Charge displacements in interfacial layers containing reaction centers.

Reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides were oriented in phospholipid interfacial layers adsorbed to a Teflon film separating two electrolyte-filled compartments of a Teflon cell. Light-induced voltage changes were measured as a function of time across electrodes immersed in the cell compartments. The experimental system is characterized both experimentally and theoretically to relate the measured signals to the light-induced displacement currents in the reaction centers. Mathematical relations between the measured signals and the distances and geometries of the charge-transfer reactions are derived. At pH 8.0 the reaction centers were found to be oriented with approximately 60% of the population oriented with the donor facing the aqueous phase. The density of the reaction centers in the layer was approximately 10(11) cm-2, which is close to that found in the native system. Reconstitution of the secondary quinone, QB, in 90% of the RCs was achieved with an approximately 100-fold excess of ubiquinone in the vesicle preparation.

Oral administration of insulin in solid form to nondiabetic and diabetic dogs.

It was previously demonstrated that a biologically active insulin could cross the mucosal membrane in the gut by using surface active substances. In this report we describe studies in which insulin administered orally, in a solid formulation, was effectively absorbed in the canine model. The insulin was mixed with cholate and soybean trypsin inhibitor. It was delivered orally, as enterocoated microtablets, to nondiabetic and diabetic (pancreatectomized) dogs in a fasting state. The time interval between the administration of the drug and the beginning of a decrease in the plasma glucose levels was 60-140 min. This decrease reached a minimum level of 20-40 % of the initial values and lasted for more than 90 min following administration of the drug. In this model a pronounced increment in plasma insulin levels was shown prior to the drop of plasma glucose concentrations. It is concluded that with this novel oral insulin formulation a beneficial biological effect can be achieved in the treatment of diabetes.

A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671.

The human neuromedulloblastoma cell line TE671 is shown by single-channel recordings to express nicotinic acetylcholine receptors (AChRs) that are blocked by alpha-bungarotoxin (alpha Bgt). These AChRs do not react with antisera to the alpha Bgt-binding protein of brain or with monoclonal antibodies (mAbs) to brain nicotinic AChRs that do not bind alpha Bgt. TE671 AChRs do react with autoantibodies to muscle AChRs from myasthenia gravis patients and with mAbs to muscle AChRs, including mAbs specific for extrajunctional AChRs. AChRs. AChRs purified from TE671 cells are composed of 4 kinds of subunits corresponding to those of muscle AChR. Sequences of cDNAs for the ACh-binding alpha subunit and the delta subunit of this AChR further identify it as muscle AChR. Expression of TE671 AChR can be up-regulated by nicotine and dexamethasone, and down-regulated by forskolin.

Thermodynamics of oligosaccharides binding to a dextran-specific monoclonal IgM.

The binding thermodynamics of seven different oligosaccharide haptens to the dextran-specific IgM secreted by the murine plasmacytoma MOPC-104E were studied by direct calorimetric measurements. The enthalpy change values observed for the binding process range between -5 and -16 kcal/mol depending on the hapten and the temp of measurement. The antibody-hapten interactions were characterized by a positive heat capacity change [delta Cp approximately 300 cal/(mol.degree)] and a resultant process of enthalpy-entropy compensation. The calculated change in unitary entropy of the reaction, delta Su, ranged between -20 and -30 eu (4 degrees C), corresponding to an expected entropy loss due to immobilization of the hapten molecules. The entropy of binding increased with rising temp, thus compensating for the decreasing enthalpy contribution to the free energy of binding. The data are consistent with a hapten binding induced conformational transition to a more relaxed state in the immunoglobulin molecule.

Pseudomonas aeruginosa cytochrome oxidase. Product inhibition by low thermodynamic driving force.

The steady-state kinetics of Pseudomonas aeruginosa cytochrome oxidase were studied. Reduced cytochrome c551 and azurin from the same bacteria were used as the electron-donating substrates, while dioxygen served as the electron acceptor. Oxidized cytochrome c551 and azurin exhibited product inhibition of the reaction. However, apo-azurin and azurin derivatives in which the copper was substituted by the redox-inert ions Ni2+, Co2+, Cd2+ and Zn2+, did not show any effect on the kinetics. These observations implied that complex formation between the substrates or the products and the enzyme is not a rate-limiting step and is not the cause for product inhibition. The integrated rate law for a reaction scheme in which we assumed that complex formation was not rate limiting was fitted to the complete reaction traces. The results suggested that it is the low thermodynamic driving force, expressed in the small differences in redox potential between the substrates and heme c of the enzyme, which cause the observed product inhibition.

