First order dissociation rates between a subpopulation of high affinity rabbit anti-fluorescyl IgG antibody and homologous ligand

The first order dissociation rates of liganded subpopulations of purified hyperimmune rabbit antifluorescyl IgG antibodies were determined using the method of Green (1963). The dissociation of radiolabeled fluorescyl ligands was measured in the presence of excess unlabeled homologous hapten. Results with three different antibody preparations indicated dissociation rates of 1.3 × 10[su?3, 2.4 × 10^?4 and 2.2 × 10^?6 sec^?1 for the liganded subpopulations. Assuming an association rate of 4.6 × 10⁸M^?1 sec^?1 (Levison et al., 1971), average equilibrium constants ranging from 3.1 × 10¹¹M^?1 to 2.1 × 10¹⁴M^?1 were calculated. These values are among the highest reported for antibody-hapten interactions.     

Affinity of the interaction between Fcgamma receptor type III (FcγRIII) and monomeric human IgG subclasses. Role of FcγRIII glycosylation

Binding of the Fc region of IgG antibodies to low affinity Fcγ receptors (FcγR) triggers important effector functions in the immune system. The type IIIb FcγR (FcγRIIIb or CD16) is a heavily glycosylated protein anchored to the membrane of neutrophils by a glycosylphosphatidylinositol link. This receptor contributes to cell activation by IgG immune complexes. To better understand the nature of the ligand-receptor association, we have studied the affinity and kinetics of the interaction between human IgG subclasses and two soluble forms of FcγRIIIb (sFcγRIIIb or sCD16) corresponding to the 188 N-terminal amino acids of the extracellular region of the receptor, a glycosylated one made in eucaryotic cells (euc.sCD16) and a non-glycosylated one (proc.sCD16) made in Escherichia coli. Experiments using a BIAcore^TM instrument, to measure protein binding in real time, showed that monomeric human IgG1 and IgG3, but not IgG2, IgG4, IgA and divalent antigen-binding fragments (F(ab′)₂) of IgG1, bound to immobilized euc.sCD16 with an affinity constant (K_A) of 1.3 ± 0.6 × 10⁶ M^?1 and 2.6 ± 0.4 × 10⁵ M^?1, respectively. The affinity constant of proc.sCD16 for human IgG1 was in the same range (1.1 ± 0.2 × 10⁶ M^?1), whereas that for human IgG3 was twofold higher (4.2 ± 0.4 × 10⁵ M^?1). The specificity of the non-glycosylated receptor for human IgG subclasses bound to Sepharose was IgG1 > IgG3 ? IgG4 ? IgG2. Thus, the extracellular polypeptide of FcγRIIIb dictates the interaction of the receptor with IgG subclasses although glycosylation plays an inhibitory role in the interaction with human IgG3.     

Concentration dependence of the binding constant of antibodies

By simultaneous dilution of the antigen (AG) and the antibody (AB) used in an immune precipitation reaction, at the equivalence AG/AB ratio, it was found that the association constant, K_a, increased from 1.7 × 10⁷ l/M at the highest concn of AG and AB, to 6.5 × 10¹¹ l/M at 100-fold dilution of both. An important (but not the only) reason for this increase is taken to be the fact that the AB solution was polyclonal.     

The Rna Binding Subset of Adenosine Antibodies

Hapten specific antibodies were elicited against adenosine-BSA conjugate (Ado*-BSA) in rats. By radiohapten assay, two major populations of hapten specific antibodies were identified. They were directed against either adenosine trialcohol(A^ox-red) or morpholino-adenosine(Morph-Ade). The association constant (Ka) for the ³H-A^ox-red binding population was determined to be 1.02 × 10⁷ M^-1. Majority of the A^ox-red specific antibodies were crossreactive with Morph-Ade, but they did not cross-react at all with adenosine(A) or deoxyadenosine(dA). ³H-A binding subsets were actually Morph-Ade specific and constitute less than 10% of the hapten specific antibodies. The Ka value for the ³H-A binding was determined to be 4.5 × 10⁶ M^-1. A very minor and highly cross-reactive subset of Morph-Ade specific antibodies participate in RNA binding. Though a larger proportion of Morph-Ade specific antibodies interact with ³H-A, only a fraction of them bind to RNA. The extreme crossreactivity and hence, the highly adaptive nature of the binding sites of the antibodies interacting with RNA, might be a stringent requirement for recognising adenine residues of nucleic acids in solution.     

