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In the IR spectra of water on polyethylene, polypropylene and polytetrafluoroethylene frequent and substantial negative peaks occurred and are explained by a theoretical analysis. New states, FEXs, Fast-formed EXcited states are formed by the combination of an IR photon and water and are now made manifest by inefficient energy transfer to the polymer combined with limited energy transfer to the small (0.5-1.0 μL) water sample., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Infrared spectra of small amounts of water on the "hydrophobic" polymers, polyethylene (PE), polypropylene (PP) and polytetrafluoroethylene (PTFE), include many negative infrared bands that become positive with increasing temperature. The new species that the bands represent must arise through absorption of the incident radiation to form short-lived femtosecond states that disappear by (a) induced (Einstein) emission, thus leading to negative IR bands, or (b) fragmentation with loss of vibrational energy or (c) are replaced by an infrared excited state. In addition, we must note that polyethylene, polypropylene and polytetrafluoroethylene carry water! and many water oligomers (chair, boat, and prism hexamer) are easily observed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Dimer formation with dipole neutralization produces species such as low polarity water (LPW) compatible with hydrophobic surfaces (Phys. Chem. Chem. Phys. 2015, 17, 24895-24900) Dimerization and dipole neutralization occurs for N-methylacetamide on polyethylene, a behavior drastically different from its contortions in acetonitrile on AgBr:AgCl planar crystals (AgX) (ChemPhysChem 2014, 15, 3598-3607). The weak infrared absorption of the amide dimer on polyethylene is shown experimentally. Dimerization of palmitic acid is shown along with some of the many ramifications for intracellular systems. Polyoligomers of water are present on polyethylene surfaces. Some high resolution spectra of three of the polyoligomers of water are shown along with a mechanistic scheme for polyoligomer formation and dissolution. The structures of some of the oligomers are known from spectroscopic studies of water on AgX., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The bridge between water repelling and water-attracting regions is recognized here as low polarity water, a novel "neutral" form of water; its identity as a dipole-dipole water dimer is supported by spectroscopic evidence of its presence in thin films of water on a polyethylene surface. High resolution (0.5 cm(-1)), low signal energies (Sg 100) and short scans (0.1 s) are used to ensure that all peaks are detected. Thin films may be trapped between two polyethylene windows, affirming the low polarity of such water; the spectra of the trapped films ("sandwich") are similar to those from a subtraction procedure. Use of the "sandwich" is a new and useful technique in surface studies. In general, intermediate forms might bridge incompatibility between different regimes, from sets of molecules (chemistry and physics) to sets of organisms (biology and sociology). Thin films of water on polyethylene also display strong and transient peaks of water oligomers, cyclic pentamers and cyclic hexamers (chair and boat), bicyclic hexamers (books 1 and 2) and tricyclic hexamers (prism) that have been previously identified in thin films of water on a silver halide surface.

