Ethanol modulates calmodulin-dependent Ca2+-activated ATPase in synaptic plasma membranes

The effects of ethanol in vitro on calmodulin-dependent Ca²⁺-activated ATPase (CaM–Ca²⁺-ATPase) activity were studied in synaptic plasma membranes (SPM) prepared from the brain of normal and chronically ethanol-treated rats. In SPM from normal animals, ethanol at 50–200 mM inhibited the Ca²⁺-ATPase activity. Lineweaver-Burk analysis indicates that the inhibition was the result of a decreased affinity of the enzyme for calmodulin, whereas the maximum activity of the enzyme was not changed. Arrhenius analysis indicates that the enzyme activity was influenced by lipid transition of the membranes, and ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were resistant to the inhibitory effect of ethanol on the enzyme activity. The resistance to ethanol inhibition was correlated with a higher enzyme affinity for calmodulin and a higher transition temperature, as compared with normal SPM. Since the calmodulin-dependent Ca²⁺-ATPase in synaptic plasma membranes is believed to be the Ca²⁺ pump controlling free Ca²⁺ levels in synaptic terminals, its inhibition by ethanol could therefore lead to altered synaptic activity.Abbreviations used ATPase adenosine triphosphatase - CaM calmodulin - CaM–Ca²⁺-ATPase calmodulin-dependent Ca²⁺-activated ATPase - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - EtOH ethanol - Hepes N—2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SPM synaptic plasma membranes - TFP trifluoperazine - Tris tris(hydroxymethyl)aminomethane - K_m Michaelis constant - T_d transition temperature - V_max maximum velocity     

Detailed analysis of heterogeneity of [3H]naloxone binding sites in rat brain synaptosomes

The experiments reported in this paper address the question of heterogeneity of [³H]naloxone binding sites in rat brainstem synaptosomal preparations at 23°C in the presence of 100 mM sodium chloride. Kinetic analysis in the presence of 0.4, 4 and 10 nM [³H]naloxone gave pseudo-first order association rate of 0.9±0.04, 1.23±0.08 and 1.06±0.08 min^–1, respectively. The dissociation of a 1 nM [³H]naloxone receptor complex was biphasic with dissociation rate constants of 1.8 and 0.4 min^–1. On the other hand, dissociation of 10 nM [³H]naloxone was monophasic with ak _d of 1.1 min^–1. Two subpopulations of binding sites were also observed by steady state binding studies, with Kd values of 0.5 and 3.4 nM and a ratio of high to low affinity sites of 1:9. Heterogeneity of [³H]naloxone binding sites could be seen by displacement studies performed with opioid eptides and alkaloids. We suggest that our data best fits a model with two independent naloxone binding sites wherein either one or both undergo a multi-step interaction with ligand.     

Interaction of dimeric and monomeric enkephalins with NG108-15 hybrid cells

The binding of the enkephalin dimer [d-Ala², Leu⁵-NH-CH₂-]₂ (DPE₂) is characterized by (1) its high affinity for receptors on NG108-15 hybrid cells, the affinity constantK=4.7×10⁹ M^–1 is up to 8-fold that of monomers (0.6 to 1.0×10⁹ M^–1), and (2) a maximal binding capacity equal to one half that of the monomers. Kinetic studies showed that DPE₂ binds with a 2-fold higher rate, k₁=6.3×10⁷ M^–1min^–1, than monomers (2.4 to 3.8×10⁷ M^–1min^–1), and dissociates at a slower rate than monomers. Dissociation of DPE₂ was consistently bi- or multiphasic but increased about 12% only after 3 hr of dissociation in the presence of a large excess of unlabeled enkephalin. The dissociation kinetics of monomers varied with enkephalin and experimental conditions used. Consistent with the value for the maximal binding capacity, the kinetic studies are interpreted in support of the hypothesis that DPE₂ binds by cross-linking two subunits of one receptor.     