Time resolved kinetics of the guinea pig Na–Ca exchanger (NCX1) expressed in Xenopus oocytes: voltage and Ca2+ dependence of pre-steady-state current investigated by photolytic Ca2+concentration jumps

Kinetic properties of the Na–Ca exchanger (guinea pig NCX1) expressed in Xenopus oocytes were investigated by patch clamp techniques and photolytic Ca²⁺ concentration jumps. Current measured in oocyte membranes expressing NCX1 is almost indistinguishable from current measured in patches derived from cardiac myocytes. In the Ca–Ca exchange mode, a transient inward current is observed, whereas in the Na–Ca exchange mode, current either rises to a plateau, or at higher Ca²⁺ concentration jumps, an initial transient is followed by a plateau. No comparable current was observed in membrane patches not expressing NCX1, indicating that photolytic Ca²⁺ concentrations jumps activate Na–Ca exchange current. Electrical currents generated by NCX1 expressed in Xenopus oocytes are about four times larger than those obtained from cardiac myocyte membranes enabling current recording with smaller concentration jumps and/or higher time resolution. The apparent affinity for Ca²⁺ of nonstationary exchange currents (0.1 mM) is much lower than that of stationary currents (6 μM). Measurement of the Ca²⁺ dependence of the rising phase provides direct evidence that the association rate constant for Ca²⁺ is about 5 × 10⁸ M⁻¹ s⁻¹ and voltage independent. In both transport modes, the transient current decays with a voltage independent but Ca²⁺-dependent rate constant, which is about 9,000 s⁻¹ at saturating Ca²⁺ concentrations. The voltage independence of this relaxation is maintained for Ca²⁺ concentrations far below saturation. In the Ca–Ca exchange mode, the amount of charge translocated after a concentration jump is independent of the magnitude of the jump but voltage dependent, increasing at negative voltages. The slope of the charge–voltage relation is independent of the Ca²⁺ concentration. Major conclusions are: (1) Photolytic Ca²⁺ concentration jumps generate current related to NCX1. (2) The dissociation constant for Ca²⁺ at the cytoplasmic transport binding site is about 0.1 mM. (3) The association rate constant of Ca²⁺ at the cytoplasmic transport sites is high (5 × 10⁻⁸ M⁻¹s⁻¹) and voltage independent. (4) The minimal five-state model (voltage independent binding reactions, one voltage independent conformational transition and one very fast voltage dependent conformational transition) used before to describe Ca²⁺ translocation at saturating Ca²⁺ concentrations is valid for Ca²⁺ concentrations far below saturation.     

Non-covalent association of heavy and light chains of human immunoglobulin G: Studies using light chain labelled with a fluorescent probe

The fluorescent probe, 5-iodoacetamidofluorescein (5-IAF), was specifically attached to the COOH-terminal cysteine residue of the L chains of two monoclonal human IgKκ proteins. The induced circular dichroism and fluorescence properties of the bound 5-IAF probe were used to study changes in its microenvironment upon reassociation of autologous or heterologous L chains and their domains with Fd' fragments. Recombination of 5-IAF-L with Fd' at pH 5.4 was accompanied by a red-shifted visible difference spectrum, an increase in the magnitude of the induced optical activity and an enhancement of fluorescence. These results were compatible with the transfer of the 5-IAF moeity to a less polar and more asymmetric environment. Equilibrium and kinetic data obtained from difference spectroscopy and fluorescence studies indicated that 5-IAF-L bound with high affinity to Fd' in a 1:1 ratio with a second-order rate constant of 1618 M^?1 sec^?1 at 25°C. Using the same approach, it was shown that the labelled constant region fragment (5-IAF-C_κ) bound to Fd' with an association constant of 5 × 10⁶ M^?1 and a forward rate constant of 30 M^?1 sec^?1. When 5-IAF-C_κ was recombined with a preformed Fd'V_κ complex, however, the intensity of the visible difference spectrum, the fluorescence emission, the binding affinity and the forward rate constant of the reaction, were significantly increased. In contrast, no spectroscopic changes were observed when V_κ was recombined with a preformed Fd'-5-IAF-C_κ complex. These data suggest that: (1) the high affinity interaction between Fd' and L results from the summation of relatively weak interactions between paired domains; (2) the binding of Vκ to Fd' modulates the reactivity of Cγl toward Cκ, probably through conformation changes transmitted via V_H-Cγl contacts; and (3) conversely, once 5-IAF-Cκ is bound to Fd', the addition of the complementary Vκ domains does not induce any detectable change in the vicinity of the fluorescent probe.     