The simplest (minimal) peptide model is HCONHCH3. An increase in the π-helix content with increased substitution in the acyl portion suggested the examination of N-methyl-trimethylacetamide) (NMT). NMT displays spectra, in which there is evolution of a set of helices defined by their amide I maxima near 1686 (3(10)), 1655 (first π), and, most importantly, at 1637 cm(-1) (π). Expanded thin-film infrared spectroscopy (XTFIS) shows pauses or slow stages, which are identified as static phases followed by dynamic phases with the incremental gain or loss of a helix turn. In addition, absorbance at 1637 cm(-1) suddenly increases at 82.1 s (30% over 0.3 s), indicating a phase change and crystallization of the π-helix, along with a coincidental decrease in the absorbance for the first π-helix. A sharp peak occurs at the maximum of the phase change at 82.5 s, representing a pure NMT π-helix. The spectra then undergo a decreasing general absorption loss over 150 s, with the π-helix evolving further to an antiparallel β-sheet fragment. The spectral quality arises from the immobilization of polar molecules on polar surfaces. The crystal structure is that of an antiparallel β-sheet., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A peptide model is a physical system containing a CONH group, the simplest being HCONHCH3 , N-methylformamide (NMF). We have discovered that NMF and N-methylacetamide (NMA), which form hydrogen-bonded oligomers in thin films on a planar AgX fiber, display infrared (IR) spectra with peaks like those of polypeptide helices. Structures can be assigned by their amide I maxima near 1672 (3(10)), 1655 (3(10)), 1653 (α), 1655 (π), and 1635 cm(-1) (π), which are the first IR data for the π-helix. Sharp peaks are an outcome of immobilization of polar species on the polar surface of silver halides. We report the first use of expanded thin-film IR spectroscopy, in which plots of every spectrum over the amide I-II range show pauses or slow stages in the increase or decrease of absorption. These are identified as static phases followed by dynamic phases, with the incremental gain or loss of a helix turn. A general theory can be stated for such processes. Density functional calculations show that the NMA α-helix pentamer (crystal structure geometry) is transformed into a π-helix-like form. For the first time, an entire sequence (3(10)-helix, α-helix, π-helix, quasiplanar species) of spectra has been recorded for NMA., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Evaporation of water on a planar AgX surface leads to a strongly bound monolayer for which IR spectra display the marker peaks for modest numbers of oligomers. From 700-1800 spectra for each isotopomer, H(2)O(16) and H(2)O(18), a pair was selected with moderate intensity at 1616 cm(-1) (a peak reported for the cyclic trimer of water) from the monolayer portion of the experiment. Every selected spectrum had lesser peaks for other oligomers. The sum of a spectroscopic pair reveals the vibrational spectra of the cyclic trimers of H(2)O(16) and H(2)O(18). All fundamentals in the mid-IR are seen including the bending, OH stretching, and intramolecular H-bonding regions, the last never previously recognized. The relative prevalence of cyclic trimer can be attributed to the "low" water concentration on the surface. In addition, a ponderal effect leads to higher concentrations of cyclic trimer in the H(2)O(18) spectra than in the H(2)O(16) spectra and allows observation of combination bands in the H(2)O(18) spectra, representing a new type of isotope effect. The spectroscopic results for the two water isotopomers are much more extensive than those obtained through matrix isolation. Remarkably complete spectra of the cyclic trimer are obtained for the first time, especially for H(2)O(18). DFT calculations with the cyclic trimer on a simplified model for the AgCl surface yield spectra consistent with the experimental spectrum. The technique can be extended to other oligomers of water and many other OH compounds.

N-methylformamide (NMF), the simplest model for peptides, exhibits hyperplectic (both simple and complex) behavior as revealed by thin film infrared spectroscopy on planar AgX [AgCl:AgBr] fiber. IR spectra (0.1 s scans) of 10 microg NMF/dichloromethane(DCM) under N(2) flow first show NMF monomer, dimers, and trimers, which then form surface-organized NMF oligomers as pseudocrystals (P(n)) of increasing length and intensity to P(12). After 4 s, P(12) decays in 1.5 to 4 s steps via P(11), P(10), P(9), P(8), P(7), P(6), and P(5) to P(4) and P(3). The nature of P(n) (n = 5-12) is explained using a model based on the crystal structure of NMF and consisting of a matrix of 7 x 7 helices, alternating R(ight) and L(eft) with TDC (transition dipole coupling) in groups with 2, 3, or 4 neighbors. The total (10) dipolar couplings are matched to the 10 maxima of P(n) and prove the value of the model. P(4) (spectrum 325) fits a 5 x 5 matrix without corners. P(3) is transformed into the very weakly absorbing cyclic hexamer, shown to be very stable and swelling in DCM with increased intensity but without wavelength changes.

Acetonitrile and [D3]acetonitrile in the vicinal region of a planar AgX fiber contain linear dipole-dipole linked oligomers as shown by 1) comparison of infrared band intensity ratios in the gaseous and condensed phases and 2) remarkable plots of absorbance (C--N stretch) versus time during evaporation from an AgX planar fiber element. The plots (CH3CN 2252 cm(-1), CD3CN 2262 cm(-1)) reveal the presence of octamers, hexamers, tetramers, and dimers along with some heptamer, trimer, and monomer structures. A novel isotope effect arises from the somewhat smaller size of the CD3CN resulting in an increase in the CN band intensity. The organized oligomers may be termed pseudocrystals and are the main components responsible for absorption intensity in the infrared spectrum of acetonitrile, on the AgX planar fiber or in an IR cell.

