Ab Complex Research Articles

Expression and reconstitution of calcineurin A and B subunits.

Calcineurin consists of two subunits, a catalytic A subunit of 60 kDa and a regulatory B subunit of 19 kDa. Both the A and B subunits of rat brain calcineurin were expressed in E. coli. The B-subunit was readily overexpressed in the pET-21a vector with yields of > 70 mg of purified B subunit per 1 culture, representing > 17% of the soluble E. coli protein. About 8 mg of purified A subunit was obtained. The enzyme activities of the A-subunit and the reconstituted AB complex were found to be comparable to that of the bovine brain enzyme. The reconstitution of the AB complex was studied and shown to be rapid.

FcγRII on Human B Cells Can Mediate Enhanced Antigen Presentation

The ability of FcγR on monocytes and macrophages to enhance presentation of Ab-bound Ag has been well established. In contrast, studies suggest that FcγRII on B cells functions primarily to suppress B cell responses. We found that presentation of tetanus toxoid (TT) by human EBV-transformed B cells was enhanced by 100- to 1000-fold, when TT was targeted to FcγRII on B cells by using a conjugate consisting of TT covalently linked to anti-human FcγRII mAb 41H16. This enhanced presentation could be blocked by mAb 41H16, heat-aggregated human IgG, or anti-MHC Class II mAb. Similarly, multimeric immune complexes composed of TT and anti-TT mAb, SA13, and 9F12 also enhanced presentation of TT. Furthermore, FcγRII on purified human peripheral blood B cells could also enhance Ag presentation. These studies provide the first clear evidence that FcγRII can participate in enhanced Ag presentation by human B cells, and suggest that FcγRII has the potential to play a stimulatory role in human B cell responses to Ag–Ab complexes.

Role of conserved amino acid residues in the complementarity determining regions on hapten-antibody interaction of anti-(4-hydroxy-3-nitrophenyl)acetyl antibodies

Monoclonal antibodies (mAbs) specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) were prepared at various times after immunization and the amino acid sequences of VH and Vλ1 in these mAbs were deduced from cDNA nucleotide sequences. Replacements due to somatic mutation were not found in day 7 mAbs but were found in those of days 14, 84 and 294. The affinity of day 7 mAbs to NP-glycine(NP-Gly) was in the order of 10 4 M −1 and it increased about 8000-fold with time after immunization. The extrinsic circular dichroism (CD) spectrum of the NP-ϵ-aminocaproic acid (NP-Cap)/Ab complex was unique for each mAb, although the spectra were grouped into two types, which tended to shift from one type to another with time, suggesting a variation in the micro-environments around NP-Cap in the combining sites. All these data indicate that the structure of the combining site was altered by somatic mutation; however, the fine-specificity measured by cross-reactivity with hapten analogues did not change significantly with time. We examined the amino acid residues in CDRs responsible for recognition of NP-haptens by comparing the amino acid sequences of anti-NP mAbs. Analyses revealed the presence of several conserved amino acid residues in CDRs of VH and Vλ1, such as Tyr-32H, and Tyr-60H, in addition to a core segment involving Arg-50H. It was predicted that these conserved residues determine Ab-specificity and that the amino acid residues introduced by somatic mutation may act by refining the orientation of specificity determining residues and resulting in an increase in affinity.

Calorimetric and Spectroscopic Studies of Complex Formation of 2,4,5-Trichloroaniline with Proton Acceptors in CCl4

Abstract Spectroscopic and calorimetric investigations of interactions between trichloroaniline (A) and organic base: tetrahydrofuran or hexamethylphosphoric triamide (B) in CCl4 solution were carried out at 298.15 K. The equilibrium constants and the enthalpies for AB and AB2 type complex formation processes were calculated from a combination of both calorimetric and spectroscopic data. The results obtained suggest that, as in the case of earlier investigated complexes of pentafluoroaniline with the same organic bases, the enthalpy of formation of the first hydrogen bond in the AB2 type associates is higher than that of the second one.

