Effects Of Specific Ligands Research Articles

In silico modelling of chelate stabilized tetrylene derivatives.

The steric and electronic effects of specific ligands can play crucial roles in stabilizing unsaturated tetrylene species. In this work, hybrid density functional theory (DFT) methods, quantum theory of atoms in molecules (QTAIM) investigations and natural bond orbital (NBO) calculations are employed to evaluate the stabilization of low-valent E(ii) centers (E = Si, Ge, Sn, Pb) through the chelating effect generated by an electron-rich ligand containing the P[double bond, length as m-dash]C-P[double bond, length as m-dash]X moiety (X = O or S). Based on several types of analyses, such as the bond dissociation energy (BDE) or the interplay between attractive (i.e., charge-transfer) and repulsive (i.e., Pauli-exchange) effects, we highlight that the stabilization energy induced by chelation is up to ca. 70 kcal mol-1 for silylenes, yet slightly decreases within the heavier analogues. Moreover, it is emphasized that chelate-stabilized silylenes can form highly stable hybrid metal-metalloid complexes with transition metals (e.g., gold). Due to push-pull effects occurring in the X→Si(ii)→Au fragment, the Si(ii)→Au bonding is significantly stronger than the X→Au, P(sp2)→Au or π(C[double bond, length as m-dash]P)→Au donor-acceptor bonds, which are potentially formed by the electron-rich P[double bond, length as m-dash]C-P[double bond, length as m-dash]X unit with the AuCl fragment. These findings are supported by energy decomposition analysis (EDA) calculations.

Nicotine facilitates nicotinic acetylcholine receptor targeting to mitochondria but makes them less susceptible to selective ligands.

Several nicotinic acetylcholine receptor (nAChR) subtypes are expressed in mitochondria to regulate the internal pathway of apoptosis in ion channel-independent manner. However, the mechanisms of nAChR activation in mitochondria and targeting to mitochondria are still unknown. Nicotine has been shown to favor nAChR pentamer assembly, folding, and maturation on the way of biosynthesis. The idea of the present work was to determine whether nicotine affects the content, glycosylation, and function of mitochondrial nAChRs. Experiments were performed in isolated liver mitochondria from mice, that either consumed or not nicotine with the drinking water (200μL/L) for 7days. Mitochondria detergent lysates were studied by sandwich or lectin ELISA for the presence and carbohydrate composition of different nAChR subunits. Intact mitochondria were examined by flow cytometry for the binding of fluorescently labeled α-cobratoxin and were tested in functional assay of cytochrome c release under the effect of either Ca2+ or wortmannin in the presence or absence of nAChR-selective ligands, including PNU-282987 (1nM), dihydro-β-erythroidine (DhβE, 1μM), PNU-120596 (0.3, 3, or 10μM) and desformylflustrabromine hydrochloride (dFBr, 0.001, 0.3, or 1μM). It was found that nicotine consumption increased the ratio of mitochondrial vs non-mitochondrial nAChRs in the liver, enhanced fucosylation of mitochondrial nAChRs, but prevented the binding of α-cobratoxin and the cytochrome c release-attenuating effects of nAChR-specific agonists, antagonists, or positive allosteric modulators. It is concluded that nicotine consumption in vivo favors nAChR glycosylation and trafficking to mitochondria but makes them less susceptible to the effects of specific ligands.

Comparative analysis of esterase and paraoxonase activities of different serum albumin species

Enzymatic activities of three types of serum albumin—rat, bovine and human—were analyzed comparatively using a mathematical model. Kinetic and equilibrium constants of carboxylesterase and paraoxonase activities of albumin in Sudlow’s sites I and II were determined. The effects of specific ligands, ibuprofen and warfarin, on enzyme kinetics in these sites were studied. Ibuprofen was found to have an inhibitory effect both on carboxylesterase and paraoxonase albumin activities, whereas warfarin specifically inhibited only carboxylesterase albumin activity.