Surface-enhanced infrared absorption spectroscopy (SEIRA) of methanol, ethanol, 1-propanol, and 2-propanol in thin films on planar silver halide (AgX) fibers under slow N(2) flow using 1 sec scans reveals structure in absorbance-time plots. The absorption intensities show extra enhancements (3x) in the absorbance (O--H stretch) ascribed to oligomers present at the AgX surface (molecule enhanced, thus MOSEIRA). This is above those due to amplification (40x, 20 reflections) and enhancement (30x, image dipoles or surface phonon polaritons). In the case of ethanol an excellent initial pentamer spectrum evolves over 8-10 min to a mixture of pentamer, tetramer, and trimer spectra that within another minute forms small oligomers and monomers. We use a new type of cell for infrared spectroscopy containing an AgX planar fiber. The optical configuration leads to a vicinal region at the surface defined by evanescent waves. Within this region are surface-induced organized species such as ethanol oligomers. The planar AgX fiber supports 20 reflections and transmits light over a wide visible-infrared wavelength range. Short scan times permit the study of volatile substrates or solvents, including the effects of solvent polarity.

The Staudinger reaction of azides has now been followed by NMR and other spectroscopic techniques. syn-(Azidomethyl,methyl)(methyl,methyl)bimane (1) and Ph(3)P form a triazaphosphadiene intermediate 2 and then the bimane P-triphenyliminophosphorane 3. The iminophosphorane reacts with an acyl chloride to yield an iminophosphonium salt 4 which then forms the oxazaphosphetane 13. The latter undergoes an electrocyclic reversion to form the phosphine oxide and the chloroimines 7E and 7Z, the last being hydrolyzed to the (acylamido)bimane 6. This set of reactions constitutes the "iminophosphorane pathway". A significant diversion of the reaction path to an (N-alkylamino)phosphonium chloride 8 occurs through reaction of 4 with H(2)O present in the CDCl(3) and through reaction of 3 with HCl. A different azide (alpha-azido-o-xylene 1b) produces the (acylamido)-o-xylene as the sole product. A less sterically hindered phosphine (tri-2-furylphosphine) reacts more slowly to form the iminophosphorane 3a from the azidobimane 1. Reaction of the bimane P-tri-2-furyliminophosphorane with acyl chloride gives only the (acylamido)bimane 6. If the acyl chloride is mixed with 1, followed by addition of the Ph(3)P, the triazaphosphadiene adduct 5 is formed via the triazaphosphadiene. The adduct 5 is converted rapidly into a six-membered cyclic compound 11. The latter either loses nitrogen to yield 6 via 7Z and 7E and the phosphine oxide or loses chloride 10 through a novel chloride-induced elimination reaction from its protonated form. The change in procedure thus results in a dramatic change in the reaction pathway, a reaction set that constitutes the "triazaphosphadiene adduct pathway". In the case of alpha-azido-o-xylene, alpha-chloro-o-xylene (10b) is the only product. The reactions of the azides 1 or 1b with tri-2-furylphosphine also produce chlorides as the major products accompanied by some acetamido derivatives. The nucleophile-induced reaction explains a "surprising result" (formation of ester rather than amide) reported by Sahlberg et al. (Sahlberg, C.; Jackson, A. M.; Claesson, A. Acta Chem. Scand. 1988, B42, 556-562). The intramolecular "aza-Wittig" reaction may depend on the nucleophilicity of the triazaphosphadiene. A comprehensive mechanistic scheme for the Staudinger reaction of azides is conveniently divided into the following: (A) formation of the triazaphosphadiene (Scheme 1), (B) reactions of the triazaphosphadiene (Scheme 2), and (C) reactions via the iminophosphorane (Scheme 3). Some approximate kinetic parameters are reported for some of the reactions.

para-Sulfonylbenzoyloxybromobimane (sBBr) was shown to be similar to the fluorescent labeling agent monobromobimane (mBBr) in reacting rapidly and selectively with thiols to produce stable derivatives which are readily separated by HPLC. Chromatography of the sBBr derivative provides a useful means of confirming the identification of an unknown thiol based upon the chromatography of its mBBr derivative and can be useful for quantitative determination of polycationic thiols for which chromatography of the mBBr derivative is unsatisfactory. Unlike mBBr, which readily penetrates cells, sBBr was found not to be taken up by cells. These characteristics allow sBBr to be used, in conjunction with mBBr, to quantify the export of thiols from cells, as illustrated for GSH and the radioprotective drug WR1065, from V79 cells. Simultaneous determination of GSH and glutathione disulfides in cell culture medium could be achieved by labeling of thiols with sBBr followed by reduction of disulfides with dithiothreitol, labeling of the resulting thiols with mBBr, and HPLC analysis for both glutathione derivatives.