Rotational mobility of the fibrinogen receptor glycoprotein IIb/IIIa or integrin alpha IIb beta 3 in the plasma membrane of human platelets.

Integrin alpha IIb beta 3 or glycoprotein IIb/IIIa (GPIIb/IIIa, 228 kDa) is a Ca(2+)-dependent, noncovalent heterodimer of glycoproteins IIb (GPIIb or alpha IIb, 136 kDa) and IIIa (GPIIIa or beta 3, 92 kDa), which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. We have determined the microsecond-range rotational motions of alpha IIb beta 3 in resting platelets, in isolated plasma membranes, and reconstituted in 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayers. The measurements were based on the time-resolved phosphorescence anisotropy [r(t)] of erythrosin-labeled F(ab) fragments [Er-F(ab)] of monoclonal antibodies bound to alpha IIb beta 3. In general, the r(t) decays were satisfactorily fitted to the sum of the two exponential terms and a constant, from which the initial anisotropy (r(in) approximately 0.05-0.11), the short (phi 1 approximately 1-14 microseconds) and the long (phi 2 approximately 15-60 microseconds) rotational correlation times, and the limiting anisotropy (r infinity approximately 0.02-0.07) were obtained. The observed values depended on the platelet preparation, temperature, Ca2+ concentration, and the antibody used. In accordance with data on the order parameter and the viscosity of the lipid bilayer of the platelet plasma membrane, phi 2 and r infinity of the alpha IIb beta 3-Er-F(ab) complexes in the three preparations decreased with the increase of temperature, the r(t) curves being fully reversible within the interval from 5 to 35 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamics of eutectic mixtures. Addition compounds and associated mixtures

Abstract

Verification of a model of a F(ab) complex with phenylarsonate by oligonucleotide-directed mutagenesis.

Abstract A model of the combining site of a mouse antibody specific for p-azophenylarsonate was tested by oligonucleotide-directed mutagenesis of the proposed hapten-contacting residues, Arg96 in the L chain, and Asn35, Trp47, Tyr50, Ser95, and Tyr100b in the H chain. The affinity and relative affinity for p-azophenylarsonate-N-acetyl-L-tyrosine of mutant antibodies expressed in transfectomas were determined by fluorescence quenching and by inhibition ELISA, respectively. The results show that alteration of the proposed contacting residues has drastic effects on hapten binding, and that the hydroxyl groups of Tyr50 and Tyr100b appear to orient the phenyl rings for optimal aromatic-aromatic interactions with the phenyl ring of the hapten. They further indicate a tight packing of the contacting residues around the hapten, which cannot accommodate changes in the positions of the functional groups of Asn35 and Ser95.

Tandem chromatographic-immunological analyses.

The function of conventional immuno-logical assays is to determine the presence and amount of known substances. Cross-reactivity of antibodies with unknown species of similar structure is a problem that can be circumvented by using chromatography in a second level of discrimination. Tandem LC-IA systems increase the selectivity and the sensitivity of assays by removing interfering species. For cases in which the goal is to prove that similar species are absent from a sample or to identify antigen variants, conventional assays fail. LC-IA systems are adept at dealing with these tasks. The necessary steps of metering reagents, separating Ag:Ab complex from other species, and enhancing detection in an immunological assay are easily accommodated by the LC system. In fact, the LC system provides a wide variety of new, high-sensitivity, high-speed methods for carrying out immunological assays. On the basis of the enormous versatility, selectivity, and sensitivity that LC-IA brings to analytical chemistry, we believe this technique will become increasingly important for monitoring analytes in complex biological matrices.

Interaction of GroE with an all-beta-protein.

