His¹,¹²⁵I-Tyr¹⁰,Nle²⁷]hGHRH(1–32)-NH₂ increased linearly with protein concentration (10–45 μg protein/tube). Binding reached equilibrium after 90 min at 30°C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites (K_d = 1.04 ± 0.19 nM, B_max = 3.9 ± 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC₅₀) for porcine GHRH (2.8 ± 0.51 nM), rat GHRH (3.1 ± 0.69 nM), [N-Ac-Tyr¹, -Arg²]hGHRH(3–29)-NH₂ (3.9 ± 0.58 nM), and [ -Thr⁷]GHRH(1–29)-NH₂ (189.7 ± 14.3 nM), consistent with their binding to a GHRH receptor. Nonhydrolyzable guanine nucleotides inhibited binding. These data describe a selective and reliable method for a competitive GHRH binding assay that for the first time utilizes rapid filtration to terminate the binding assay.     

Adenine nucleotide translocation in yeast mitochondria. Effect of inhibitors of mitochondrial biogenesis on the ADP translocase

1. Optimal test conditions for adenine nucleotide translocation in Candida utilis mitochondria are a standard medium, consisting of 0.63 M mannitol, 2 mM EDTA (or ethylene glycol tetraacetic acid, EGTA), 10 mM morpholinopropane sulfonic acid (pH 6.8), and a temperature of 0 °C.

2. Adenine nucleotide translocation in C. utilis mitochondria is an exchange-diffusion process. The whole pool of internal adenine nucleotides is exchangeable, ADP being the most readily exchangeable nucleotide. The rate of mitochondrial ADP exchange, but not the K_m value, depends on growth conditions. At 0 °C, the rate is about 3 to 4 nmoles ADP/min per mg protein for mitochondria obtained from yeast grown in the presence of 1.5% glucose; it rises to 11.5 nmoles when glucose is replaced by 3% ethanol in the growth medium. The K_m value for ADP is 2 μM. The Q₁₀ is about 2 between 0 and 20 °C. Among other exchangeable adenine nucleotides are ATP, dADP and the methylene and the hypophosphate analogues of ADP. Unlike mammalian mitochondria, C. utilis mitochondria are able to transport external UDP by a carboxyatractyloside-sensitive process.

3. Under conditions of oxidative phosphorylation (phosphate and substrate present in an aerated medium), added ADP is exchanged with internal ATP. A higher ATP/ADP ratio was found in the extramitochondrial space than in the intramito-chondrial space. The difference between the calculated phosphate potentials in the two spaces was 0.9–1.7 kcal/mole.

4. Atractyloside, carboxyatractyloside, bongkrekic acid and palmityl-CoA inhibit mitochondrial adenine nucleotide translocation in C. utilis as they do in mammalian mitochondria, but 2 to 4 times less efficiently. The inhibition due to atractyloside or palmityl-CoA is competitive with respect to ADP whereas that due to bongkrekic acid and carboxyatractyloside is non-competitive. Carboxyatractyloside and atractyloside inhibitions are additive. The apparent K_d for the binding of [³⁵S]-carboxyatractyloside and [¹⁴C]bongkrekic acid is 10–15 nM and the concentration of sites 0.4–0.6 nmole/mg protein in both cases. [³⁵S]Carboxyatractyloside binding is competitively displaced by atractyloside and vice versa.

5. Binding of [¹⁴C]ADP has been carried out with mitochondria depleted of their endogenous adenine nucleotides by incubation with phosphate and Mg²⁺ at 20 °C. The amount of bound [¹⁴C]ADP which is atractyloside removable is 0.08–0.16 nmole/mg protein.

6. The rate of ADP transport is quite different in mitochondria isolated from C. utilis, according to whether it is grown on glucose, or on ethanol or in the presence of chloramphenicol; for instance, it decreases by 10 times when 3% ethanol in the growth medium is replaced by 10% glucose and by 5 times when chloramphenicol is added to the medium. These variations are accompanied by parallel variations in cytochrome aa₃. The number of atractyloside-sensitive ADP binding sites is not modified by the above conditions of culture, nor the number of [³⁵S]carboxyatractyloside binding sites. The affinity for ADP is apparently not significantly modified, nor the size of the endogenous adenine nucleotide pool. In contrast to glucose repression or chloramphenicol inhibition, semi-anaerobiosis in C. utilis lowers significantly the mitochondrial binding capacity for carboxyatractyloside. Strict anaerobiosis in S. cerevisiae results in a practical loss of the cytochrome oxidase activity, and also of the carboxyatractyloside and ADP binding capacity. Transition from anaerobiosis to aerobiosis restores the cytochrome oxidase activity and the ADP and carboxyatractyloside binding capacities.     