Monoclonal antibodies specific to the beta and gamma subunits of the Torpedo acetylcholine receptor inhibit single-channel activity.

The functional role of individual ACh receptor subunits in the mechanism of the nicotinic ACh receptor channel was examined using subunit-specific monoclonal antibodies (mAbs) as probes. Single-channel recordings from the Torpedo californica purified ACh receptor reconstituted in planar lipid bilayers were used as the assay to evaluate the influence of distinct mAbs on the ion conduction and gating characteristics of the ACh receptor channel. The mAbs that bind to the main immunogenic region on an extracellular domain of the alpha subunits do not perturb the open-channel conductance or lifetimes. A mAb that binds to extracellular domains of alpha and beta subunits and two mAbs that bind to the cytoplasmic surface of the beta and gamma subunits inhibit single-channel activity. Thus, mAbs with primary specificity for beta and gamma subunits affect channel gating. This approach may specify the functional roles of distinct structural domains in the ACh receptor molecule.

The structure of the voltage-sensitive sodium channel. Inferences derived from computer-aided analysis of the Electrophorus electricus channel primary structure.

A variety of computer-aided analyses was applied to the recently derived amino acid sequence of the Electrophorus electricus sodium channel protein in order to extract structural information such as hydrophobicity, periodicity, and secondary structure predictors. We propose a schematic model for the arrangement and folding of the polypeptide chain within the bilayer. The model consists of 4 homologous regions, each containing 8 membrane-spanning (probably alpha-helical) structures. Several of these structures are amphipathic with a repeat of 3.5 residues, 4 of which (one from each homologous region) are postulated to form a negatively charged channel lining. Gating currents are proposed to arise from voltage-dependent separation of multiple ion pairs buried within the hydrophobic, intramembranous protein interior.

The effect of an applied electric field on the charge recombination kinetics in reaction centers reconstituted in planar lipid bilayers.

Reaction Centers (RCs) from the photosynthetic bacterium Rhodopseudomonas sphaeroides were incorporated in planar bilayers made from monolayers derived from liposomes reconstituted with purified RCs. The photocurrents associated with the charge recombination process between the reduced primary quinone (QA-) and the oxidized bacteriochlorophyll donor (D+) were measured as a function of voltage (-150 mV less than V less than 150 mV) applied across the bilayer. When QA was the native ubiquinone (UQ) the charge recombination was voltage independent. However, when UQ was replaced by anthraquinone (AQ), the recombination time depended on the applied voltage V according to the relation tau = 8.5 X 10(-3) eV/0.175S. These results were explained by a simple model in which the charge recombination from UQ- proceeds directly to D+ while that from AQ occurs via a thermally activated intermediate state, D+I-QA, where I is the intermediate acceptor. The voltage dependence arises from an electric field induced change in the energy gap, delta G0, between the states D+I-QA and D+IQA-. This model is supported by the measured temperature dependence of the charge recombination time, which for RCs with AQ gave a value of delta G0 = 340 +/- 20 meV. In contrast, delta G0 for RCs with UQ as the primary acceptor, is sufficiently large (approximately 550 meV) so that even in the presence of the field, the direct pathway dominates. The voltage dependence shows that the electron transfer from I- to QA is electrogenic. From a quantitative analysis of the voltage dependence on the recombination rate it was concluded that the component of the distance between I and QA along the normal to the membrane is about one-seventh of the thickness of the membrane. This implies that the electron transfer from I to Q contributes at least one-seventh to the potential generated by the charge separation between D+ and QA-.

Resolution of two distinct electron transfer sites on azurin.