Molecular chaperones are involved in protein folding both in vivo and in vitro. The Escherichia coli chaperone GroEL interacts with a number of nonnative proteins. A common structural motif of nonnative proteins, which is recognized by GroEL, has not yet been identified. In order to study the role of beta-sheet secondary structure on the interaction of nonnative proteins with GroEL, we used the F(ab) fragment of a monoclonal antibody as a model substrate protein. Here we show that GroEL interacts functionally with this all-beta-protein during reactivation. Antibody fragments refold spontaneously in good yield from the guanidine-denatured state. Functional refolding to the native state is inhibited transiently by GroEL, but there is no complete folding arrest in the absence of Mg-ATP and GroES. The yield of these unspecifically released GroEL-bound F(ab) fragments corresponds to that of the spontaneous reactivation in the absence of chaperones. However, the refolding kinetics in the presence of GroEL are considerably slower. The addition of Mg-ATP to the GroEL.F(ab) complex results in an immediate release of bound substrate protein and a significant increase in the amount of reconstituted antibody fragments compared to spontaneous reactivation. GroES is not essential for functional GroEL-mediated refolding of the F(ab) fragment but affects the reactivation yield to a small extent. Interestingly, stimulation of the GroEL-mediated F(ab) refolding depends primarily on the binding and not on hydrolysis of adenosine triphosphates. Previous results indicate the binding of alpha-helices to GroEL. The results presented in this paper suggest that beta-sheet secondary structural elements are recognized by GroEL. We therefore conclude that the interaction of a nonnative protein with GroEL depends mainly on the nature of the early folding intermediate but not on a specific element of secondary structure.

Azaborolyl-Komplexe, 27 / Azaborolyl Complexes, 27

Alkenyl substituted 1H-1,2-azaborolyl (Ab) complexes are promising candidates for stereoselective reactions, due to the chirality of the AbM moiety and the intramolecular coordination of the alkenyl group to the metal atom. The synthesis of [1-tert-butyl-2-methy1-3-(4-penten- l -y1)-η5-1H-1,2-azaborolyl]-bis(triphenylphosphine)rhodium (3) is achieved by the reaction of AbLi (2) [from AbH (1)] with (Ph3P)3RhCl. In benzene solution one PPh3 ligand of 3 is substituted by the C=C group of the pentenyl substituent. Simultaneously the η5-coordination of the Ab ring changes to an allylic η3-coordination forming 4. Steric reasons seem to be responsible for this intramolecular rearrangement. The shorter 2-propen-1-yl substituent is unable to act in the same way and therefore only forms the stable complex [1-tert-butyl-2-methyl-3-(2-propen- l -yl)-η5-1H-1,2-azaborolyl]-bis(triphenylphosphine)rhodium (7). Mass spectrometry, 1H, 11B and 31P NMR data were used to characterize the novel complexes.

Thermodynamics of hydrogen-bonded complex formation in chloroform + cyclic ether mixtures

The molar excess enthalpies H E for the binary mixtures chloroform + oxolane, + 1,3-di-oxolane, +oxane, + 1,4-dioxane, and + 1,3-dioxane have been measured as a function of mole fraction at atmospheric pressure and 298.15 K using a Picker flow microcalorimeter. The imprecision at the maximum value of H E is about ±0.5%. Values of H E were strongly negative for all mixtures. The magnitude and the symmetry of the H E curves depended on ring size and number of oxygen atoms. These results and the literature vapour pressure data for the chloroform + oxolane, chloroform + 1,3-dioxolane, and chloroform + 1,4-dioxane mixtures have been analyzed with the ideal associated solution model. The A+ B+ AB+ AB 2 model, where complexes AB and AB 2 are expected to form by the simultaneous chemical equilibria A+B ⇄ AB and A+2B ⇄ AB 2, was used for the diether mixtures. The simpler association model A+ B+ AB with formation of the AB complex was employed for the monoether mixtures. The equilibrium constants and enthalpies of formation of the AB and AB 2 complexes have been calculated from this analysis.