Characterization of rat brain opioid receptors by [Tyr-3,5-3H]1,d-Ala2, Leu5-enkephalin binding

[Tyr-3,5-³H]¹,d-Ala², Leu⁵-enkephalin ([³H]DALA) was used for labeling the opioid receptors of rat brain plasma membranes. The labeled ligand was prepared from [Tyr-3,5-diiodo]¹,d-Ala², Leu⁵-enkephalin by catalytic reductive dehalogenation in the presence of Pd catalyst. The resulting [Tyr-3,5-³H]¹,d-Ala², Leu⁵-enkephalin had a specific activity of 37.3 Ci/mmol. In the binding experiments steady-state level was reached at 24°C within 45 min. The pseudo first order association rate constant was 0.1 min^–1. The dissociation of the receptor-ligand complex was biphasic with k_–1-s of 0.009 and 0.025 min^–1. The existence of two binding sites was proved by equilibrium studies. The high affinity site showed aK _D=0.7 nM andB _max=60 fmol/mg protein; the low affinity site had aK _D=5 nM andB _max=160 fmol/mg protein. A series of opioid peptides inhibited [³H]DALA binding more efficiently than morphine-like drugs suggesting that labeled ligand binds preferentially to the subtype of opioid receptors. Modification of the original peptides either at the C or N terminal ends of the molecules resulted in a decrease in their affinity.     

Specific binding of kainic acid to purified subcellular fractions from rat brain

The subcellular distribution of kainic acid (KA) binding sites in rat brain has been studied using a microcentrifugation assay. KA did not bind to myelin or brain cytosol and had few or no binding sites in the nuclear fraction. However, it bound to microsomal components (K _d =128–136 nM; 2.5–4.8 pmol/mg protein), purified synaptic plasma membranes (SPM) (K _d =45–71 nM; 5.8–6.5 pmol/mg), and purified cell-body and intraterminal mitochondria (K _d =11–31 nM; 0.4–1.1 pmol/mg). Bound KA could be totally displaced byl-glutamate orl-aspartate, but several putative antagonists of these amino acids (nuciferin, compound HA-966, 2-amino-4-phosphonobutyrate, and 2-amino-3-phosphonoproprionate) failed to displace KA or did so at very high concentrations (4 mM). Glutamic acid diethyl ester (GDEE) andd,l--aminoadipate (-AA) were more effective (IC₅₀, 0.2–0.8 mM) and showed differential effects in their capacity to displace KA bound to the various subcellular fractions. Thus, GDEE only displaced 40–60% of the KA bound by SPM or mitochondria and did not prevent the binding of KA to microsomes. -AA, on the other hand, was more effective in preventing the binding of KA at high concentrations and displaced between 80 and 100% of the drug. Both compounds showed biphasic curves of KA displacement from synaptic plasma membranes and mitochondria. The overall results indicate the presence of multiple binding sites for KA in brain cells and suggest that KA does not act exclusively at synaptic glutamate receptors. The mechanism of KA action is most likely quite complex, and the drug probably acts at multiple binding sites affecting a number of processes.     

Cell wall strength of Hansenula polymorpha in continuous cultures in relation to the recovery of methanol oxidase (MOX)

Summary The changes in cell wall strength of Hansenula polymorpha have been investigated in continuous cultures with respect to the recovery of methanol oxidase (MOX). Cultures grown on several substrate mixtures that enable induction of MOX have been compared with cultures grown on methanol as the sole inducer. The effects of dilution rate (D) on lysis properties have been studied. The cell wall strength was consistently influenced by growth media and D. Media containing glycerol/methanol showed the slowest lysis kinetics, with a large fraction of non-degradable cell wall material. In continuous cultures grown on a mixture of glucose and methanol both the resistance to zymolyase and the mean cell wall thickness increased at D<0.1 h^–1. The yield of MOX by zymolyase lysis is reproducible and up to 100% higher than that of the standard ultrasonic treatment. The lysis kinetics indicated that zymolyase punctures the cell wall; since the release rate of MOX is lower than that of protein, the cell contents will leak through. At D-values>0.2 h^–1, both protein and MOX release rates increase, reflecting a change in lysis mechanism due to the increased fraction of thin daughter cells. Kinetic analysis of zymolyase lysis using both physical and enzymatic methods provides information for achieving optimal recovery of MOX.Abbreviations and symbols C _MOX MOX activity [MOX units·g X^–1] - D dilution rate [h^–1] - MOX methanol oxidase - k_c decay rate constant of A 610 nm [min^–1] - k_d decay constant of MOX activity [min^–1] - k_dis dissociation rate constant [min^–1] - k_MOX release rate constant of MOX activity [min^–1] - k_p release rate constant of protein [min^–1] - R recovery efficiency of enzyme [-] - St stability of enzyme [-]     