Trehalose induces functionally active conformation in the intrinsically disordered N-terminal domain of glucocorticoid receptor

Glucocorticoid receptor (GR) is a classic member of the nuclear receptor superfamily and plays pivotal roles in human physiology at the level of gene regulation. Various constellations of cellular cofactors are required to associate with GR to activate/repress genes. The effects of specific ligands on the AF2 structure and consequent preferential binding of co-activators or co-repressors have helped our understanding of the mechanisms involved. But the data so far fall short of fully explaining GR actions. We believe that this is because work so far has largely avoided detailed examination of the contributions of AF1 to overall GR actions. It has been shown that the GR containing only the N-terminal domain (NTD) and the DNA-binding domain (GR500) is constitutively quite active in stimulating transcription from simple promoters. However, we are only beginning to understand structure and functions of GR500 in spite of the fact that AF1 located within the NTD serves as major transactivation domain for GR. Lack of this information has hampered our complete understanding of how GR regulates its target gene(s). The major obstacle in determining GR500 structure has been due to its intrinsically disordered NTD conformation, frequently found in transcription factors. In this study, we tested whether a naturally occurring osmolyte, trehalose, can promote functionally ordered conformation in GR500. Our data show that in the presence of trehalose, GR500 is capable of formation of a native-like functionally folded conformation.

Photochemical properties and shape evolution of CdSe QDs in a non-injection reaction

Highly monodispersed CdSe quantum dots (QDs) were prepared without an injection procedure. A series of Cd salts of long chain fatty acids, including Cd-myristate (C14), Cd-palmitate (C16) and Cd-stearate (C18) was prepared, and all metallic precursors and surfactants were mixed together followed by increasing the temperature in a controlled manner. The reaction resulted in highly monodisperse and bright zinc blende QDs. In addition, the effects of specific ligands which have been known to lead anisotropic growth of the nanocrystals in the injection method were investigated. The use of alkyl phosphonic acid and alkyl amine was found to produce extremely monodisperse CdSe QDs with a high quantum yield. This procedure was proven to be able to yield a large quantity of zinc blende CdSe QDs (2 g) in a one-pot reaction. The use of a controlled amount of tetradecylphosphonic acid and octadecylamine resulted in tetrapod- and match-shaped QDs, the first reported by a non-injection method. These results clearly demonstrate that appropriate combination of precursors can provide high quality of CdSe nanocrystals in terms of quantum yield, monodispersity and shape control by a non-injection method.

733: Mixed bacteria generate a differential intra-amniotic inflammatory response compared to infections with a single microbial species

inflammatory response compared to infections with a single microbial species Antonette Dulay, Catalin Buhimschi, Lydia Shook, Guomao Zhao, Mert Bahtiyar, Christian Pettker, Anna Sfakianaki, Heather Lipkind, Sonya Abdel-Razeq, Irina Buhimschi Yale University, Ob/Gyn & Reprod. Sci., New Haven, CT OBJECTIVE: Amniotic fluid (AF) TLR2 mediated innate immune signaling occurs following their engagement by Gram positive (GP), Mycoplasma and Ureaplasma spp. TLR4 recognizes Gram negative (GN) microbial ligands. Because cross-talk among TLR2 and TLR4 may generate a differential immune reaction, we explored in vivo and in vitro the intensity of an inflammatory response elicited by single vs mixed spp. STUDY DESIGN: We studied 133 preterm AF samples from amniocentesis in the following groups: single GN species (n 37, GA 26 3w), single GP (n 24, GA 26 3w), Mycoplasma or Ureaplasma (n 30, GA 26 4w), mixed TLR2 ligands (n 10, GA 26 2w), and mixed TLR2 and TLR4 ligands (n 32, GA 25 1w). IL6, TNF , IL8, and the anti-inflammatory IL10 were assessed by ELISA. Data was corrected for GA. A HEK293 Blue-4 cell line was used to address specific ligand effects with particular focus on TLR4 activation. HEK cells were incubated (n 5) with TLR4 (LPS), TLR2 (Lipoteichoic acid [LTA], Pam3Cys [PAM], Mycoplasma FSL-1) & mixed ligand groups (LPS PAM, LPS FSL-1, PAM LTA). RESULTS: In vivo, compared to all other possible combinations, single GN infections had the highest AF IL6, IL8, TNF and IL10 levels (P .001). There was no difference in the inflammatory response among various single TLR2 engaging spp. Conversely, mixed GPs augmented all cytokine responses at levels above single TLR2-engaging spp. (P .016). When GNs cohabitate in AF with GP microbes the cytokine response is above that elicited by single or mixed GPs but less than GNs alone (all P .05). In vitro, LPS induced the highest TLR4 activation (P .001), with the lowest response seen for FSL. LPS response was significantly dampened by PAM (12-fold decrease) and FSL-1 (10-fold decrease). LTA PAM gave a 2-fold increase in cell activation over either alone (all P .05). CONCLUSION: GN bacteria generate a robust AF inflammatory response, that could be either potentiated or dampened through their interaction with various GPs or Ureaplasma/Mycoplasma spp.