The lymphocytosis promoting action of pertussis toxin can be mimicked in vitro: Holotoxin but not the B subunit inhibits invasion of human T lymphoma cells through fibroblast monolayers

Pertussis toxin is known to elicit lymphocytosis in whooping cough patients and experimental animals, by blocking the extravasation of lymphocytes and stimulating their release from lymphoid organs such as the thymus. The mechanisms responsible for these unique effects of PT are not fully understood. The effect of pertussis toxin (PT) on the invasive behavior of human CCRF-CEM T lymphoma cells has been investigated with the use of a monolayer invasion assay (MIA). We had previously found that invasion of murine T lymphoma cells in this model system was correlated with their ability to extravasate and form metastases after i.v. injection in syngeneic animals. We now show that human CEM cells can also penetrate through a precultured confluent monolayer of murine 10T1/2 fibroblast-like cells within a few hours. In a quantitative MIA run over 24 h, PT at concentrations above 10 −14 M inhibited invasion of the CEM cells. In addition, PT stimulated the release (‘evasion’) of CEM cells that had invaded under the monolayer before the toxin was added. The A subunit of PT was totally inactive, the B subunit had a small residual effect, and reconstitution of the AB complex partially restored the activity. The invasion-inhibiting activity of two different holotoxin preparations and of the subunits perfectly matched their activity in the Chinese hamster ovary cell clustering assay, which is known to depend on a functional AB complex. We suggest that inhibition of monolayer invasion by PT can be used as an in vitro model system to investigate the cellular and molecular mechanisms underlying the lymphocytosis-promoting action of the toxin. Furthermore, the method is sufficiently sensitive to be used for titration of toxin activity. Our data indicate that the ADP-ribosylating activity of the A subunit is indeed required, and that the promotion of lymphocytosis is not elicited by the binding of the B subunit alone.

Subunit interactions control protein phosphatase 2A. Effects of limited proteolysis, N-ethylmaleimide, and heparin on the interaction of the B subunit

Protein phosphatase 2A consists of a heterotrimeric complex composed of a catalytic subunit (C) and two associated subunits (A and B). Limited tryptic digestion of the heterotrimeric ABC form resulted in the selective degradation of the Mr = 55,000 B subunit to a 48-kDa polypeptide. The cleavage sites were determined to be within a 3-7-kDa region of the COOH terminus. Proteolysis led to dissociation of the B subunit from the enzyme complex and correlated with an increase in cardiac myosin light chain, smooth muscle myosin light chain peptide, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) phosphatase activity. Purification of the digestion products and native gel electrophoresis indicated that dissociation of the B subunit was responsible for the increase in phosphatase activity. Kinetic analyses with several substrates revealed that dissociation of the B subunit resulted in a 2-7-fold increase in Vmax and a 1.6-5 fold increase in Km. Proteolytic dissociation of the B subunit increased the sensitivity of protein phosphatase 2A to inhibition by okadaic acid. Inhibition of the trypsinized enzyme was very similar to that observed for the purified AC form of protein phosphatase 2A. Incubation of the ABC complex with N-ethylmaleimide resulted in dissociation of the C subunit and generation of an AB complex. Selective release of the C subunit indicated that the B subunit interacts directly with the A subunit and that one or more free sulfhydryls are required to maintain the heterotrimeric structure of protein phosphatase 2A. Treatment of the enzyme with heparin resulted in an increase in specific activity that was due to the release of the B subunit from the complex. These results provide evidence that the B subunit binds directly to the A subunit to modulate enzyme activity and substrate specificity and that the COOH-terminal region of this protein is important for interaction with the AC complex. Dissociation of the B subunit by polyanionic substances related to heparin may represent a mechanism for regulating the activity of this enzyme.

Measurement of ligand-induced activation in single viable T cells using the lacZ reporter gene.