Structural requirements for recognition of vasopressin by antibody; Thermodynamic and kinetic characteristics of the interaction

The thermodynamic and kinetic properties of two conventional antivasopressin antisera were studied. Values for binding affinity were determined by equilibrium measurements to be Kd = 1.4 and 2.7 × 10^?12M. The kinetic parameters were independently determined. The association rate constants (kas) were calculated by a pseudo-first-order analysis of binding kinetics (ka = 3.1 and 1.7 × 10⁷M^?1 sec^?1). The dissociation rate constants (kds) were measured by dissociating antibody-labelled antigen complexes with large excess of unlabelled antigen (kd = 1.6 and 1.7 × 10^?5 sec^?1). A fairly close agreement was achieved between equilibrium and kinetic evaluation of the affinity.The heterogeneity cannot be assessed through equilibrium experiments because of the very low concentrations of reagents to be handled. However, kinetic studies strongly suggested that the molecular heterogeneity with respect to affinity of the antisera is restricted to a narrow range (5 × 10^?13M to 7 × 10^?12M).Despite their very similar physicochemical properties these two antisera exhibited different fine specificities: the study of cross-reactivities with various analogues of the original hapten showed that one antiserum—5—is clearly directed against the C-terminal moiety of the molecule. The antigenic determinant is a sequential one and composed of the last four aminoacids Cys-Pro-Arg-GlyNH₂, while the other antiserum is not so sensitive to modifications of the last residue GlyNH₂.     

Characterization of high-affinity antifluorescyl IgM antibodies

High-affinity IgM rabbit antibodies were elicited using the fluorescein hapten system. Purity and identification of IgM and IgG anti-fluorescyl antibodies was determined by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. Ultracentrifugation studies verified that liganded antibodies were of a high mol. wt (901 kd).Comparative analyses of IgM and IgG anti-fluorescyl antibody active sites substantiated the observation that purified IgM preparations, similar to their IgG counterparts, possessed high-affinity antibody active sites. Dissociation rate data confirmed that some IgM molecules within the purified antibody populations possessed association constants at 10¹¹M^?1, comparable with values of 10¹¹M^?1 or greater obtained for some anti-fluorescein IgG populations studied in this laboratory. A diffusion-controlled association rate of 4.6 × 10⁸M^?1 sec^?1 (Levison et al., 1971) was assumed in dissociation rate calculations. This is the first report of purified IgM antibodies possessing exceptionally high affinities.     

The muscarinic agonist pilocarpine inhibits DNA and interferon-γ synthesis in peripheral blood mononuclear cells

Lymphocyte basal DNA synthesis and proliferative responses to phytohemagglutinin (PHA) showed a dose-dependent (5 × 10⁻⁵ − 5 × 10⁻³ M) inhibition by the muscarinic agonist pilocarpine, in contrast to the basal enhancing effect produced by the M₂ muscarinic-nicotinic agonist carbacol. The effect of pilocarpine was reversed by both atropine (1 × 10⁻⁶ M) and pirenzepine (1 × 10⁻⁷ − 1 × 10⁻⁸ M), M₁ − M₂ and M₁ muscarinic antagonists, respectively. The effect of pilocarpine may thus be specific for the M₁ muscarinic receptor. Pilocarpine also inhibited interferon-gg (IFN-γ)-PHA induced production, but was unable to reverse the pokeweed mitogen (PWM)-induced DNA synthesis. Distinct immunoregulatory activities are suggested for cholinergic muscarinic receptors M₁ and M₂.     

