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1.
The fluorescent probe l-anilinonaphthalene-8-sulfonate (ANS) has been used to investigate the properties of plasma membranes derived from normal hepatocytes and from hepatoma tissue culture (HTC) cells as well as used to study the effects of Ca2+ and procaine on these membrane systems. The interaction of ANS with hepatocyte plasma membranes (50 nmol/mg protein; KD = 120,μM) resulted in a marked enhancement of fluorescence and a 20-nm blue shift. Both Ca2+ and procaine further increased the fluorescence intensity. Binding studies showed no alteration in the number of ANS binding sites but a significant decrease in KD (40–50 μm). Procaine was also shown to completely displace Ca2+ from the membrane. The interaction of ANS with HTC cell plasma membranes again resulted in an enhancement in fluorescence intensity but with different binding properties (102 nmol/mg protein; KD = 74 μM) from the hepatocyte system. The addition of Ca+2 resulted in the formation of high and low affinity ANS binding sites as shown by Scatchard plot analysis with KD values of 15 μm and 50 μm. The effect of procaine on ANS fluorescence in the normal and transformed cell membranes was indistinguishable; however, in the latter system procaine only displaced 60% of the bound Ca2+. These studies suggest several structural and binding alterations between plasma membranes derived from hepatocytes and HTC cells. 相似文献
2.
Jacqueline M. Skeer David B. Sattelle 《Archives of insect biochemistry and physiology》1993,23(3):111-124
This study has identified specific, stereoselective phenylalkylamine (PAA, (±)- [3H]verapamil) binding sites of low-affinity and high-density in cockroach (Periplaneta americana) nervous system and skeletal muscle membranes. Scatchard transformation of equilibrium binding data revealed a single population of binding sites in both tissues with dissociation constants (Kd) of 273 nM and 377 nM and binding capacities (Bmax) of 23 pmol·mg protein?1 and 37pmol·mg protein?1 for cockroach nervous tissue and skeletal muscle membranes, respectively. The PAA binding site in cockroach nervous tissue membranes was found to be dihydropyridine (DHP)-insensitive, whereas the corresponding site in cockroach skeletal muscle membranes was DHP-sensitive. This property of a DHP-sensitive PAA receptor distinguishes the binding sites identified in cockroach skeletal muscle from those in cockroach nervous tissue and indicates that pharmacologically distinct putative Ca2+ channel subtypes are present in insect nerve and muscle. © 1993 Wiley-Liss, Inc. 相似文献
3.
5'-Azido-N-1-Napthylphthalamic Acid, a Photolabile Analog of N-1-Naphthylphthalamic Acid : Synthesis and Binding Properties in Curcurbita pepo L
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A photolabile analog of N-1-naphthylphthalamic acid (NPA), 5′-azido-N-1-naphthylphthalamic acid (Az-NPA), has been synthesized and characterized. This potential photoaffinity label for the plasma membrane NPA binding protein competes with [3H]NPA for binding sites on Curcurbita pepo L. (zucchini) hypocotyl cell membranes with K0.5 = 2.8 × 10−7 molar. The K0.5 for NPA under these conditions is 2 × 10−8 molar, indicating that the affinity of Az-NPA for the membranes is only 14-fold lower than NPA. While the binding of Az-NPA to NPA binding sites is reversible in the dark, exposure of the Az-NPA treated membranes to light results in a 30% loss in [3H]NPA binding ability. Pretreatment of the membranes with NPA protects the membranes against photodestruction of [3H]NPA binding sites by Az-NPA supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment. 相似文献
4.