Partial trypsin digestion as an indicator of mis-folding of mutant alanine:glyoxylate aminotransferase and chaperone effects of specific ligands. Study of a spectrum of missense mutants

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficiency results in primary hyperoxaluria type 1 (PH1). More than 75 PH1 mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have previously demonstrated that many such mutants expressed by transcription/translation are subject to generalized degradation by the proteasome and a specific limited trimming by an endogenous ATP-independent protease activity. Here, we report the results of partial digestion using trypsin as a mimic for the endogenous non-proteasomal protease and the use of N-terminal protein sequencing to determine the sensitive site. Partial trypsin digestion also provided an indicator of proper folding of the mutant enzyme. For selected mutations the sensitivity to trypsin could be ameliorated by addition of pyridoxal phosphate or aminooxy acetic acid as specific pharmacological chaperones.

Effect of Cu(II) ligands on electroless copper deposition rate in formaldehyde solutions: An EQCM study

The electroless copper deposition rate for 6 CuII complexes decreases in the ligand sequence: nitrilotriacetic acid (NTA) > N,N,N′,N′-tetrakis-(2-hydroxypropyl)-ethylenediamine (Quadrol) > glycerol > L(+)-tartrate ~ sucrose > \(\hbox{\sc {DL}}(_+^-)\)-tartrate. Both CuII complex stability and specific ligand effects were found to influence the Cu deposition process. The specific ligand effects are most obvious in the case of Quadrol (high kinetic activity at a high CuII complex stability), glycerol and sucrose (additional reaction of Cu2O formation by interaction of CuII with ligand). According to the EQCM data for 11 CuII complexes (including data from the former study) the higher kinetic activity is demonstrated by complexes with ligands containing amino groups; this factor is more important for Cu deposition rate than copper complex stability. A potential dependence of the Cu reduction partial current on the electrode potential has been extracted from the EQCM data in the complete electroless plating bath. An increase in CuII reduction rate was found to occur in electroless plating solution for CuII complexes with NTA and Quadrol compared with that in formaldehyde-free solutions. Possible reasons for the acceleration of the partial CuII reduction reaction and the overall process kinetics are discussed using a hypothetical reaction sequence involving intermediate copper oxy-species and active Cu* formation as well as development of the preferred Cu surface structure.