Living polymerization of [o-(trifluoromethyl)phenyl]acetylene by a new catalyst system,MoOCl4–Et3Al–EtOH (1 : 1 : 4)

A new MoOCl₄-based living polymerization catalyst, MoOCl₄–Et₃Al–EtOH (mole ratio 1 : 1 : 4)/anisole, has been developed. Polymerization of [o-(trifluoromethyl)phenyl]acetylene by this catalyst in anisole at 30°C yielded a polymer having very narrow molecular weight distribution (M_w/M_n = 1,02), a four-fold excess of ethanol over MoOCl₄ was necessary to attain narrow molecular weight distributions. Multistage polymerization experiments clearly showed the living nature of the polymerization, which was maintained in the temperature range of 0 to 30°C. The absolute number-average molecular weight of the polymer measured by vapor pressure osmometry could be correlated with the number-average molecular weight measured by gel permeation chromatography as follows: M_n(VPO) = 1,48 × M_n(GPC). The propagation rate constant (k_p) at 30°C is 1,5 mol·L^–1·s^–1.     

Concurrent Initiation by Air in the Atom Transfer Radical Polymerization of Methyl Methacrylate

The effect of air in atom transfer radical polymerization (ATRP) of methyl methacrylate (MMA) was studied. Air initiated polymerization was clearly noticed by the appearance of a low molecular weight peak in the synthesis of high molecular weight poly(isobutylene)‐graft‐poly(methyl methacrylate) (M _n = 5.0 × 10⁵ g/mol). The concentration of chains initiated by oxygen (air) was ≈8 × 10^?4 mol/L, determined using the Gladstone‐Dale relationship. The tentatively proposed mechanism for air initiated polymerization was supported by kinetic studies. Similar to typical ATRP systems, the rate of air initiated polymerization increased with temperature, [MMA], amount of air, and activity of the catalyst complex. Polymers with lower polydispersities (M _w/M _n = 1.13) were obtained in the presence of Cu(II) as compared to Cu(I) catalyst complex system.

Kinetic plots for the air initiated bulk polymerization of MMA at (?) 20 °C, (?) 50 °C, and (?) 90 °C.     

The binding of soluble immune complexes of guinea pig IgG2 to homologous peritoneal macrophages Determination of the avidity constants at 4 °C.

Soluble immune complexes of guinea pig IgG₂ anti-DNP (2,4-dinitrophenyl) antibodies and DNP₁₉BSA (bovine serum albumin) were prepared at a variety of antibody-to-antigen ratios. The mean sizes of the complexes were determined by gel filtration, and equilibrium binding of the complexes to homologous peritoneal macrophages was measured at 4°C. Scatchard plots of the binding data were linear indicating that the complexes were binding to a uniform population of functionally independent membrane receptors. The avidity constants for complex binding to macrophages increased from 15.4 × 10⁷ M^?1 for complexes containing an average of two antibody molecules to 59.0 × 10⁷ M^?1 for complexes containing an average of four, compared with an association constant for monomeric IgG₂ of 0.21 × 10⁷ M^?1. Calculation of the molar-free energy changes accompanying monomer and immune complex binding revealed not only that the intrinsic binding activity of mononer was sufficient to account for the complex binding activities by simple cooperation of linked antibodies, but also that the cross- linking of antibodies with antigen imposed a strain on the antibody-receptor interaction, resulting in complex binding energies that were lower than the values expected from a simple cooperative mechanism. Complex inhibition by monomeric IgG₂ of unrelated antibody specificity was studied, and it was observed that high concentrations of monomer led to an increase in macrophage discrimination between complexes of different size. The relevance of this finding to the in vivo clearance of circulating complexes is discussed.     

Monoclonal Antibodies Against Human Chorionic Gonadotropin (hCG): II. Affinity and Ability to Neutralize the Biological Activity of hCG

ABSTRACT: Monoclonal antibodies (MCA) against hCG have been characterized with regard to their affinity and their ability to neutralize the biological activity of hCG in vivo. The production and specificities of these reagents were described in the preceeding paper of this series. Equilibrium association constants (K_a) of the MCA, determined by radioimmunological saturation assays, ranged from less than 1 × 10⁸M⁻¹ up to 3.7 × 10⁹M⁻¹ whereas values for conventional polyclonal antisera against hCG ranged from 8.9 × 10⁹M to 1.8 × 10¹⁰M⁻¹. The ability of MCA to neutralize the biological activity of hCG was tested in a rat bioassay in vivo; 9 of 13 different MCA preparations tested could neutralize hCG. Surprisingly, this property did not correlate with affinity or specificity, and was not restricted to those MCA recognizing the hormone specific β-subunit. It could be demonstrated that determinants on each individual subunit as well as epitopes formed by both subunits are involved in the expression of the biological activity of hCG.     

