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.

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 a-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.

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-.

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.

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.