Pseudomonas aeruginosa azurin is stoichiometrically and specifically labeled upon reduction by Cr(II)aq ions, yielding a substitution-inert Cr(III) adduct on the protein surface. We investigated the effect of this chemical modification on the reactivity of azurin with two of its presumed partners in the redox system of the bacterium. The Pseudomonas cytochrome oxidase catalyzed oxidation of reduced native and Cr(III)-labeled azurin by O2 was found to be unaffected by the modification. The kinetics of the electron exchange reaction between native or Cr(III)-labeled azurin and cytochrome c551 were studied by the temperature-jump method. Though similar chemical relaxation spectra were observed for native and modified systems, they differ quantitatively. Analysis of the concentration dependences of the relaxation times and amplitudes showed that both obey the same mechanism but that the specific reaction rates of the Cr(III)-modified protein are attenuated. This decreased reactivity of Cr(III)-labeled azurin toward one of its physiological partners suggests the involvement of the labeled region in the electron transfer reaction with cytochrome c551. Furthermore, the presence of a second active site, involved in the reduction of cytochrome oxidase, is suggested by the results.

A heterologous immunoglobulin chain recombinant carries a distinct site for dinitrophenyl and obeys the common hapten binding mechanism.

A heterologous recombinant of the immunoglobulin alpha heavy chain derived from MOPC-460 and the lambda light chain from MOPC-315 was prepared. This H460L315 hybrid binds N epsilon-(2,4-dinitrophenyl)-L-lysine (DNPL) with an affinity of 1.6 X 10(4) M-1 (7 degrees C). This Ig-hapten complex exhibits an absorption spectrum which is different from those observed for each of its parent-DNPL complexes. Very small quenching is caused in the intrinsic fluorescence of the hybrid upon hapten binding, as contrasted by the large quenching of the parent molecules. Chemical relaxation kinetic measurements show that H460L315 exists in solution in two conformations which exchange with a relaxation time of 20 ms (7 degrees C). This transition is accompanied by a change in the fluorescence quantum yield of the protein. DNPL binds to both conformers at comparable fast, though not diffusion-controlled, rates. The equilibrium between the two conformers is shifted upon hapten binding, and the two complexes exchange at a faster rate than the free protein conformers. Thus H460L315 carries a new binding site for DNPL but follows the common mechanism of hapten binding as that observed for other immunoglobulins. These properties of the hybrid should be closely related to the interactions between its constituting domains.

Proton magnetic resonance relaxation in Pseudomonas aeruginosa cytochrome oxidase solutions.

We have measured the temperature and frequency dependence of solvent proton magnetic relaxation rates in solutions of Pseudomonas aeruginosa cytochrome oxidase (EC 1.9.3.2) in its native low spin oxidized, its reduced, and its carbonyl reduced derivative. In solutions of the native oxidized enzyme, a large paramagnetic enhancement of the proton NMR relaxation rates, propagated to the solvent by the fast exchange mechanism, is observed. The ratio (T1/T2)pmg = 1.25 +/- 0.10 at 24 MHz demonstrates that dipole-dipole interaction of the neighboring paramagnets is the dominant relaxation mechanism. Measurements of proton relaxation in solutions of cytochrome oxidase from which the hemes D have been extracted demonstrates that hemes C do not contribute to the observed paramagnetic effects. The electron spin relaxation time of the ferric hemes D of 3.2 +/- 0.4 ns is calculated from the frequency dispersion data. This is the longest value reported for hemoprotein solutions so far. These features of a low spin ferric hemoprotein are similar to those found recently both for the microbial and for the microsomal cytochrome P-450. The calculated distances between the exchanging proton(s) and heme D iron ions demonstrate the high accessibility of the environment of heme D from the solvent side, also for molecules not penetrating the inner coordination sphere.

A common mechanism of hapten binding to immunoglobulins and their heterologous chain recombinants.

Kinetics and thermodynamics of binding of the hapten beta-D-(1-6)-galactotriose to the homogeneous IgA T-601 and to heterologous recombinants of heavy and light chains prepared from mouse myeloma IgA's X-24, J-539, and T-601, which all have the same galactan specificity, have been studied by the chemical relaxation method. All the immunoglobulin-hapten systems investigated were found to exhibit two relaxation times. The reciprocal value of the fast time increased linearly, while that of the slow time leveled off with increasing hapten concentration. This behavior indicates the presence of a fast bimolecular association and a slower monomolecular step. The data obtained for homologous and hybrid immunoglobulins were all found to fit a mechanism where the proteins exist in two conformations and hapten binding shifts their equilibrium to the higher affinity conformer. Furthermore, the kinetic and thermodynamic parameters for the hapten binding by the hybrids were found to be similar to those of their parent proteins. These results strongly suggest that this conformational transition is an inherent property of the tertiary domain structure of the antibody, probably involving changes in the interactions between heavy- and light-chain domains.