Analysis of Kinetics of Dihydroethidium Fluorescence with Superoxide Using Xanthine Oxidase and Hypoxanthine Assay

Superoxide (O₂ ^?) is an important reactive oxygen species (ROS), and has an essential role in physiology and pathophysiology. An accurate detection of O₂ ^? is needed to better understand numerous vascular pathologies. In this study, we performed a mechanistic study by using the xanthine oxidase (XOD)/hypoxanthine (HX) assay for O₂ ^? generation and a O₂ ^? sensitive fluorescent dye dihydroethidium (DHE) for O₂ ^? measurement. To quantify O₂ ^? and DHE interactions, we measured fluorescence using a microplate reader. We conducted a detailed reaction kinetic analysis for DHE–O₂ ^? interaction to understand the effect of O₂ ^? self-dismutation and to quantify DHE–O₂ ^? reaction rate. Fluorescence of DHE and 2-hydroethidium (EOH), a product of DHE and O₂ ^? interaction, were dependent on reaction conditions. Kinetic analysis resulted in a reaction rate constant of 2.169 ± 0.059 × 10³ M^?1 s^?1 for DHE–O₂ ^? reaction that is ~100× slower than the reported value of 2.6 ± 0.6 × 10⁵ M^?1 s^?1. In addition, the O₂ ^? self-dismutation has significant effect on DHE–O₂ ^? interaction. A slower reaction rate of DHE with O₂ ^? is more reasonable for O₂ ^? measurements. In this manner, the DHE is not competing with superoxide dismutase and NO for O₂ ^?. Results suggest that an accurate measurement of O₂ ^? production rate may be difficult due to competitive interference for many factors; however O₂ ^? concentration may be quantified.     

Rous sarcoma virus p19 binds to specific double-stranded regions of viral RNA: effect of p19 on cleavage of viral RNA by RNase III.

The extent of binding of purified RSV(Pr-C) p19 and p12 to a variety of RNAs was measured using a sensitive nitrocellulose filter binding assay which is capable of detecting binding reactions with association constants as low as 3 × 10⁶ liters × mol^?1 (Hizi, A., Leis, J. P., and Joklik, W. K. 1977). RSV p19 bound 60 and 34 S RSV (Pr-C) RNA with association constants of 5.1 × 10¹¹ and 1.8 × 10¹⁰ liters × mol^?1. RSV p19 bound preferentially to specific double-stranded regions of the RNA since: (a) The association constant for Neurospora nuclease-digested 34 S RNA was the same as for untreated RNA; (b) the association constant for 34 S RNA partially digested with Escherichia coli RNase III (which is specific for double-stranded RNA regions) was 30-fold lower than for untreated RNA; (c) p19 prevented cleavage of 34 S RSV-RNA by E. coli RNase III; (d) p19 bound cell precursor RNAs containing RNase III-sensitive sites, but not mature RNAs lacking RNase III-sensitive sites. On the other hand, purified RSV p12 bound all RNAs tested with association constants roughly proportional to their molecular weights. A possible function for p19 in regulating the processing of viral RNA and its subsequent translation has been proposed.     

Serine protease inhibitors (serpins) in human seminal plasma: Concentrations and inhibition of acrosin