We have used the bacterial beta-galactosidase gene (lacZ) as a reporter gene for the rapid measurement of T-cell antigen receptor (TCR)-mediated activation of individual T cells. The reporter construct contained the lacZ gene under the control of the nuclear factor of activated T cells (NF-AT) element of the human interleukin 2 enhancer [Fiering, S., Northrop, J. P., Nolan, G. P., Matilla, P., Crabtree, G. R. & Herzenberg, L. A. (1990) Genes Dev. 4, 1823-1834]. The activity of the intracellular lacZ enzyme was analyzed by flow cytometric measurement of fluorescein accumulation in cells loaded with the fluorogenic beta-galactosidase substrate fluorescein di-beta-D-galactopyranoside. As a model system, the T-cell hybridoma BO4H9.1, which is specific for the lysozyme peptide (amino acids 74-88)/Ab complex, was transfected with the NF-AT-lacZ construct. lacZ activity was induced in 50-100% of the transfectant cells following exposure to pharmacological agents, to the physiological peptide/major histocompatibility complex ligand, or to other TCR-specific stimuli. Interestingly, increasing concentrations of the stimulus increased the fraction of lacZ+ cells, but not the level of lacZ activity per cell. Even under widely varying levels of stimulus, the level of lacZ activity in individual lacZ+ cells remained within a remarkably narrow range. These results demonstrate that TCR-mediated activation can be readily measured in single T cells and strongly suggest that, once committed to activation, the level of NF-AT transcriptional activity in individual T cells is independent of the form or concentration of stimulus. This assay is likely to prove useful for the study of early activation events in individual T cells and of TCR ligands.

Polymeric complexes of the antiseptic katamin AB and their biological activity

It is possible to evaluate the extent of the course of the reaction under consideration and to calculate the values of the degrees of the electrostatic binding (Qel) of ketamin AB by the investigated copolymers from the amount of the isolated acid. The compositions of the synthesized carboxyl-containing co~olymers of VP, the values of the intrinsic viscosities of their aqueous-salt solutions ([n]~1N KCI), as well as the values of Qel are presented in Table i. The analysis of the data in Table I indicates that the degree of the electrostatic binding of katamin AB with the copolymers (I)-(III) increases with the increase in the relative molar content of katamin AB in the solution (~) in the range from 0 to I. The comparison of the values of Qel for the copolymers (I)-I and (1)-3, (II)-I and (II)-3, and (III)-i and (III)-3 correspondingly indicates that the portion of the molecules of the antiseptic connected by an ionic bond to the polymer-carrier increases with the increase in the amount of the ionogenic carboxyl groups in the copolymer at a constant value of ~. Among the copolymers of close composition (I)-2, (II)-1, (III)-i and (I)-3, (11)-2, and (111)-2 at the same value of ~, the values of Qel found for the copolymers of VP-~A are significantly less than those of the polymers VP-+[AA and VP-AA. The observed phenomenon is caused by differences in the microstructure of the copolymers of VP utilized. It is known [9] that the copo!ymer s of VP-~A and VP-AA are characterized by a high degree of the microInstitute of High-Molecular Compounds, Academy of Sciences of the USSR, Leningrad. R.

Thermodynamic behaviour of binary systems of 1,4-dioxane with 1,2-dichloroethane, dichloromethane, trichloroethene, tetrachloroethene and cyclohexane. Vapour pressures and excess molar Gibbs energies