The finding that molt-inhibiting hormone (MIH) regulates vitellogenesis in the hepatopancreas of mature Callinectes sapidus females, raised the need for the characterization of its mode of action. Using classical radioligand binding assays, we located specific, saturable, and non-cooperative binding sites for MIH in the Y-organs of juveniles (J-YO) and in the hepatopancreas of vitellogenic adult females. MIH binding to the hepatopancreas membranes had an affinity 77 times lower than that of juvenile YO membranes (KD values: 3.22 × 10-8 and 4.19 × 10-10 M/mg protein, respectively). The number of maximum binding sites (BMAX) was approximately two times higher in the hepatopancreas than in the YO (BMAX values: 9.24 × 10-9 and 4.8 × 10-9 M/mg protein, respectively). Furthermore, MIH binding site number in the hepatopancreas was dependent on ovarian stage and was twice as high at stage 3 than at stages 2 and 1. SDS-PAGE separation of [125I] MIH or [125I] crustacean hyperglycemic hormone (CHH) crosslinked to the specific binding sites in the membranes of the J-YO and hepatopancreas suggests a molecular weight of ~51 kDa for a MIH receptor in both tissues and a molecular weight of ~61 kDa for a CHH receptor in the hepatopancreas. The use of an in vitro incubation of hepatopancreas fragments suggests that MIH probably utilizes cAMP as a second messenger in this tissue, as cAMP levels increased in response to MIH. Additionally, 8-Bromo-cAMP mimicked the effects of MIH on vitellogenin (VtG) mRNA and heterogeneous nuclear (hn) VtG RNA levels. The results imply that the functions of MIH in the regulation of molt and vitellogenesis are mediated through tissue specific receptors with different kinetics and signal transduction. MIH ability to regulate vitellogenesis is associated with the appearance of MIH specific membrane binding sites in the hepatopancreas upon pubertal/final molt. 相似文献
5.
Localization of 3,5,3'-L-triiodothyronine receptor in rat kidney mitochondrial membranes 总被引:3,自引:0,他引:3
Binding of triiodothyronine(T3) to submitochondrial fractions from rat kidney was studied. Both inner and outer mitochondrial membranes were purified by sucrose gradient centrifugation. Both membranes had specific binding sites for T3. Scatchard analysis of T3 binding by membranes gave different affinity constants between inner and outer membranes. In studies with gel filtration of soluble T3 receptors, four main T3 binding activities in outer membranes and two main T3 binding activities in inner membranes were isolated. The results indicate that both inner and outer mitochondrial membranes have specific binding sites for T3 and that each membrane has a specific structure in T3 receptor. 相似文献
6.
N. Bojji Reddy W. King Engel 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,585(3):343-359
[3H]Dihydroalprenolol, a potent ß-adrenergic antagonist, was used to identify the adenylate cyclase-coupled ß-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed [3H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [3H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [3H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions.The binding of [3H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K1) and dissociation (K2) rate constants of 2.21 · M? · min?1 and 3.21 · 10?1, respectively, were obtained. The dissociation constant (Kd) of 15 nM for [3H]dihydroalprenolol obtained from saturation binding data closely agreed with the (Kd) derived from the ratio of dissociation and association rate constants (K2/K1).Several β-adrenergic agents known to be active on intact skeletal muscle also competed for [3H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [3H]dihydroalprenolol binding sites with a potency of isoproterenol > epinephrine > norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (?)-isomers being more than potent than (+)-isomers. Phenylephrine, an α-adrenergic agonist, showed no effect either on [3H]dihydroalprenolol binding or on adenylate cyclase. Known ß-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [3H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The (Ki) values for the antagonists determined from inhibition of [3H]dihydroalprenolol binding agreed closely with the (Ki) values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [3H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the ß-adrenergic receptor. 相似文献
7.
Chihiro Nishimura Seitaro Ohkuma Jun-ichi Tamura Kinya Kuriyama 《Neurochemistry international》1982,4(5):413-418
The specific binding of GABA (γ-aminobutyric acid) agonist 3H-muscimol, to synaptic membranes from the rat brain showed a significant increase, when the membranous preparations were treated with a low concentration (10?4–10?5M) of mercurial sulfhydryl reagents such as p-chloromercuribenzoate and mercuric chloride. This activation in GABA receptor binding was bicuculline-sensitive, and was partially restored by subsequent treatments with 10 mM cysteine, penicillamine, or mercaptoethanol. Scatchard analysis of the binding revealed that this activation was due to the increase in the affinity of both high and low affinity bindings sites but not in the Bmax values. On the other hand, the treatment of synaptic membranes with hydrophilic sulfhydryl reagents such as N-ethylmaleimide and iodoacetate had no effect on the binding. These hydrophilic sulfhydryl reagents, however, induced an increase of the binding following the pretreatment of synaptic membranes with 0.01% Triton X-100 or 0.5 U/mg prot. of phospholipase A2 (EC 3.1.1.4.). These results suggest that mercurials-sensitive sulfhydryl groups, which are normally masked by membrane lipids, may play a modulating role in GABA receptor binding at central synapses. 相似文献
8.