We illustrate the use of polycrystalline silver halide fibers (2-20 microns transparency range) for attenuated total internal reflection Fourier transform infrared (IR) spectroscopic measurements of microsamples (10 micrograms of protein). A powerful adjunct technique is a simple method for carrying out deuterium for proton exchange. Spectra of trypsin, soybean trypsin inhibitor, and their complex are easily obtained. Two kinds of difference spectra (DS) are revealing: DS1 (changes in protein on combination with ligand), IR of the trypsin-soybean trypsin inhibitor complex (T.SBTI complex)--sigma [IR of trypsin (T) + IR of soybean trypsin inhibitor (SBTI)], the small values at all wavelengths indicating no conformational change of the proteins upon complexation, and DS2 (changes in materials on deuteration), IR of protioprotein--IR of deuterioprotein, which reveals the infrared bands affected by deuteration. The rate and the extent of the exchange are additional valuable parameters readily measured with this technique. In the present instance, the rate and the amount of the exchange for T.SBTI complex after 30 min was substantially less than that expected from the simple sum of the same parameters for the two individual proteins, T and SBTI. The enzymatic activity of trypsin on the fiber survived for more than a day, no autodegradation being detected by SDS-gel electrophoresis.

As sensory perception is the channel by which we learn about the nature of the world around us it has been the subject of philosophical inquiry since classical antiquity. With the emergence of science in its modern sense in the 17th century, experimental investigation began to be possible but it was only with the relatively recent advent of molecular biology that sensory perception can be studied at its basic level.

A detailed three-dimensional structure of a molecule is a necessary, but not sufficient prerequisite to understanding its behaviour. Crystal structures are lacking for protein receptors and ion channels. However, the amino acid sequences for representatives of the most important superfamilies of neuroactive proteins (ligand-gated ion channels, voltage-gated ion channels and G-protein receptors) are known. To address the problem of three-dimensional structure and, subsequently, the dynamic properties of these molecules, a holistic approach was applied to the nicotinic acetylcholine receptor (nAChR), the sodium channel of nerves (NaC) and rhodopsin (Rh) to build 'working' structures. For nAChR and Rh, the chains are placed within an envelope derived from image analysis of electron micrographs. Physical organic principles suggest how to juxtapose binding groups as well as structures for ion channels, leading to the identification of the most common feature of channels, the positively charged 'S4' segment and the ever-present amphiphilic segment of nAChR proteins, which has homologous counterparts in the glutamate receptor and glutamate-binding proteins. The cyclic GMP-gated NaC (cGMP-NaC) is related to the eel and rat neuronal NaC, and has a novel positively charged control sequence.

Red cells of different species respond differently to the treatment with the membrane mobility agent, A2C, with respect to both the A2C interaction and the subsequent cell-cell interaction. Depending on whether both, one or neither of the processes are effective, some red cells (e.g., nucleated Leghorn hen red cells, rat red cells) fuse easily, some (human red cells) show morphological changes but do not fuse, and others (nucleated Rock hen red cells) show little or no response. Mixed fusion (i.e., between fusible cells of different species) is readily obtained, indicating that no species-specific recognition sites are required for A2C-induced fusion. the potential for fusion is a transferable characteristic. In the presence of fusible cells, A2C induces both heterologous and homologous fusion of otherwise 'non-fusible' cells. Electron micrographs of fusing cells after treatment with A2C reveal 'onion-ring' structures ('whorls'), free of intramembranous protein particles but different from the smooth appearance of A(2)C particles. Whorls are considered to arise from fusion-potent membrane areas. Fusion is apparent at multiple sites along the contact line between apposed membranes. The postulated appearance of vesicle-like structures along the fusion line (Kosower, E.M., Kosower, N.S. and Wegman, P. (1977) Biochim. Biophys. Acta 471, 311-329) is confirmed by micrographs. The mechanism of this fusion process is duscussed and compared to other types of fusion process.