Measurements of vapour pressures have been made for the systems 1,4-dioxane with dichloromethane and 1,2-dichloroethane at 298.15 K, for 1,4-dioxane with trichloroethene and tetrachloroethene at 303.15 K, and for 1,4-dioxane with cyclohexane (c-C6H12) at 288.15 K. The total vapour pressure vs. liquid phase composition data have been used to obtain the activity coefficients γA and γB, and activities aA and aB of 1,4-dioxane (A) and the other component (B) in the various systems, and excess molar Gibbs energies GEm for the above systems, by using Barker's procedure. GEm is found to be positive throughout the whole composition range for mixtures containing c-C6H12 and CCl2CCl2, and negative for those containing CH2Cl2, CH2ClCH2Cl and CHClCCl2. Analysis of the experimental data of GEm, and aA and aB has shown that 1,4-dioxane forms AB and AB2 complexes with CH2Cl2, CH2ClCH2Cl and CHClCCl2 in the liquid state. The equilibrium constants K1 and K2 for the formation of the complexes AB and AB2 have been evaluated from the data of the activities of the two components of the systems of 1,4-dioxane with CH2Cl2, CH2ClCH2Cl and CHClCCl2. The four species A, AB, AB2 and B that are present in mutual equilibrium in mixtures of 1,4-dioxane with CH2Cl2, CH2ClCH2Cl and CHClCCl2, conform accurately to the theory of ideal associated mixtures due to McGlashan and Rastogi.

Thermal and volumetric properties of chloroform + dimethylsulfoxide: Thermodynamic analysis using the ideal associated solution model

We have made new measurements of partial molar enthalpies of solution, excess heat capacities, and excess volumes of mixtures of chloroform and dimethylsulfoxide. In combination with published vapor pressures and excess enthalpies the results of these measurements have been analyzed within the context of the ideal associated solution model to yield ΔC p ϑ and ΔVϑ for the formation of AB and A2B complexes. It is shown that the ideal associated solution model is consistent with all of the available thermodynamic data for this system, indicating that nearly all of the deviations of this system from ideal solution behavior can be accounted for in terms of chemical interactions of the two components.

Thermodynamics of complex formation in chloroform + 1,4-dioxane

We have made new measurements of excess heat capacities and excess volumes of mixtures of chloroform and dioxane. In combination with published vapor pressures and excess enthalpies the results have been analyzed using the ideal associated solution model to yield K, ΔHθ, ΔCpθ, and ΔVθ for the formation of AB and A2B complexes. It is demonstrated that the ideal associated solution model is consistent with all of the available thermodynamic data for this system, indicating that chemical interactions of the two components can account for nearly all of the deviations from ideal solution behavior. The results of our thermodynamic analysis are discussed in relation to the results of similar analyses of other systems in which there are strong chemical interactions of chloroform with electron-donor molecules. Keywords: complex formation, hydrogen bonding, ideal associated solution model, chloroform, 1,4-dioxane.

The chlorophyll ab complex, CP29, is associated with the Photosystem II reaction centre core

Photosystem II particles from spinach prepared by an octylglucoside method (Ghanotakis et al. (1987) Biochim. Biophys. Acta 891, 15–21) totally lack the antenna chlorophyll-protein complex LHC II, but do contain CP47, CP43 and a third chlorophyll-protein complex, as shown by non-denaturing SDS electrophoresis. This latter core-associated complex is shown to be CP29 on the basis of: (i) the apparent molecular weight of the holocomplex; (ii) the presence of Chi b as well as Chi a in excised gel slices of the complex; and (ii) the cross reaction of the apoproteins with polyclonal antibodies to barley CP29 and with monoclonal antibodies to the N-terminus of the apoprotein of pea CP29. The PS II particles are also enriched in a 23 kDa polypeptide of unknown function.

Thermodynamic investigation of molecular complexation in binary acetone + bromoform mixture

Molar excess volumes, VE, and molar excess enthalpies, HE, have been measured for acetone (A) + bromoform (B) mixtures at 298.15 and 308.15 K. Results of these measurements suggest the formation of a 1:1 acetone–bromoform molecular complex. The equilibrium constant, K, the enthalpy of formation, ΔH, of the AB complex as also the molar volume change, ΔV, accompanying the complexation reaction [Formula: see text] have been evaluated from the measured HE and VE data. The K value so obtained has further been substantiated by the K value determined for the AB molecular entity from nmr studies on this mixture. Partial molar enthalpy of B (at infinite dilution) in A and vice versa have also been evaluated from the measured HE data at 298.15 K and used to calculate a consistent molar enthalpy of AB complex formation.