Calcium-binding properties and ATPase activities of rat liver plasma membranes 总被引:1,自引:0,他引:1
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A M Chambaut F Leray-Pecker G Feldmann J Hanoune 《The Journal of general physiology》1974,64(1):104-126
Plasma membranes from rat liver purified according to the procedure of Neville bind calcium ions by a concentration-dependent, saturable process with at least two classes of binding sites. The higher affinity sites bind 45 nmol calcium/mg membrane protein with a KD of 3 µM. Adrenalectomy increases the number of the higher affinity sites and the corresponding KD. Plasma membranes exhibit a (Na+-K+)-independent-Mg2+-ATPase activity which is not activated by calcium between 0.1 µM and 10 mM CaCl2. Calcium can, with less efficiency, substitute for magnesium as a cofactor for the (Na+-K+)-independent ATPase. Both Mg2+- and Ca2+-ATPase activities are identical with respect to pH dependence, nucleotide specificity and sensitivity to inhibitors. But when calcium is substituted for magnesium, there is no detectable membrane phosphorylation from [γ-32P] ATP as it is found in the presence of magnesium. The existence of high affinity binding sites for calcium in liver plasma membranes is compatible with a regulatory role of this ion in membrane enzymic mechanisms or in hormone actions. Plasma membranes obtained by the procedure of Neville are devoid of any Ca2+-activated-Mg2+-ATPase activity indicating the absence of the classical energy-dependent calcium ion transport. These results would suggest that the overall calcium-extruding activity of the liver cell is mediated by a mechanism involving no direct ATP hydrolysis at the membrane level. 相似文献
9.
Yves Giudicelli Daniele Lacasa Brigitte Agli 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,715(1):105-115
The binding characteristics of the β-adrenergic agonist to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37°C (second order rate constant k1=1.37·107·M?1·min?1). Dissociation of specific binding by 0.5 mM (?)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k2 of 0.106·min?1 and 0.011·min?1.[3H]Hydroxybenzylisoproterenol binding was saturable (Bmax=690±107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [3H]hydroxybenzylisoproterenol binding sites, one having high affinity (KD=3.5±0.7 nM) but low binding capacity (10% of the total sites) and one haveing low affinity (KD=101±20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [3H]hydroxybenzylisoproterenol binding with the following order of potency=(?)-propranolol>(?)-isoproterenol>(?)-norepinephrine≈ (?)-epinephrine>>(+)-isoproterenol=(+)-propranolo, which is consistent with binding to β1-adrenergic receptors. Competition curves of [3H]hydroxybenzylisoproterenol binding by the β-agonist (?)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by β-antagonist (?)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [3H]hydroxybenzyl-isoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (?)-isoproterenol, then number of [3H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (?)-isoproterenol to stimulate adenylate cyclase correlate closely with the ability of (?)-isoproterenol to displace [3H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the β-antagonist [3H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [3H]hydroxybenzylisoproterenol can be successfully used to label the β-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [3H]dihydroalprenolol in these cells. 相似文献
10.