Treatment of a line of Friend leukemia cells with a dispersion of the membrane mobility agent, A2C, yields cells that undergo successive nuclear divisions without cytokinesis, resulting eventually in cells with as many as 30 nuclei. Neither the DNA replication rate of the cells nor the generation time is different after treatment; in addition, the multiple nuclei divide synchronously, and the chromosome number corresponds to the number of nuclei in the cell. Inhibition of cytokinesis is not observed if the cells are washed with reagent-free medium within 1 hr of treatment, but is observed if washing is delayed for 24 hr. Membrane mobility agent loaded with the fluorescent probe, Flomol F20C, leads to fluorescent membrane; fluorescence disappears from the membrane after a change of medium within 1 hr, but not after a change of medium within 24 hr. Some stages in the overall development resemble those seen for cytochalasin B inhibition of cytokinesis, although the mechanisms may well be different for the inhibition promoted by membrane mobility agent. The inhibition of cytokinesis by A2C provides a potentially interesting means of studying cytokinesis and the regulation of differentiation.

Two plausible, transmembrane ion channel elements (These 'elements' are alpha-helical sequences of 24 amino acids in which polar, hydrophilic side chains occupy one side and hydrophobic side chains the other) have been identified in the serine chemoreceptor-methyl-accepting chemotaxis protein (MCP) (SerR) of E. coli and the aspartate chemoreceptor-MCP (AspR) of S. typhimurium. That the chemoreceptor might serve as, or activate, an ion channel is supported strongly by the occurrence of membrane depolarization, specific peptide methylation and neurotoxin inhibition of response in the chemotaxis of S. aurantia (E.P. Greenberg, refs. 13-18).

Revised assignments for the bilayer helices of the beta-, gamma- and delta-subunits of the acetylcholine receptor are presented. A new feature of the model is extensive charge matching between the polar groups of the ion channel elements of different subunits.

Based on the nicotinic acetylcholine receptor model [(1987) Eur. J. Biochem. 168, 431-449], a partial model is constructed for the exobilayer portion of the GABAA receptor, an approach justified by the superfamily relationship of the two receptors [(1987) Nature 328, 221-227]. The model predicts successfully the excess positive charge on interior strands which constitute the ligand-responsive portion of the receptor. Binding to GABA expands the exobilayer portion of the receptor, opening a pathway to a chloride channel. Separate binding sites for antianxiolytics (benzodiazepines) and hypnotics (barbiturates) are suggested, with prolongation of chloride entry projected as a consequence of stabilization of the open form. The anion-exchange protein (AEP) of membranes (band 3 of red blood cell membranes) is similar in some respects to the gamma-aminobutyric acid (GABAA) receptor. Both proteins are inhibited and labeled by diisocyanatostilbenedisulfonate (DIDS), both transport Cl- and HCO-3, and both are membrane proteins. Starting with the lysines known to be labeled in band 3 protein, searches of the amino acid sequences of the GABAA receptor alpha- and beta-subunits reveal at least 4 reasonably homologous sequences. The relationship between AEP and GABAA receptor leads to the idea that the chloride/bicarbonate channel may be the ancestor of all ligand-gated channels, with ligand gating by gamma-aminobutyric acid and acetylcholine arising later in evolution.

Receptors on normal human peripheral blood lymphocytes can be divided into two classes by means of the capping response exhibited in the presence of the reagents, diamide or colchicine (microtubule-related) and A2C (microtubule-independent). Diamide and colchicine promote capping of concanavalin A (Con A) receptors. Diamide capping is reversible, while colchicine capping is not reversible under the conditions used. A2C does not promote the capping of Con A receptors. In contrast, diamide and colchicine do not affect the rate at which either anti-immunoglobulin (anti-Ig) or wheat germ agglutinin (WGA) receptors cap, but A2C effectively enhances cap formation for both anti-Ig and WGA receptors. The simplicity of the classification method promises to be of use in the investigation of membrane receptors.