Allosteric cooperative interactions among redox sites of Pseudomonas cytochrome oxidase.

Anaerobic reductive spectrophotometric titrations of Pseudomonas aeruginosa cytochrome oxidase were performed. Both types of hemes (C and D) of the dimeric enzyme were monitored. The reduction process was found to involve cooperative allosteric and spectroscopic interactions between the two subunits. The model fitting the data best involves the following features. (1) The redox potential of heme C is about 60 mV higher than that of heme D. (2) In the electron uptake, a positive cooperativity of about 30 mV exists between the two D-type hemes residing in the two subunits. (3) A negative cooperativity of the same magnitude (30 mV) is found between the two C-type hemes bound to two subunits. (4) No interaction was found between heme C and D in the same subunit or in the different subunits. (5) It is suggested that the reduction of the heme, of each kind, has about twice the spectral change compared to that observed upon reduction of the second one. The possible significance of this model for the mechanism of action of the enzyme is discussed

Hapten-linked conformational equilibria in immunolglobulins XRPC-24 and J-539 observed by chemical relaxation.

The interaction of oligogalactan haptens with the murine myeloma proteins XRPC-24 and J-539 has been investigated by the fluorescence temperature-jump method. The relaxation spectrum is composed of two processes, the faster representing hapten assocaition and the slower a protein isomerization. In both cases the concentration dependence of relaxation times and amplitudes was consistent with the general mechanism formulated by Lancet and Pecht (1976, Proc. Natl. Acad. Sci. U.S.A. 73:3549), in which the equilibrium between two conformations of the protein is shifted by hapten binding. The intact proteins and their Fab fragment had identical kinetic behavior, indicating that the conformational changes are located in the Fab region. Temperature dependence analysis for protein J-539 permitted the calculation of activation parameters and led to a consistent energy profile for all the elementary steps. The conformational states are separated by large activation barriers, but have similar free energies. The results suggest that hapten-induced conformational changes in immunoglobulins are more general phenomena than was previously thought.

Binding site of a dextran-specific homogeneous IgM: thermodynamic and spectroscopic mapping by dansylated oligosaccharides.

The hapten binding properties of the homogeneous mouse IgM secreted by MOPC-104E were investigated. Hapten-association constants were determined either by equilibrium and displacement equilibrium dialysis or by fluorometric titrations of the protein with the fluorescent derivatives of the haptens. For the latter type of measurements, several oligosaccharides were derivatized to the corresponding dansylhydrazones. The synthesis, generally applicable to oligosaccharides with free reducing ends, is described. Analysis of the thermodynamic parameters for the binding of 18 haptens forms the basis for proposing a model of the binding site of MOPC-104E. This model is supported and refined by results of the measurements of linear and circular polarization of the fluorescence of the dansylated haptens. The binding site is proposed to consist of a cavity with about 12-A depth, complementary to a terminal nonreducing nigerosyl group. At the entrance to this cavity, a further subsite is identified forming interactions of lower specificity with an additional glucose unit.

Circular dichroism and fluorescence studies of homogeneous antibodies to type III pneumococcal polysaccharide.

The near-ultraviolet circular dichroism (CD) of three homogeneous anti-type III pneumococcal antibodies in the absence and the presence of the specific hexasaccharide ligand was studied. In addition recombinations and hybridizations of H and L chains derived from two of these antibodies were carried out and the CD spectra of bound and free reconstituted IgG molecules were measured. The results indicate that the CD spectra of the native antibodies in the 260-310-nm range are very similar in shape and sign and exhibit a positive band at 285 nm. The homologous reconstituted antibody molecules exhibited CD spectra very similar in shape and sign to those of the native antibody molecules although recombinant molecules are no longer stabilized by interchain disulfide bonds. Upon addition of the hexasaccharide ligand, a significant decrease in amplitude of the CD spectra (18-21%) occurred in all three native antibodies and their Fab fragments as well as in the homologous recombinant molecules. No CD spectral changes could be detected upon interaction of the hapten ligand with the heterologous recombinants. All homogeneous antibodies studied exhibited fluorescence quenching upon oligosaccharide binding and a blue shift of the emission maximum. This property allowed the determination of the binding constant of one selected antibody to be made. Taken together, CD and fluorescence spectroscopic data suggest that oligosaccharide ligands induced detectable conformational changes in the Fab fragment of the antibody.