Diego Ruano Jesus Benavides Alberto Machado Javier Vitorica 《Journal of neurochemistry》1995,64(2):867-873
Abstract: The aging-associated changes in hippocampal benzodiazepine (ω) receptor isotypes have been investigated in rats of the Wistar and Fischer 344 strains. Displacement experiments of [3H]flunitrazepam binding by zolpidem demonstrated that in hippocampal membranes from adult (3-month-old) Wistar strain rats, high (type I; ω1)-, intermediate (type IIM; ω2)-, and low (type IIL; ω5)- affinity sites for this imidazopyridine account for 27.1 ± 7.5, 44.2 ± 7.5, and 28.8 ± 5.1%, respectively. In hippocampal membranes from aged (24-month-old) rats of the same strain, the relative abundance of these sites was 42.8 ± 9.3, 26.3 ± 4, and 36.0 ± 5.9%, respectively. In contrast, no significant difference was observed in the whole benzodiazepine (ω) binding site density between adult and aged rats. The increase in type I (ω1) binding site density in the hippocampus of aged rats was also demonstrated in saturation experiments with [3H]zolpidem. This aging-induced increase in [3H]zolpidem binding was also observed in hippocampal membranes from Fischer 344 rats. Moreover, in both rat strains, GABA induced a greater enhancement of [3H]zolpidem (5 nM) binding to type I (ω1) sites (GABA shift) in aged than in adult hippocampal membranes. Quantitative autoradiographic analysis of [3H]zolpidem binding to coronal brain sections from adult and aged Fischer 344 rats demonstrated that the aging-associated increases in the density of type I (ω1) binding sites were restricted to the hippocampus. Moreover, increases in binding density were larger in the dentate gyrus and in the CA2 field than in the CA1 and CA3 fields. 相似文献
11.
《The International journal of biochemistry》1994,26(2):201-206
- 1.1. A non-radioisotopic method utilizing a biotin-avidin approach was used to characterize lactoferrin binding to the clonal MAC-T bovine mammary epithelial cell line.
- 2.2. Binding of lactoferrin to MAC-T cells and isolated membranes was specific and saturable.
- 3.3. Unlabeled lactoferrin competed for and displaced biotin-labeled lactoferrin from binding sites on mammary epithelial cells. In contrast, unlabeled transferrin did not compete.
- 4.4. Scatchard analysis of lactoferrin binding to MAC-T cell crude membranes was nonlinear, revealing two classes of binding sites with association constants (Ka) of 2.36 × 107 and 3.36 × 106M−1.
- 5.5. Binding of lactoferrin to MAC-T cells may be associated with the initial events which result in decreased MAC-T cell proliferation.
12.
J Hyttel 《Life sciences》1978,23(6):551-555
The subcellular localization of dopamine-sensitive adenylate cyclase was studied in rat brain striatum and compared to the distribution of dopamine binding sites. The highest specific activity of adenylate cyclase activities sensitive to dopamine was associated almost exclusively with synaptic membranes (mithchondrial fraction; P2). Using [3H] haloperidol and [3H] apomorphine as markers for the dopamine receptor, specific binding was observed in both the mitochondrial (P2) and microsomal (P3) fractions. Data for the mitochondrial fraction revealed a heterogeneity of binding sites. Two saturable sites for [3H] haloperidol were observed with Kd values of 2.5nM and 12.5nM respectively. Overall, the localization of multiple binding sites in the crude synaptosomal fraction correlates well with the localization of dopamine-sensitive adenylate cyclase in this fraction. 相似文献
13.