Ligand-induced Effects on Pyruvate Dehydrogenase Kinase Isoform 2

Tryptophan fluorescence was used to analyze binding of ligands to human pyruvate dehydrogenase isoform 2 (PDHK2) and to demonstrate effects of ligand binding on distal structure of PDHK2 that is required for binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase. Ligand-altered binding of PDHK2 to L2 and effects of specific ligands on PDHK2 oligomeric state were characterized by analytical ultracentrifugation. ATP, ADP, and pyruvate markedly quenched the tryptophan fluorescence of PDHK2 and gave maximum quenching/L0.5 estimates: approximately 53%/3 microM for ATP; approximately 49%/15 microM for ADP; and approximately 71%/approximately 590 microM for pyruvate. The conversion of Trp-383 to phenylalanine completely removed ATP- and ADP-induced quenching and > or = 80% of the absolute decrease in fluorescence due to pyruvate. The W383F-PDHK2 mutant retained high catalytic activity. Pyruvate, added after ADP, quenched Trp fluorescence with an L0.5 of 3.4 microM pyruvate, > or = 150-fold lower concentration than needed with pyruvate alone. ADP-enhanced binding of pyruvate was maintained with W383F-PDHK2. Binding of PDHK2 dimer to L2 is enhanced when L2 are housed in oligomeric structures, including the glutathione S-transferase (GST)-L2 dimer, and further strengthened by reduction of the lipoyl groups (GST-L2(red)) (Hiromasa and Roche (2003) J. Biol. Chem. 278, 33681-33693). Binding of PDHK2 to GST-L2(red) was modestly hindered by 200 microM level of ATP or ADP or 5.0 mM pyruvate; a marked change to nearly complete prevention of binding was observed with ATP or ADP plus pyruvate at only 100 microM levels, and these conditions caused PDHK2 dimer to associate to a tetramer. These changes should make major contributions to synergistic inhibition of PDHK2 activity by ADP and pyruvate. Ligand-induced changes that interfere with PDHK2 binding to GST-L2(red) may involve release of an interdomain cross arm between PDHK2 subunits in which Trp-383 plays a critical anchoring role.

Stability constants of aza-oxa-crown ether complexes with silver(I) in nonaqueous polar solvents

Complex formation between the silver(I) ion and monoaza- and diaza-crown ethers has been studied in five polar, nonaqueous solvents using potentiometric techniques. The stability constants are spread over seven orders of magnitude, and the major contributor to this variation is the solvation of the silver(I) cation. The ring-size dependence of the stability constants is determined both by the correspondence of the diameters of the silver(I) ion and the ring-cavity size of the macrocycles, and by specific ligand effects including association complex formation between the ligands and the solvent molecules and conformational effects in the free ligands. In the 1:1 complexes of the silver(I) ion with monoaza- and diaza-crown ethers the dominant interaction is between the silver(I) ion and the ligand's nitrogen atoms, but the ether–oxygen atoms also interact with the silver(I) ion. In the AgL 2 + complexes (L=monoaza-crown ether), however, the monoaza-crown ethers coordinate as monodentate ligands, as with simple mono-amine complexants. In these complexes the ethoxy-chains prevent direct interaction between the silver(I) cation and the solvent molecules, with the result that in dipolar aprotic solvents the free energies of transfer of the ligands and the complexes are equivalent, i.e. the so-called cryptate hypothesis holds for the AgL 2 + complexes.

Inhibitors of arachidonic acid metabolism potentiate tumour necrosis factor-α-induced apoptosis in HL-60 cells

We investigated whether and how could various modulators of arachidonic acid metabolism affect apoptosis induced by tumour necrosis factor-α (TNF-α) in human myeloid leukaemia HL-60 cells. These included arachinonyltrifluoromethyl ketone (AACOCF 3; cytosolic phospholipase A 2 inhibitor), indomethacin (cyclooxygenase inhibitor), MK-886 (3-[1-(4-chlorobenzyl)-3- t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethyl propanoic acid; 5-lipoxygenase-activating protein inhibitor), nordihydroguaiaretic acid (general lipoxygenase inhibitor), and arachidonic acid itself. Incubation of HL-60 cells with nordihydroguaiaretic acid resulted in apoptosis and it was characterised by mitochondria membrane depolarisation, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. Indomethacin and nordihydroguaiaretic acid synergistically potentiated TNF-α-induced apoptosis, while arachidonic acid, AACOCF 3 and MK-886 did not modulate its effects. Furthermore, indomethacin potentiated apoptosis in cells treated with a differentiating agent, all-trans retinoic acid, which induces resistance to TNF-α. However, the observed effects were probably not associated either with the cyclooxygenase- or lipoxygenase-dependent activities of indomethacin and nordihydroguaiaretic acid, respectively. Since indomethacin may reportedly activate peroxisome proliferator-activated receptors (PPARs), the effects of specific ligands of PPARs on apoptosis were studied as well. It was found that selective PPARs ligands had no effects on TNF-α-induced apoptosis. The findings suggest that arachidonic acid metabolism does not play a key role in regulation of apoptosis induced by TNF-α in the present model. Nevertheless, our data raise the possibility that indomethacin could potentially be used to improve the treatment of human myeloid leukaemia.