Acrosin is a trypsin-like serine protease present in its zymogen form in the acrosome of spermatozoa. The activated enzyme is thought to digest a pathway for the sperm through the zona pellucida of the ovum during the process of fertilisation. We have shown that in a purified system boar acrosin was inhibited by human protein C inhibitor with an apparent second order rate constant (k_app) of 3.7×10⁴M⁻¹s⁻¹ 1. Protein C inhibitor is present in high concentrations in seminal plasma and endogenous protein C inhibitor was found in the immediate vicinity of disrupted acrosomal membranes of washed human spermatozoa. This serpin could therefore function as a scavenger of prematurely activated acrosin in the male reproductive tract.Since little is known about the interaction of acrosin with other serpin type inhibitors, we analysed in this study the interaction of boar acrosin with other purified human serpins. Antithrombin III, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, and α₁-antitrypsin inhibited acrosin activity. The following apparent k_apps were calculated: Antithrombin III: 19.5×10⁴ M⁻¹s⁻¹, plasminogen activator inhibitor-1: 21.5×10⁴M⁻¹s⁻¹, plasminogen activator inhibitor-2: 3.3×10⁴M⁻¹s¹, α₁-antitrypsin: 0.09×10⁴M⁻¹s¹. α₂-antiplasmin and heparin cofactor II did not inhibit acrosin. SDS-stable acrosin/serpin complexes were only seen with antithrombin III; all other acrosin inhibitors were cleaved by the enzyme.As determined by enzyme-linked immunosorbent assays, the concentrations of protein C inhibitor, plasminogen activator inhibitor-1, and plasminogen activator inhibitor-2 in individual seminal plasma samples from healthy donors were 5.33±0.47 μM, 88±24 pM and 163±17 pM (means±SD), respectively. The concentrations of antithrombin III in seminal plasma were ⩽50 nM (semiquantitative immunoblotting). Therefore considering both, the k_app calculated for the inhibition of boar acrosin in a purified system and the concentration of each serpin in seminal plasma, protein C inhibitor seems to be the best candidate to function as a physiological acrosin inhibitor in the male reproductive tract.     

Pressure studies on the metallation of a supra‐molecular porphyrin complex

The complex formation of meso‐tetrakis‐(1‐methylpyridinium‐4‐yl)porphine p‐toluenesulfonate (TMPyP) with zinc ions was examined at various pressures. By the application of high pressure, the second‐order reaction rate constant for the metallation was increased to give the negative volume of activation (ΔV^{). This result suggested that the activated complex is more strongly hydrated than the reactants. By the presence of anionic polyelectrolytes such as polyacrylic acid sodium salt (NaPAA, polymers with five molecular weight ranges from 1.1×10³ to 6.6×10⁵ were used) and poly(l‐glutamic acid sodium salt) (M_w = 3.7×10⁴), the metallation reaction was largely accelerated, whereas neither poly(l‐lysine HBr salt) (M_w = 3.6×10³) nor poly(ethylene glycol) (M_w = 1.1×10⁴) affected the reaction at all. The acceleration effect of polyanions could be intuitively attributed to the accumulation of the cationic TMPyP in the vicinity of the polyanion to afford a supra‐molecular porphyrin complex (polyanion‐TMPyP), resulting in the increase in the collision probability of TMPyP with a zinc ion which was also accumulated in the polyanion domain. The degree of acceleration by the presence of NaPAA was the largest when the molecular weight was 2.5×10³. The volume of activation (ΔV^{) in the presence of polyanions was less negative in comparison with that for the spontaneous reaction, due to the more pronounced dehydration of the activated complex than the reactants in the vicinity of the polyanion. A good correlation between the ΔV^{ value and the entropy of activation (ΔS^{) for the metallation in the presence of various polyanions supported that the acceleration effect could be related to the solvation effects.     

Modulation by 310-1310-1310-1stimulation of IgE-mediated14C-serotonin release from rat mast cells

The formaldehyde method was used to examine the effects of clonidine and methoxamine on IgE-mediated¹⁴C-serotonin release from rat mast cellsin vitro. Clonidine (10^?11?10^?8 M) caused dose-related enhancement of the mediator release 7min after the antigen challenge yohimbine (10^?6 M) blocked this enhancement by clonidine (10^?6 M), but prazosin (10^?6 M) did not. Methoxamine did not enhance this immunological release reaction at concentrations up to 10^?6 M. PGE₁ (2×10^?8?2×10^?5 M), isoproterenol (10^?10?10^?8 M), dopamine (4×10^?8?4×10^?8 M) and aminophylline (6×10^?6?6×10^?4 M) caused dose-related inhibition of this mediator release 1 min after antigen challenge. Clonidine (10^?13?10^?12 M), but not methoxamine (10^?8?10^?6 M), reversed dose-dependently this inhibition of mast cells by PGE₁ (2×10^?6 M), isoproterenol (10^?8 M), dopamine (4×10^?6 M); yohimbine (10^?8 M) antagonized this reversing action of clonidine (10^?12 M), but prazosin (10^?10 M) did not. Neither clonidine (10^?14?10^?11 M) nor methoxamine (10^?8?10^?6 M) reversed the inhibitory action of aminophylline (2×10^?4 M). These results suggest that clonidine enhances IgE-mediated¹⁴C-serotonin release from rat mast cells and also antagonizes the inhibition of mast cells by PGE₁, isoproterenol and dopamine, but not by aminophylline in this immunological reaction through α₂-adrenergic receptors, and that the inhibition of adenylate cyclase of mast cells is one of the biochemical actions of α₂-adrenergic mechanisms.     