Acute modulation of myocardial function by angiotensin 1–7

Angiotensin 1–7 is a bioactive heptapeptide of the renin–angiotensin system. Its cardiovascular actions have recently acquired growing relevance, mainly due to its counter-regulatory actions in the angiotensin cascade. The aim of the present study was to evaluate the actions of angiotensin 1–7 on myocardial function. Increasing concentrations of angiotensin 1–7 (10⁻⁹ to 10⁻⁵ M) were added to rabbit right papillary muscles: (1) in baseline conditions with intact endocardial endothelium (EE); (2) after selective removal of the EE with Triton X-100 (1 s, 0.01%); (3) with intact EE in the presence of the Mas receptor antagonist A-779, the AT₁ receptor antagonist ZD-7155, the AT₂ receptor antagonist PD-123,319 or the nitric oxide synthesis inhibitor NG-nitro-l-arginine (l-NA). Concerning the effects on contractility, we observed a significant decrease on active tension, dT/dt_max, peak shortening and dL/dt_max of −10.5 ± 3.6%, −8.0 ± 3.0%, −5.3 ± 2.6% and −5.7 ± 2.3%, respectively. There was no change on relaxation parameters, namely dT/dt_min or dL/dt_min. Time to half relaxation was significantly decreased. The presence of ZD-7155 or PD-123,319 did not change these effects. However, angiotensin 1–7 effects on myocardial properties were abolished after selective EE removal and in the presence of A-779 or l-NA. In conclusion, in this animal species, angiotensin 1–7 through its binding to Mas receptor induces a negative inotropic effect modulated by the EE and nitric oxide and independent of AT₁ or AT₂ receptors activation. As the effects described in the present work were influenced by the endocardial endothelium, they may be disrupted in situations associated to endothelial dysfunction, as in heart failure or myocardial ischemia.     

Calmodulin selectively modulates the guanylate cyclase activity by repressing the lipid phase separation temperature in the inner half of the bilayer of rat brain synaptosomal plasma membranes

The association of [¹²⁵I-]calmodulin with rat brain synaptosomal plasma membranes, when incubated for 1 h at 25° in the presence or in absence of 20 M Ca²⁺, follows a sigmoid path with a Hill coefficient h=1.79±0.12 and h=1.72±0.11, respectively. The total association of calmodulin with the membrane increased approx. 60%–80% at all the range of calmodulin concentrations used in the presence of 20 M Ca²⁺. A three fold increase of guanylate cyclase activity was shown in the presence of low concentrations of calmodulin (up to 10 mM); higher concentrations (up to 40 mM) however, led to a progressive inhibition of the enzyme activity with respect to maximal stimulation. Calmodulin increased the lipid fluidity of synaptosomal plasma membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r_o/r)-1]^–1. Arrhenius-type plots of [(r_o/r)-1]^–1 indicated that the lipid separation of the membrane at 22.7±1.2° was perturbed by calmodulin such that the temperature was reduced to 16.3±0.9° and 15.5±0.8° in the absence or in the presence of 20 M Ca²⁺. Arrhenius plots of guanylate cyclase and acetylcholinesterase activities exhibited brak points at 25.7±1.4° and 22.3±1.0° in control synaptosomal plasma membranes, respectively. The break point for the guanylate cyclase was reduced to 16.3±0.9° in calmodulin treated synaptosomal plasma membranes whereas that of acetylcholinesterase remained unaffected (21.1±0.9°). The allosteric properties of guanylate cyclase by Mn-GTP (as reflected by changes in the Hill coefficient) were modulated by calmodulin while those of acetylcholinesterase by fluoride (F^–) were not altered. We propose that calmodulin achieves these effects through asymmetric perturbations of the membrane lipid structure and that increase in membrane fluidity of the inner leaflet of the membrane induced by calmodulin may be an early key event to the process of neurotransmitter release.     