Folding of the five polypeptide subunits (alpha 2 beta gamma delta) of the nicotinic acetylcholine receptor (AChR) into a functional structural model is described. The principles used to arrange the sequences into a structure include: (1) Hydrophobicity----membrane crossing segments (2) amphipathic character----ion-carrying segments (ion channel with single group rotations) (3) molecular shape (elongated, pentagonal cylinder)----folding dimensions of exobilayer portion (4) choice of acetylcholine binding sites----specific folding of exobilayer segments (5) location of reducible disulfides (near agonist binding site)----additional specification of exobilayer arrangement (6) genetic homology----consistency of functional group choices (7) noncompetitive antagonist labeling----arrangement of bilayer helices. The AChR model is divided into three parts (a) exobilayer: 11 antiparallel beta-strands from each subunit (b) bilayer: 4 hydrophobic and 1 amphiphilic alpha-helices from each subunit and (c) cytoplasmic: one (folded) loop from each subunit. The exobilayer strands can form a closed "flower" (the "resting state") which is opened ("activated") by agonists bound perpendicular to the strands. Rearrangement of the agonists to a strand-parallel position and partial closing of the "flower" leads to a desensitized receptor. The actions of acetylcholine and succinoyl and suberoyl bis-cholines are clarified by the model. The opening and closing of the exobilayer "flower" controls access to the ion channel which is composed of the 5 amphiphilic bilayer helices. A molecular mechanism for ion flow in the channel is given. The unusual photolabeling of intrabilayer serines in alpha, beta and delta, but not in gamma-subunits near the binding site for non-competitive antagonists (NCAs) is explained. The dynamic behavior of the AChR channel and many experimental results can be interpreted in terms of the model.

Antibodies to the synthetic peptide (carrier-coupled) corresponding to amino acids 210-223 of the primary sequence of eel Na channel (C1+ peptide) were generated. The antipeptide antibodies were used to identify functional roles as well as the accessibility from the external membrane surface of the C1+ domains. Rabbit antipeptide antibodies bound specifically to the C1+ synthetic peptide and to an eel membrane fraction bearing a high density of Na channels. When applied to the external surface of cultured dorsal root ganglion cells obtained from newborn rats, the antibodies modify Na channel inactivation by shifting the steady-state Na current-inactivation parameter, h infinity, curve to more negative potentials in fast and slow Na currents. The rate of inactivation of the slow channel is shown to be increased. The antibodies do not have a significant effect on activation of the channels. Part of the amino acid sequence corresponding to C1+ peptide is therefore accessible, in the mammalian Na channel, from the external membrane surface and is associated with the inactivation gate.

The mechanism of action of one class of sodium channel opening agents (batrachotoxin, veratridine, aconitine and grayanotoxin) is proposed to involve complexation of a triad of agent oxygen atoms with the epsilon-ammonium ion of a channel lysing side chain, holding open the mouth or exit of the ion channel. This idea complements the oxygen triad model derived by structural considerations (Masutani, T., Seyama, I., Narahashi, T. and Iwasa, J. (1981) J. Pharm. Exp. Therap. 217, 812) and extended by crystal structure comparisons (Codding, P.W. (1983) J. Am. Chem. Soc. 105, 3172). The mechanism is based on results for acetylcholine receptor ion channel gating, structure and function, using single group rotation (SGR) theory (cf. Kosower, E.M. (1983) Biochem. Biophys. Res. Commun. 111, 1022 and in press (1983); FEBS Lett. (1983) 155, 245; ibid. 157, 144; Biophys. J. (1983) 45, in press).

A modified structural model of rhodopsin is presented. Seven (alpha-helical) segments of 24 largely hydrophobic amino acid residues are assembled with exobilayer connecting strands into an aligned set, using the sequences of human red, green, and blue iodopsins (cone pigments) and human and bovine rod rhodopsins. (Aligned set numbering is used in this article). The inner region of the heptahelical hydrophobic domain includes one His-Glu (Asp) ion pair (red, green rod) near the retinylidene moiety in addition to an iminium ion Asp-99 pair. The negative charges posited in the "point-charge model" to cause the shift of the retinylidene iminium ion light absorption to longer wavelengths in the protein ("opsin shift") are Asp-99 (red, green rod), Glu-102 (red, green), and Glu-138 (rod). Blue iodopsin lacks both an ion pair and a counter charge to the iminium ion in the inner region, a fact that explains its absorption relative to rod rhodopsin. The spectroscopic difference between rod rhodopsin and the red/green iodopsins is due to the influence of Glu-102 in the latter. The red-green difference is due to the net effect of seven OH groups around the chromophore, all such groups being found within one helix turn of the retinylidene location. The tryptophan, which rotates as the retinylidene group isomerizes, may be Trp-142 or Trp-177. The geometric change (the rhodopsin "photoswitch") resulting from cis-trans isomerization in the first excited electronic state (S1), ultimately leads to RX (photoactivated rhodopsin, metarhodopsin II) and changes the activity of exobilayer groups, possibly causing dissociation of Lys-83 and Arg-85 from the carboxylate groups at positions 263 and 265.