Kinetics and molecular weight evolution during controlled radical polymerization

Automatic, continuous online monitoring of polymerization reactions (ACOMP) was applied to the controlled radical polymerization (CRP) of butyl acrylate (BA) using N‐tert‐butyl‐1‐diethylphosphono‐2,2‐dimethylpropyl nitroxide (SG1), to determine monomer conversion, evolution of molecular weight, reduced viscosity, and rate constants. The conversion is roughly first order, but depends only on the initial ratio of free SG1 to initiator; i.e., it is zeroeth order in initiator concentration. While it was found that the weight‐average molecular weight M _w, and viscosity‐average mass increase in approximately linear fashion with conversion, their values are finite at zero conversion. Although ACOMP involves no chromatographic separation columns, a useful measure of polydispersity evolution was found from combining M _w and viscosity‐based masses. CRP is contrasted with monitoring results for conventional free‐radical polymerization. Distinct light‐scattering signatures are expected, and found experimentally, for the two cases. The CRP kinetic findings allowed the determination of the equilibrium constant between active and dormant species at 120 °C (K_eq = 1.53 × 10⁻¹⁰ M ), as well as the corresponding kinetic constant of deactivation (k_deact = 2.8 × 10⁺⁷ L · mol⁻¹ · s⁻¹) and activation (k_act = 4.2 × 10⁻³ s⁻¹). Cross‐checks on the monitoring results were made with conventional Gel Permeation Chromatography (GPC), and kinetic behavior was also analyzed in the light of numerical integration software.

Raw data for PBA CRP reaction #6 in Table 1 vs. detector time, which lags the reaction time by 800 s. Shown are viscosity, RI, UV and 90 degree light‐scattering signals, together with the time at which stabilization, monomer flow, and polymerization began. The temperature curve is offset to the left 800 s with respect to the detector signals.     

Human placentae membrane receptor for IgG—i. Studies on properties and solubilization of the receptor

Characterization of the human placental membrane receptor for human ¹²⁵I-IgG is described. The receptor bound specifically both monomers and aggregates of human IgG. Human colostral IgA, bovine, sheep, pig, and horse IgG were not bound. No effect of pH in the range 6.6–7.4, ionic strength in the range 0.1–0.5, and temperature between 4 and 45°C on the binding was found. A water-soluble fraction containing the active receptor (glycoprotein fraction-PGP) was obtained from the placental membranes using lithium diiodosalicylate. The solubilized receptor interacted with IgG better at 4°C than at 20°C or 37°C. The results on replacement of monomeric IgG by aggregated IgG, and vice versa, suggest that both monomers and aggregates of human IgG, were bound to the same receptor sites. The apparent association constant for monomeric human IgG was 0.86 ± 0.2 × 10⁷ mole^?1, and 2.0 ± 0.16 × 10¹⁵ IgG molecules were bound per l mg of the membrane protein. Formaldehyde (0.1%), 2-mercaptoethanol (50 mM), and periodate (4 mM) showed no effect on the binding properties of the membrane-bound and on the solubilized receptor, as well. Higher concentrations of periodate (10 mM or 20 mM) decreased the binding of IgG to membranes but showed no effect on the water-soluble receptor. Both the membrane-bound and the solubilized receptor were sensitive to papain. Pronose abolished the receptor activity after prolonged proteolysis only. Neuraminidase did not affect the activity of the receptor. The decrease of the binding activity of the membrane-bound receptor by trypsin and phospholipase C was due to a release of a material containing an active receptor. No effect of trypsin or phospholipase C on the activity of solubilized receptor was observed. The results obtained suggest a protein character of the placental Fc receptor. After electrophoresis of ¹²⁵I-labeled solubilized receptor in polyacrylamide gel in the presence of SDS, 2 major protein peaks with molecular weights of 74,000 and 104,000 and 3 minor peaks with molecular weights of 56,000, 144,000, and 163,000 were found.