Specific binding of a Verticillium dahliae phytotoxin to protoplasts of cotton, Gossypium hirsutum

Summary The occurrence of specific, high-affinity binding sites for a protein-lipopolysaccharide (PLP) phytotoxin purified from culture filtrates of a virulent Vertidllium dahliae isolate has been demonstrated in cotton protoplasts. Binding of the ¹²⁵I-radiolabelled PLP-complex to protoplasts from cotyledon tissue was saturable and with an affinity (K_d = 17.3 nM) comparable with the concentration required for biological activity. A single class of binding site, accessible at the surface of the intact protoplasts, was found and the maximal number of binding sites were estimated as 2.41 × 10^–16 moles per protoplast. The binding affinity to protoplasts proved near identical to that found with purified plasma membrane fractions from roots. When cultivars exhibiting resistance or susceptibility towards the pathogen were compared, no significant differences were found in the affinity of binding, but five times as many binding sites per protoplast and sixteen times as many binding sites per mg membrane protein were found in the resistant cultivar.Abbreviations PLP protein-lipopolysaccharide - k_d dissociation constant - B_max maximal number of binding sites - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Differential effect of ethanol on muscarinic cholinergic binding to rat and locust neural membranes

We have compared the effect of ethanol, a membrane perturbant, on the muscarinic binding sites in neural membranes from a vertebrate (rat) and an insect (locust). The binding of the muscarinic antagonist [³H]quinuclidinyl benzilate ([³H]QNB) to both rat and locust neural membranes was inhibited by ethanol at 10–500 mM concentrations; but this inhibition was greater in the locust. Ethanol (500 mM) increased the apparent dissociation constant (K d) of [³H]QNB binding to rat membranes from 0.13±0.01 nM in control to 0.20±0.02 nM; there was also an small but significant reduction in the number of binding sitesB _max. In locust, 500 mM ethanol reduced theB _max of [³H]QNB binding from 590±30 in control to 320±40 pmol/g protein; no significant alteration in theK _D was detected. The dissociation rate constant (k _off) of [³H]QNB increased from 0.020±0.003 in controls to 0.031±0.004 (min^–1) in the presence of 500mM ethanol, the association rate constant (k _on) did not change significantly. In locust, 500 mM ethanol did not affect eitherk _on ork _off. Competition experiments revealed that the binding affinities of both the agonist carbamylcholine and the antagonist atropine to the rat membranes were reduced in the presence of ethanol. In contrast, ethanol caused no alteration in the binding affinities of these ligands to the locust membranes. This differential effect of ethanol on rat and locust muscarinic binding suggests a difference in the hydrophobic domains and/or the membrane interactions of the muscarinic receptors in the two species.     

Studies on lithium transport across the red cell membrane

Summary Binding of³H-saxitoxin to Na⁺ channels was studied in subcellular fractions prepared from rat brain homogenates. Saxitoxin binding to synaptosomes was saturable with an apparent dissociation constant of about 1nm; about 1 pmol/mg protein was bound at saturating saxitoxin concentrations. A linear, nonsaturable component of saxitoxin binding accounted for less than 3% of the total binding at 30nm. Saxitoxin binding to synaptosomes was unaffected by depolarization with elevated K⁺ concentrations, or by activation of the Na⁺ channels with batrachotoxin plus a purified polypeptide toxin from the scorpionLeiurus quinquestriatus. A procedure is described for preparing a membrane fraction that contains 70–80% of the total saxitoxin binding activity of the crude homogenate. The specific activity of this fraction was about 4 to 6 pmol/mg protein. About 60–70% of the saxitoxin binding sites were solubilized by incubating these membranes with the nonionic detergent Triton X-100; the detergent-solubilized binding sites eluted at a position corresponding to a mol wt of about 700,000 on gel filtration chromatography. Both membrane-bound and solubilized saxitoxin binding were assayed by a new cation exchange column method. The binding of saxitoxin to both membrane-bound and detergent-solubilized binding sites was saturable with an apparent dissociation constant of about 2nm. Dissociation of the saxitoxin-receptor complex followed a single exponential decay with a rate constant at 0° of 0.1 min^–1 for membrane bound and 0.2 min^–1 for detergent-solubilized binding sites. The measured association rate constant was 6×10⁸ m ^–1 min^–1 at 0° for membrane-bound saxitoxin binding sites.     