The binding of [3H]ploridzin by isolated luminal membranes of the rabbit proximal tubule and by slices of rabbit kidney cortex was studied.Kinetic analyses of the relationship between the concentration of phloridizin in the incubation medium and the binding of phloridzin to the membrane indicated two distinct classes of receptors sites. One class, comprising high affinity sites, reached saturation at 20–25 μM phloridzin, had a K(phloridzin) of 8 μM, and 8·10+2 nmoles interacted with 1 mg of brush border protein. The other class, comprising low affinity sites, had a K(phloridzin) of 2.5 mM, and the number of binding sites was 1.25 nmoles/mg Na+ was required for the binding of phloridzin at the high affinity sites. Na+ decreased the apparent Ki for phloridzin; the apparent V of binding was not altered. Binding at the low affinity sites was independent of Na+. Ca2+ was necessary for maximal binding at the high affinity sites. Binding of phloridzin at high affinity sites was more sensitive to N-ethylmalcimide and mersalyl than was binding at low affinity sites. Binding at high affinity sites, but not at low affinity sites, was temperature dependent.d-Glucose was a competitive inhibitor of the high affinity binding of phloridzin. The apparent K1 was 1 mM. D-Glucoe inhibited non-competitively at the low affinity sites. l-Glucose had no influence on phloridzin binding. Phloretin was a competitive inhibitor of high affinity phloridzin binding with an apparent Ki of 16 μM. Phloretin inhibited low affinity bindings of phloridizin non-competitively. Binding of phloridzin at high affinity sites was completely reversible. Binding at low affinity sites was only partially reversed. Phloridzin bound at high affinity sites on the brush border was displaced by phloridzin and phloretin but not by d-glucose.The mechanism of the high affinity binding of phloridzin was distinguished from that of the initial interaction of d-glucose with the membrane. Binding of phloridzin required Na+, whereas the interaction of d-glucose with the membranes had a prominent Na+-independent component.Intact renal cells in cortical slices accumulated phloridzin. The uptake did not saturate, was Na+ independent, and was not competitively inhibited by sugars. These characteristics resemble those for the low affinity binding of phloridzin by isolated membranes. It is suggested that low affinity binding may represent an initial binding followed by uptake of the glycoside into membrane vesicles. 相似文献
14.
Brigitte Amiranoff Marc Laburthe Gabriel Rosselin 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,627(2):215-224
Specific binding sites for vasoactive intestinal peptide were characterized in plasma membranes from rat intestinal epithelial cells. At 30°C, the interaction of 125I-labelled peptide with intestinal membranes was rapid, reversible, specific and saturable. At equilibrium, the binding of 125I-labelled peptide was competitively inhibited by native peptide in the 3 · 10?11?3 · 10?7 M range concentration. Scatchard analysis of binding data suggested the presence of two distinct classes of vasoactive intestinal peptide binding sites: a class with a high affinity Kd = 0.28 nM) and a low capacity (0.8 pmol peptide/mg membrane protein) and a class with a low affinity (Kd = 152 nM) and a high capacity (161 pmol peptide/mg membrane protein). Secretin competitively inhibited binding of 125I-labelled peptide but its potency was 1/1000 that of native peptide. Glucagon and the gastric inhibitory peptide were ineffective. The guanine nucleotides, GTP and Gpp(NH)p inhibited markedly the interaction of 125I-labelled peptide with its binding sites, by increasing the rate of dissociation of peptide bound to membranes. The other nucleotides triphosphate tested (ATP, ITP, UTP, CTP) were also effective in inhibiting binding of 125I-labelled peptide to membranes but their potencies were 1/100-1/1000 that of guanine nucleotides.The specificity and affinity of the vasoactive intestinal peptide-binding sites in plasma membranes prepared from rat intestinal epithelial cells, which is in agreement with an adenylate cyclase highly sensitive to the peptide recently characterized in these membranes (Amiranoff, B., Laburthe, M., Dupont, C. and Rosselin, G. (1978) Biochim. Biophys. Acta 544, 474–481) further argue for a physiological role of the peptide in the regulation of intestinal epithelial function. 相似文献
15.
《Neurochemistry international》1986,8(2):223-227
The sensitivity of [3H]GABA and [3H]muscimol high-affinity binding sites to physiological (Krebs-Ringer's bicarbonate) and non-physiological (Tris-citrate) buffers was examined using synaptosomal membranes from bovine retinas. The maximum number of sites (Bmax) for [3H]GABA was present when the tissue was assayed in KRB. With only one exception, this effect was independent of the washing conditions used or a small change in pH. In contrast, [3H]muscimol binding sites were maximally present when the tissue was washed in Tris, regardless of the assaying conditions or the small change in pH. Neither [3H]GABA nor [3H]muscimol was displaced by ( - )baclofen. The apparent dissociation constants (Kd) of the ligands did not change under any of the conditions tested. These findings demonstrate a fundamental difference between GABA and muscimol binding sites. 相似文献
16.