Metal−Ligand Bond Distances in First-Row Transition Metal Coordination Compounds: Coordination Number, Oxidation State, and Specific Ligand Effects

Mean metal−ligand bond distances for the coordination ligands isothiocyanate, pyridine, imidazole, water, and chloride, bound to the transition metals Mn, Fe, Co, Ni, Cu, and Zn in their 2+ oxidation states, were collected from searches the Cambridge Structure Database. The metal−ligand bond distances were converted to bond orders through the bond distance-bond order technique, as suggested by Pauling. The mean bond order sums at the 2+ metal centers were found to be independent of coordination number or geometry and to be strongly ligand-dependent; the values (by ligand) are as follows: isothiocyanate = 2.56 ± 0.13; imidazole = 2.13 ± 0.04; chloride = 2.12 ± 0.07; pyridine 1.95 ± 0.10; water = 1.88 ± 0.10. The bond order sum for Fe(III) bound to chloride was found to be 3.09, approximately one bond order unit larger than for the 2+ metal centers bound to chloride. Division of the ligand-specific bond order sums by coordination number allows prediction of the M−L bond distance to within 0.017 Å, regardless of the specific coordination geometry. The physical basis for the ligand-specific variation in bond order sum is also discussed.

Acetylcholinesterase-induced respiratory burst in macrophages: evidence for the involvement of the macrophage mannose-fucose receptor

It has long been suggested that acetylcholinesterase is capable of functioning in a non-cholinergic manner. However, very little is known about the molecular structures which mediate the interaction between this protein and the cellular membrane. Previously it was demonstrated that acetylcholinesterase interacted in a carbohydrate-specific manner with peritoneal macrophages and induced the ‘respiratory burst’ [1]. This study aimed to establish whether a carbohydrate-binding site exists on the acetylcholinesterase molecule itself, or alternatively, whether the macrophage carbohydrate-binding receptor is involved. No carbohydrate binding properties intrinsic to acetylcholinesterase were detected using affinity chromatography with immobilised monosaccharides, erythrocyte agglutination and gel-diffusion techniques. The interaction between acetylcholinesterase and several monosaccharide columns observed in this study appeared to be due to ionic interactions. Moreover, it was shown that a specific inhibitor of the enzymatic activity of AChE, BW284C51, could inhibit the peritoneal cell response not only to acetylcholinesterase, but also to several other stimuli, thus exhibiting a non-specific effect on macrophages. However, the inhibitory effects of specific ligands of the macrophage mannose-fucose receptor and the inability of non-glycosylated acetylcholinesterase to interact with macrophages suggested that the effect of acetylcholinesterase on peritoneal cells is most probably mediated by the macrophage mannose-fucose receptor. The role of the mannose-fucose receptor in triggering the respiratory burst response was supported by the fact that several ligands of these receptors were capable of inducing the functional response of macrophages.

Characterization of mutant Met100Lys of cytochrome c-550 from Thiobacillus versutus with lysine-histidine heme ligation.