Ca2+-dependent regulation of calmodulin binding and adenylate cyclase activation in bovine cerebellar membranes

The binding parameters of ¹²⁵I-labeled calmodulin to bovine cerebellar membranes have been determined and correlted with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca²⁺, calmodulin binds to a finite number of specific membrane sites with a dissociation constant (K_d) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca²⁺-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be infered from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca²⁺ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca²⁺ atoms. The concentration of the active calmodulin ·Ca²⁺ species required for half-maximal activation of adenylate cyclase is very similar to the K_d of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca²⁺-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.     

Noninvasive assessment of cardiac contractility by using (dP/dt)/P of carotid artery pulses during exercise

In earlier studies we have shown that both the pressure (P) of the carotid artery pulse (CAP) and its first derivative (CAP dP/dt) could be recorded during moderate exercise. To establish that the CAP (dP/dt)/P is a noninvasive substitute for the left ventricular (LV) value, LV (dP/dt)/P, an index of cardiac contractility, we studied CAP (dP/dt)/P under various states of activity in the autonomic nervous system in 12 healthy male subjects. Increased sympathetic nerve activities yielded by passive tilting, emotional load, or cold stress increased CAP (dP/dt)/P significantly (P< 0.05). Increased parasympathetic nerve activity by ocular compression, however, did not significantly affect the value. Moderate exercise at a heart rate of approximately 150 beats·min^–1 increased it significantly from 16.7 to 25.2·s^–1 in a supine position (P<0.001) and from 16.6 to 24.8·s^–1 in an upright position (P<0.001). It increased monotonically as heart rate increased, but the slope was steeper when the heart rate was greater than approximately 100 beats·min^–1 than it was when the rate was less than 100 beats·min^–1. In conclusion, the present study indicated that CAP (dP/dt)/P can be used as a noninvasive index of cardiac contractility even in moderate exercise.     

Isolation of membrane bound light-harvesting-complexes from the dinoflagellates Heterocapsa pygmaea and Prorocentrum minimum

We have isolated Chl a-Chl c-carotenoid binding proteins from the dinoflagellates Prorocentrum minimum and Heterocapsa pygmaea grown under high (500 mol m^–2 s^–1, HL) and low (35 mol m^–2 s^–1, LL) light conditions. We compared various isolation procedures of membrane bound light harvesting complexes (LHCs) and assayed the functionality of the solubilized proteins by determining the energy transfer efficiency from the accessory pigments to Chl a by means of fluorescence excitation spectra. The identity of the newly isolated protein-complexes were confirmed by immunological cross-reactions with antibodies raised against the previously described membrane bound Chl a-c proteins (Boczar et al. (1980) FEBS Lett 120: 243–247). Spectroscopic analysis demonstrated the relatedness of these proteins with the recently described Chl-a-c ₂-peridinin (ACP) binding protein (Hiller et al. (1993) Photochem Photobiol 57: 125–131; Iglesias Prieto et al. (1993) Phil Trans R Soc London B 338: 381–392). The water-soluble peridinin-Chl a binding-protein (PCP) was not detectable in P. minimum. Two functional forms of ACP with different pigmentation were isolated. A variant of ACP which was isolated from high-light grown cells, that specifically binds increased amounts of diadinoxanthin was compared to the previously described ACPs that bind proportionately more peridinin.Abbreviations ACP Chl a-Chl c-peridinin binding protein - AEBSF 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride - DDM dodecyl -d maltoside - Deriphat 160 N-lauryl-beta-iminopropionic acid - HEPES (N-2-hydroxyethylpiparizine-N-2-ethanesulphonic acid) - HL high light (500 mol m^–2 s^–1) - LL low light (35 mol m^–2 s^–1) - ₇₃₀ fluorescence yield (emission at 730 nm) - PCP peridinin-Chl a-binding protein - PMSF phenyl-methyl-sulfonyl-fluoride - PS I Photosystem I - PS II Photosystem II     