Transferrin binding and iron uptake in mouse hepatocytes

Methods were developed for obtaining highly viable mouse hepatocytes in single cell suspension and for maintaining the hepatocytes in adherent static culture. The characteristics of transferrin binding and iron uptake into these hepatocytes was investigated. (1) After attachment to culture dishes for 18–24 h hepatocytes displayed an accelerating rate of iron uptake with time. Immediately after isolation mouse hepatocytes in suspension exhibited a linear iron uptake rate of 1.14·10⁵molecules/cell per min in 5 μM transferrin. Iron uptake also increased with increasing transferrin concentration both in suspension and adherent culture. Pinocytosis measured in isolated hepatocytes could account only for 10–20% of the total iron uptake. Iron uptake was completely inhibited at 4°C. (2) A transferrin binding component which saturated at 0.5 μM diferric transferrin was detected. The number of specific, saturable diferric transferrin binding sites on mouse hepatocytes was 4.4·10⁴±1.9·10⁴ for cells in suspension and 6.6·10⁴±2.3·10⁴ for adherent cultured cells. The apparent association constants were 1.23·10⁷ 1·mol^?1 and 3.4·10⁶ 1·mol^?1 for suspension and cultured cells respectively. (3) Mouse hepatocytes also displayed a large component of non-saturable transferrin binding sites. This binding increased linearly with transferrin concentration and appeared to contribute to iron uptake in mouse hepatocytes. Assuming that only saturable transferrin binding sites donate iron, the rate of iron uptake is about 2.5 molecules iron/receptor per min at 5 μM transferrin in both suspension and adherent cells and increases to 4 molecules iron/receptor per min at 10 μM transferrin in adherent cultured cells. These rates are considerably greater than the 0.5 molcules/receptor per min observed at 0.5 μM transferrin, the concentration at which the specific transferrin binding sites are fully occupied. The data suggest that either the non-saturable binding component donates some iron or that this component stimulates the saturable component to increase the rate of iron uptake. (4) During incubations at 4°C the majority of the transferrin bound to both saturable and nonsaturable binding sites lost one or more iron atoms. Incubations including 2 mM α,α′-dipyridyl (an Fe¹¹ chelator) decreased the cell associated ⁵⁹Fe at both 4 and 37°C while completely inhibiting iron uptake within 2–3 min of exposure at 37°C. These observations suggest that most if not all iron is loosened from transferrin upon interaction of transferrin with the hepatocyte membrane. There is also greater sensitivity of ⁵⁹Fe uptake compared to transferrin binding to pronase digestion, suggesting that an iron acceptor moiety on the cell surface is available to proteolysis.     

Sulfhydryl groups are involved in the binding of agonists and antagonists to the human mineralocorticoid receptor

To investigate the role of sulfhydryl groups in the interaction of agonists and antagonists with the human mineralocorticoid receptor (hMR) the effect of methyl methanethiosulfonate (MMTS) on free and liganded-hMR was examined. hMR was expressed in insect cells (Sf9) using the baculovirus system. Treatment of cytosol with MMTS at 4°C inhibited the binding to hMR of both [³H]aldosterone and [³H]RU26752 (a synthetic aldosterone antagonist). At 4°C, the sensitivity to MMTS of the liganded-hMR complexes was dependent upon the nature of the ligands: agonists (aldosterone, corticosterone and cortisol) rendered the hMR resistant to MMTS, whereas antagonists (progesterone and RU26752) did not protect the receptor against MMTS inactivation. Analysis of the dose- and time-dependent effects of MMTS revealed that the free hMR and the RU26752-hMR complexes displayed a similar sensitivity to MMTS and that MMTS increased the dissociation of RU26752 from the hMR. At 4°C the aldosterone-hMR complexes were not affected by MMTS treatment, whereas at 20°C MMTS increased the dissociation of aldosterone from hMR. This effect was unrelated to the dissociation of hsp90 from hMR, because the sensitivity of the aldosterone-hMR complexes to MMTS remained unchanged after covalent linkage between hsp90 and the receptor. Our results suggest that agonists and antagonists modify the receptor conformation in distinct ways that render cysteine residues of the ligand binding domain more or less accessible to the MMTS action.     

Characterization of a H-arginine-vasopressin binding site in the cingulate gyrus of the rat pup

Autoradiographic analysis of 1, 8, 16 and 26-day-old rat brains showed ³H-arginine⁸-vasopressin (³H-AVP) binding to the cingulate gyrus-dorsal hippocampus (CG) only in the 8-day-old rat brain. Saturation analysis of CG membranes prepared from pups (7–10 days) and adults (90 days) revealed a small but significant increase in binding site concentration in adults compared to pups. However, the K_d of the ³H-AVP binding site increased significantly with age. The K_d of ³H-AVP binding to pup CG membranes was 0.9±0.1 nM, while the adult CG was 5.7±1.0 nM. The pharmacological specificity of ³H-AVP binding sites in the pup and adult CG was similar, but differed markedly from the profile observed in adult septal membranes. The primary specificity difference between the pup CG and septum was the reduced potency of certain V₁ receptor antagonists. In competition experiments the CG binding site showed a reduced affinity for the V₁ antagonist, [d(CH₂)₅, Tyr(Me)]AVP. This reduced affinity for the V₁ antagonist was also documented autoradiographically using ³H-[d(CH₂)₅, Tyr(Me)]AVP. The data suggest that the ³H-AVP binding site expressed in the pup CG is not identical to the V₁ type receptor present in the periphery and brain of the adult rat.