Ronald J. Lukasiewicz Edward L. Bennett 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,544(2):294-308
High-affinity, specific binding of radiolabeled α-bungarotoxin to particulate fractions derived from rat brain shows saturability (Bmax ≈ 37fmol/mg, KDapp = 1.7 nM) and insensitivity to ionic strength, and is essentially irreversible (Kon = 5 · 106 min?1 · mol?1; Kdisplacement = 1.9 · 10?4 min?1, τ1/2 = 62 h). Subcellular distribution of specific sites is consistent with their location on synaptic junctional complex and post-synaptic membranes. These membrane-bound binding sites exhibit unique sensitivity to cholinergic ligands; pretreatment of membranes with cholinergic agonists (but not antagonists) induces transformation of α-bungarotoxin binding sites to a high affinity form toward agonist. The effect is most marked for the natural agonist, acetylcholine. These results strongly support the notion that the entity under study is an authentic nicotinic acetylcholine receptor. 相似文献
17.
Adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cells and rat brain membranes were investigated using [3H]adenosine as labelled ligand. Both the hybrid cells and brain membranes were found to have a high affinity binding site, Kd 0.8 and 3 nM respectively. The same ligand was used to demonstrate two lower affinity binding sites on brain membranes, Kds 1.4 and 29.1 μM and a single low affinity site on the hybrid cells, Kd 2.6 μM. Structure activity studies of the low affinity binding site on hybrid cells showed this to be an ‘R’ adenosine receptor of the A2 subtype. It is concluded that [3H]adenosine can be used to demonstrate both high and low affinity binding sites and that 108CC15 hybrid cells provide a valuable system for studying adenosine receptors. 相似文献
18.
The Min proteins (MinC, MinD, and MinE) form a pole-to-pole oscillator that controls the spatial assembly of the division machinery in Escherichia coli cells. Previous studies identified that interactions of MinD with phospholipids positioned the Min machinery at the membrane. We extend these studies by measuring the affinity, kinetics, and ATPase activity of E. coli MinD, MinE, and MinDE binding to supported lipid bilayers containing varying compositions of anionic phospholipids. Using quartz crystal microbalance measurements, we found that the binding affinity (Kd) for the interaction of recombinant E. coli MinD and MinE with lipid bilayers increased with increasing concentration of the anionic phospholipids phosphatidylglycerol and cardiolipin. The Kd for MinD (1.8 μm) in the presence of ATP was smaller than for MinE (12.1 μm) binding to membranes consisting of 95:5 phosphatidylcholine/cardiolipin. The simultaneous binding of MinD and MinE to membranes revealed that increasing the concentration of anionic phospholipid stimulates the initial rate of adsorption (kon). The ATPase activity of MinD decreased in the presence of anionic phospholipids. These results indicate that anionic lipids, which are concentrated at the poles, increase the retention of MinD and MinE and explain its dwell time at this region of bacterial cells. These studies provide insight into interactions between MinD and MinE and between these proteins and membranes that are relevant to understanding the process of bacterial cell division, in which the interaction of proteins and membranes is essential. 相似文献
19.
Armand Malnoë Jos A. Cox Eric A. Stein 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,714(1):84-92
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 (Kd) 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 Kd 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. 相似文献
20.
Anthony G. Lee 《Biophysical journal》2021,120(13):2710-2722
γ-Aminobutyric acid type A (GABAA) receptors in the brain are located in the outer membranes of brain cells where the concentration of cholesterol is high. Of the 25 available high-resolution structures available for GABAA receptors, none were determined in the presence of cholesterol, but four include resolved molecules of cholesterol hemisuccinate (CHS). Here, a molecular docking procedure is used to sweep the transmembrane (TM) surfaces of the receptors for cholesterol binding sites. Cholesterol docking poses determined in this way match 89% of the resolved CHS when CHS molecules deemed unlikely to represent typical bound cholesterols are excluded. The receptors are pentameric, and their TM surfaces consist of a set of five facets, each including pairs of TM helices from two adjacent subunits. Each facet contains hydrophobic hollows running from one side of the membrane to the other, within which are six potential binding sites for cholesterol, three on each side of the membrane. High-resolution structures of GABAA receptors with bound neurosteroids show that neurosteroids bind in these cholesterol binding sites, so the binding of neurosteroids and cholesterol will be competitive. 相似文献