Binding of spectinomycin by ribosomes fromEscherichia coli

The binding of the aminocyclitol antibiotic spectinomycin to 70S ribosomes and to 30S subunits fromEscherichia coli has been investigated. The association was influenced by the presence of messenger RNA. The K_d for [³H]-4 OH-spectinomycin binding to 70S ribosomes was 2×10^–7 M without mRNA (polyinosinic acid), and 1×10^–6 M with polyinosinic acid. Dissociation of the antibiotic from the ribosomes was significantly affected by the presence of a bound messenger RNA, which reduced the rate of dissociation by a factor of 5.7. The presence of mRNA did not influence the association of spectinomycin with the 30S subunit. The dissociation rate from the small subunit was comparable to the rate of dissociation from the 70S ribosome and was not affected by the presence of mRNA.     

Regulation of calmodulin content in synaptic plasma membranes by glucocorticoids

Synaptic plasma membranes (SPM) from the brain are known to have specific binding sites for several steroid hormones, but the mechanisms of membrane transduction of steroid signals is not understood. In this study, corticosterone was found to prevent temperature-dependent dissociation of endogenous calmodlin (CaM) from highly purified SPM from rat cerebral cortex. The steroid stabilizes Ca²⁺-dependent membrane binding of endogenous CaM (78% of total CaM), whereas Ca²⁺-independent binding of CaM (the other 22%) is not affected. The stabilization of membrane binding of endogenous CaM by corticosterone is concentration-dependent, with the maximal effect occurring at steroid concentration of 1 M. The EC₅₀ is estimated as 130 nM, which is almost identical to the K_d of specific binding of the steroid to SPM (120 nM) reported previously. The effect in stabilizing membrane binding of CaM is specific to corticosterone and other glucocorticoids (cortisol, dexamethasone and triamcinolone); gonadal steroids (17-estradiol, progesterone and testosterone) are ineffective. Furthermore, corticosterone administration in vivo (2 mg/kg, i.p.) produced a rapid increase of CaM content in SPM, occurring within 5 min after steroid injection and persisting for at least 20 min. Since CaM mediates a variety of biochemical processes in synaptic membranes, we hypothesize that the effect of glucocorticoids in promoting membrane binding of CaM may lead to a cascade of consequences in synaptic membrane function.Special issue dedicated to Dr. Sidney Ochs.     

Kinetics of iron passage through subcellular compartments of rabbit reticulocytes

Summary The kinetics of the separate processes of Fe₂(III)-transferrin binding to the transferrin receptor, transferrin-receptor internalization, iron dissociation from transferrin, iron passage through the membrane, and iron mobilization into the cytoplasm were studied by pulse-chase experiments using rabbit reticulocytes and⁵⁹Fe,¹²⁵I-labeled rabbit transferrin. The binding of⁵⁹Fe-transferrin to transferrin receptors was rapid with an apparent rate constant of 2×10⁵ m ^–1 sec^–1. The rate of internalization of⁵⁹Fe-transferrin was directly measured at 520±100 molecules of Fe₂(III)-transferrin internalized/sec/cell with 250±43 sec needed to internalize the entire complement of reticulocyte transferrin receptors. Subsequent to Fe₂(III)-transferrin internalization the flux of⁵⁹Fe was followed through three compartments: internalized transferrin, membrane, and cytosol.A process preceding iron dissociation from transferrin and a reaction involving membrane-associated iron required 17±2 sec and 34±5 sec, respectively. Apparent rate constants of 0.0075±0.002 sec^–1 and 0.0343±0.0118 sec^–1 were obtained for iron dissociation from transferrin and iron mobilization into the cytosol, respectively. Iron dissociation from transferrin is the rate-limiting step. An apparent rate constant of 0.0112±0.0025 sec^–1 was obtained for processes involving iron transport through the membrane although at least two reactions are likely to be involved. Based on mechanistic considerations, iron transport through the membrane may be attributed to an iron reduction step followed by a translocation step. These data indicate that the uptake of iron in reticulocytes is a sequential process, with steps after the internalization of Fe₂(III)-transferrin that are distinct from the handling of transferrin.     