The membrane mobility agent 2-(2-methoxyethoxy)-ethyl 8-cis-2-n-octyl-cyclopropyl)-octanoate promotes cap formation from wheat germ agglutinin-receptor combinations at the expense of agglutination in membranes of malignant mastocytoma cells.

The membrane mobility agent, A2C, actively promotes the fusion of hen erythrocytes under conditions similar to those used by Lucy et al. for glyceryl monooleate.

Monobromobimane labels red cell membrane protein thiol groups; bands exhibit fluorescence after sodium dodecyl sulfate acrylamide gel electrophoresis and correspond to almost all of those staining with Coomassie blue. The response of membrane protein thiol groups to oxidative challenge and the dynamics of recovery of the thiol groups may be followed. Diminished labeling is found after oxidation with diamide, with both intrachain and interchain disulfide bond formation demonstrated by sodium dodecyl sulfate acrylamide gel electrophoresis. Regeneration of thiol groups under physiological conditions (incubation with glucose) after a moderate degree of diamide oxidation is shown to be complete (with respect to thiol group content and degree and distribution of bimane label) in normal human red blood cell membranes. Even after oxidation of almost half of the membrane protein thio groups (maximum degree of oxidation achieved), regeneration of thiol groups is almost complete; a minor fraction resides in the form of disulfide-linked high molecular weight proteins (demonstrated by the electrophoretic profile) which may be reduced completely with dithiothreitol. Bimane fluorescent labeling provides a convenient and sensitive method for following membrane thiol group status under physiological conditions.

The intracellular thiol-oxidising diazenes, diazenedicarboxylic acid bis-N,N-dimethylamide and diazenedicarboxylic acid bis-N'-ethylpiperazinide, have been used in the study of red cells. A difference in the consequences of diazene oxidant treatment between the human red cell and rat red cells has been found in respect to the quantity of oxidant needed for glutathione (GSH) oxidation, to the fate of GSH, and to the reactivity of hemoglobin. In the first place, significantly more oxidant is needed for GSH oxidation in the rat red cell than in the human cell. Secondly, in the human cell, all of the GSH is converted to glutathione disulfide (GSSG), from which GSH is regenerated. In the rat cell, GSH disappears without being converted to GSSG, and GSH is not regenerated. Thirdly, a decrease in rat hemoglobin thiol groups, but no change in human hemoglobin, is found. Sterically unhindered thiol groups in the rat hemoglobin are thought to react with the usual adduct intermediate in GSH oxidation by diazene (formed from RCON = NCOR + GSH leads to RCON(SG)NHCOR) to produce mixed disulfides, from which GSH is not easily regenerated. The results support the idea that reduction of mixed disulfides of GSH and protein is not carried out directly by GSSG reductase but necessitates thiol transferase and GSH. The thiol-oxidising diazenes may be of use in mapping of exposed, reactive thiol groups in proteins.

Folding of the five polypeptide subunits (alpha 2 beta gamma delta) of the nicotinic acetylcholine receptor (AChR) into a functional structural model is described. The principles used to arrange the sequences into a structure include: (1) hydrophobicity----membrane-crossing segments; (2) amphipathic character----ion-carrying segments (ion channel with single group rotations); (3) molecular shape (elongated, pentagonal cylinder)----folding dimensions of exobilayer portion; (4) choice of acetylcholine binding sites----specific folding of exobilayer segments; (5) location of reducible disulfides (near agonist binding site)----additional specification of exobilayer arrangement; (6) genetic homology----consistency of functional group choices; (7) noncompetitive antagonist labeling----arrangement of bilayer helices. The AChR model is divided into three parts: (a) exobilayer consisting of 11 antiparallel beta-strands from each subunit; (b) bilayer consisting of four hydrophobic and one amphiphilic alpha-helix from each subunit; (c) cytoplasmic consisting of one (folded) loop from each subunit. The exobilayer strands can form a closed 'flower' (the 'resting state') which is opened ('activated') by agonists bound perpendicular to the strands. Rearrangement of the agonists to a strand-parallel position and partial closing of the 'flower' leads to a desensitized receptor. The actions of acetylcholine and succinoyl and suberoyl bis-cholines are clarified by the model. The opening and closing of the exobilayer 'flower' controls access to the ion channel which is composed of the five amphiphilic bilayer helices. A molecular mechanism for ion flow in the channel is given. Openings interrupted by short duration closings (50 microseconds) depend upon channel group motions. The unusual photolabeling of intrabilayer serines in alpha, beta and delta subunits but not in gamma subunits near the binding site for non-competitive antagonists is explained along with a mechanism for the action of these antagonists such as phencyclidine. The unusual alpha 192Cys-193Cys disulfide may have a special peptide arrangement, such as a cis-peptide bond to a following proline (G.A. Petsko and E.M. Kosower, unpublished results). The position of phosphorylatable sites and proline-rich segments are noted for the cytoplasmic loops. The dynamic behavior of the AChR channel and many different experimental results can be interpreted in terms of the model. An example is the lowering of ionic conductivity on substitution of bovine for Torpedo delta M2 segment. The model represents a useful construct for the design of experiments on AChR.