The heme iron in cytochrome c-550 from Thiobacillus versutus has a methionine and a histidine as axial ligands. In order to study the characteristics of a possible lysine-histidine ligation in a heme protein, the methionine has been replaced by a lysine. This residue acts as a ligand between pH 3 and 12. The midpoint potential of the mutant has shifted -329 mV compared to wild type, but apart from this shift the pH dependence of the midpoint potential is unchanged, suggesting that the large drop is caused by specific ligand effects and not by protein refolding. While the EPR spectrum of wild-type cytochrome c-550 shows one species with gz = 3.35, in the spectrum of the mutant two species occur with gz values of 3.53 and 3.30. The intensity ratio of both species depends on the presence of organic cosolvents. In the low frequency region (-4 to -1 ppm) of the 1H NMR spectrum of mutant ferrocytochrome c-550, four one-proton peaks replace the resonances of the ligand methionine side chain protons. Using two-dimensional NMR spectroscopy (COSY and NOESY), these protons and five others have been assigned to the lysine ligand. The spectroscopic results obtained for this mutant show similarities with those observed for the alkaline form of cytochrome c, supporting the Lys-His ligation proposed for this protein. The data are consistent with the evidence for amine ligation in cytochrome f: the EPR spectrum of M100K cytc-550 is similar to that of cytochrome f. However, the NMR spectra show significant differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Gene Expression in Choriocarcinoma by Methotrexate and Hydroxyurea*

Choriocarcinoma, a highly malignant neoplasm of trophoblastic origin, is remarkable for its marked degree of sensitivity to chemotherapeutic agents. We treated two cell lines derived from choriocarcinoma patients with two antineoplastic agents, methotrexate and hydroxyurea (HU), both of which cause nucleotide depletion and have been previously shown to be effective against choriocarcinoma, and found pleiotropic regulation of several genes. Three genes, hCG alpha-subunit, beta-subunit, and placental alkaline phosphatase, were all strongly induced by methotrexate and HU. Expression of c-myc, an oncogene associated with proliferation, was reduced to nearly undetectable levels by the drug treatment, while expression of beta 2-microglobulin, a component of the class 1 major histocompatibility locus, was unchanged. In addition, the mechanism of induction by HU of one of these genes, the hCG alpha-subunit gene, was found to occur at the level of transcription. The similar effects of methotrexate and HU, two mechanistically unrelated antimetabolites, on the induction of specific gene expression in choriocarcinoma cells suggest that these effects are due to nucleotide pool alteration, rather than specific ligand effects. Furthermore, the hCG alpha-subunit promoter contains a transcriptionally regulated HU-responsive element.

Identification and partial characterization of the xanthine oxidase transitions of the milk fat globule membrane

Differential scanning calorimetry of bovine milk fat globule membranes (MFGM) yields five to eight transitions, depending on the conditions employed during isolation and assay of the membranes. Transitions A, B, and C were shown in a previous publication to derive from lipid melting, while transition D was found to stem from the unfolding of a structural protein termed butyrophilin [K. C. Appell, T. W. Kennan, and P. S. Low (1982) Biochim. Biophys. Acta 690, 243–250] . In this report we present evidence that the E 1, E 2, and F endotherms derive from the major MFGM protein, xanthine oxidase. Support for this contention derives from (i) thermal gel analysis; (ii) thermal inactivation analysis; (iii) comparison of the calorimetric properties of endotherms I, II, and III of purified xanthine oxidase with transitions E 1, E 2, and F of MFGM; (iv) comparison of the properties of a peculiar exotherm in scans of both the purified enzyme and MFGM; and (v) examination of the effects of specific ligands, reducing agents, and pH on both the xanthine oxidase and MFGM transition. The existence of three independent endotherms (I, II, and III) in purified xanthine oxidase demonstrates that the enzyme is composed of multiple independent domains. The interconversion of transitions I (E 1) and II (E 2) with a change in the redox conditions of the medium implies that these two transitions may be manifestations of the interconvertible dehydrogenase and oxidase forms of the enzyme, respectively. The relative independence of the I/II transitions from transition III further shows that only slight interaction between the major domains of xanthine oxidase exists.