Demonstration of human platelet β-adrenergic receptors using 125I-labeled cyanopindolol and 125I-labeled hydroxybenzylpindolol

The radioiodinated pindolol analogs ¹²⁵I-labeled cyanopindolol ([¹²⁵I]CYP) and ¹²⁵I-labeled hydroxybenzylpindolol ([¹²⁵I]HBP) have been used to study binding to human platelet β-adrenergic receptors. [¹²⁵I]CYP binds to a saturable class of binding sites on platelet membranes with a dissociation constant (K_d) of 14±3 pM and maximal binding capacity (B_max) of 18±4 fmol/mg protein. Binding of [¹²⁵I]CYP is reversible and is characterized by forward and reverse rate constants of 1.8·10⁷ s^?1·M^?1 and 3.8·10^?4 s^?1, respectively. [¹²⁵I]HBP binds to a saturable class of platelet membrane sites with a K_d of 50±10 pM and B_max of 32±6 fmol/mg protein. [¹²⁵I]HBP also binds to a saturable class of sites on intact platelets with a K_d of 58±14 pM and B_max of 24±4 molecules per platelet. Binding of [¹²⁵I]CYP and [¹²⁵I]HBP is stereospecifically inhibited by propranolol and epinephrine; the (?) stereoisomers are at least 50-times more potent than the (+) stereoisomers. Binding of both radioligands is inhibited by adrenergic ligands with a potency order of propranolol ? isoproterenol > epinephrine > practolol > norepinephrine > phenylephrine. These observations indicate that [¹²⁵I]CYP and [¹²⁵I]HBP bind to platelet sites which have the pharmacological characteristics of β-adrenergic receptors but which are not typical of either the β₁ or β₂ sub-type.     

Behavior of rat hypothalamic and extrahypothalamic immuno-reactive TRF in thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC)

[³H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [³H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K_d) of 1.5 nM was observed when the [³H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K_d 20 nM) was observed when [³H] QNB concentration was above 20 nM. Using 10 nM [³H] QNB for binding, the second order association rate constant (k₁) of 0.064 nM^?1 min^?1 and first order dissociation rate constant (k₂) of 0.078 min^?1$\text{(}\text{T}\text{1}\text{2}\text{8}\text{min}\text{)}$ were observed. k₂/k₁ = K_d of 1.22 nM is in good agreement with K_d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [³H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [³H] QNB binding than the muscarinic agonist.     

Specific Binding of L-[3h]-Glutamic Acid to Rat Substantia Nigra Synaptic Membranes

Abstract

The specific binding of L-[³H] -glutamic acid (GLU) was investigated in synaptic membranes from rat substantia nigra. L-[³H]-GLU binding to the membrane preparations occurred in a reversible and saturable way. The specific binding was stimulated by the presence of CaCl₂ and was reduced by freezing and thawing the membranes. Scatchard analysis of the saturation isotherms yielded a non-linear plot suggesting that the binding reaction does not occur through a simpla bimolecular association. Assuming non-interacting binding sites, a high (K_D1, 139 nM; Bmax1, 3.5 pmoles/mg protein) and a low (K_D2, 667 nM; Bmax2, 15.1 pmoles/mg protein) affinity L-[³H]-GLU binding site were obtained. The kinetics of dissociation of bound L-[³H]-GLU was biphasic; the respective dissociation rate constant (k-1) being 0.20 min^?1 and 0.013 min^?1. A series of amino acid receptor agonists and antagonists were tested as inhibitors of L-[³H]-GLU specific binding. Quisqualic acid, L-GLU and D-α-aminoadipate (D-α-AA) were the most potent inhibitors. DL-2-amino-4-phosphonobutyrate (APB), N-Methy1-D-aspartate (NMDA) and D-GLU were moderate inhibitors, whereas diamino-pimelic acid (DAPA) and glutamate diethyl ester (GDEE) exhibited the lowest relative potency. Kainic acid (KA), γ-aminobutyric acid (GABA) and bicuculline were not able to modify at any concentration used the specific binding of L-[³H]-GLU. These data demonstrate the presence of specific GLU binding sites in synaptic structures at substantia nigra level and support the idea that excitatory amino acids may play a role in synaptic transmission in this brain region.