Two diamide analogues, diazene dicarboxylic acid bis (n'-methylpiperazide) or DIP, and its bis-N'-methyl iodide salt, or DIP + 2, were tested for their ability to penetrate cultured Chinese hamster cells and oxidize intracellular glutathione. DIP penetrated the cells at a reasonable rate at 18 degrees C, 160 nmoles being required to oxidize the endogenous glutathione of 2 X 10(6) cells, but it penetrated very slowly at 0 degrees C. DIP +2 did not effectively oxidize glutathione in Chinese hamster cells, possibly because it did not enter the cells. DIP became toxic after about 10 min of exposure, but its toxicity could be moderated by using anoxic conditions. DIP, but not DIP +2, sensitized anoxic Chinese hamster cells to X-radiation by a factor of 1-5, an effect that was due entirely to removal of the shoulder from the survival curve.

Diamide, which in concentrations of 10(-5) M and higher oxidizes glutathione intracellularly, produces a dose-related increase in the frequency of miniature end-plate potentials (MEPPs). With high enough doses, quantal release is blocked, apparently through exhaustion. The early phase of MEPP frequency increase is accompanied by an increase in EPP amplitude that may reach more than 10-fold and is therefore not produced by depolarization of axon terminals. Subsequently, EPP amplitude is reduced and falls to zero, associated with failure of invasion of the nerve action into the terminals while the MEPP frequency remains elevated. Both facilitation and PTP follow the time course of change in EPP amplitude. The increase in MEPP frequency with diamide does not require external Ca2+ but raising external Ca2+ increases the MEPP rate in the presence of diamide. External Ca2+ is necessary for EPP appearance and also potentiates the diamide effects. Conversely diamide reduces the requirements for Ca2+ in releasing ACh. Diamide substitutes for external Ca2+ in K+ evoked MEPP release and in the absence of external Ca2+, diamide-evoked MEPP release is increased by raising external Mg2+ levels. The action of diamide may be dependent on the actual release of Ca2+ from intracellular stores or it may work through mimicking some of the actions of Ca2+. The action of diamide bears close resemblance to the effects of prolonged stimulation of the motor axon at 10 Hz.

Rat, but not human, erythrocytes undergo fusion promoted by the membrane-mobility agent 2-(2-methoxyethoxy)-ethyl cis-8-(2-octylcyclopropyl)octanoate (A2C). The difference in behavior is correlated with rat erythrocyte membrane protein degradation caused by Ca2+-activated proteases. The human erythrocyte is deficient in such protease activity. Membrane protein degradation is a necessary, but not sufficient, requirement for membrane fusion. Membrane protein degradation probably releases membrane components from certain constraints. In addition, the motion of membrane components precedes fusion and must be promoted by reagents such as A2C, leading to the creation of fusion-potent lipid areas. This sequence of chemical and physical events occurs in other fusion processes.

Chemical logic and single group rotation (SGR) theory are applied to the primary structure determined by Noda et al. [(1984) Nature 312, 121-127] to construct a molecular model of the sodium channel of Electrophorus electricus. Both structural and dynamic aspects of the channel are accounted for, including gating current, sensitivity to changes in membrane potential, channel opening, a binding site for sodium, selectivity for sodium over potassium, capacity for rapid sodium flow, sensitivity to batrachotoxin (or other toxins) and inactivation.