A diphenylmethane derivative selective for the anti-estrogen binding site may help define its biological role

By employing as a probe the new compound, N,N-diethyl-2-[(4-phenylmethyl)-phenoxy]-ethanamine·HCl (N,N-DPPE), which preferentially binds the antiestrogen binding site, it is demonstrated that this site appears to contribute to the growth inhibitory action of tamoxifen on MCF-7 human breast cancer cells, even at lower concentrations of this anti-estrogen (1 × 10 −7 M to 1 × 10 −6 M) at which the major effect is clearly mediated via estrogen receptor. The combination of N,N-DPPE and tamoxifen is additive and this effect is not abolished by 17β-estradiol. This suggests that the anti-estrogen binding site is not simply a passive reservoir for binding tamoxifen, but may itself mediate the cytotoxic effects of specific ligands.

Macrophage membrane potential changes associated with gamma 2b/gamma 1 Fc receptor-ligand binding.

We have studied the effects of specific ligands of the receptor for the IgG Fc fragment (FcR) on the membrane potential (delta psi) of the macrophage cell line J774 by the [3H]tetraphenylphosphonium ion equilibration technique. We observe a membrane depolarization with binding of FcR ligands that is dependent on the degree of receptor crosslinking. Binding of the FcR by monovalent ligands is not sufficient to induce a significant drop in delta psi, but a sustained depolarization lasting approximately equal to 20 min occurs with insoluble multivalent ligands. This FcR-mediated depolarization can be inhibited by substitution of Na+ from the cell incubation medium with monovalent choline cation, indicating that depolarization is due to Na+ influx into the cell. The extracellular Ca2+ does not play a significant role in membrane depolarization. The depolarization response is not triggered by monoclonal antibodies directed against three other major macrophage surface antigens. The cell depolarization mediated by FcR ligands is followed by a prolonged hyperpolarization that can be partially blocked by ouabain and quinine, indicating that the hyperpolarization response is a result of a combination of a Na+,K+-ATPase activity and a Ca2+-activated K+ conductance. These data support our hypothesis that the mouse macrophage IgG FcR is a ligand-dependent ion channel.

Mouse macrophage Fc receptor for IgG gamma 2b/gamma 1 in artificial and plasma membrane vesicles functions as a ligand-dependent ionophore.

We tested the effect of specific ligands to the mouse macrophage IgG gamma 2b/gamma 1 Fc fragment receptor (FcR) on ion permeability of plasma membrane vesicles prepared from J774 macrophages by nitrogen cavitation. The monoclonal antibody directed against IgG gamma 2b/gamma 1 FcR (gamma 2b/gamma 1 FcR), 2.4G2 IgG, and soluble and immobilized immunocomplexes induces a dramatic cation flow through plasma membrane vesicles, as measured by [3H]tetraphenylphosphonium+ accumulation. Challenge with the monovalent 2.4G2 Fab also produces an ion flow but the effect is smaller by a factor of 2, and three other monoclonal antibodies directed against major surface antigens of mouse macrophages produce no net ion flow. Membrane vesicles incubated with FcR ligands do not discriminate between Na+ and K+ and show low permeability to Ca2+. gamma 2b/gamma 1 FcR was purified by using monoclonal 2.4G2 and reconstituted into proteoliposomes. Under these circumstances, the purified receptor increased the cation permeability of the proteoliposomes in the presence of specific ligands. The data indicate that the gamma 2b/gamma 1 FcR of J774 macrophages functions as a ligand-dependent ionophore. The ion influx into macrophages mediated by the FcR may play an important role as a signal for internalization of membranes and stimulus-secretion coupling.

High affinity lipid binding sites on the peripheral membrane enzyme pyruvate oxidase. Specific ligand effects on detergent binding.

High affinity lipid binding sites on the peripheral membrane enzyme pyruvate oxidase. Specific ligand effects on detergent binding.