Two photochromic activators of the electrogenic membrane of the electroplax of Electrophorus electricus are described. monium)methyljazobenzene dibromide (Bis-Q), one of the most potent ever reported, is active at concentrations of less than 10-7 M. Its cis isomer, which is obtained from the trans by exposure to light of 330 nm, is practically devoid of activity. Photoregulation of the potential of the membrane takes place in the presence of Bis-Q, presumably because of the conversion of the active trans isomer to the inactive cis isomer in the single-cell electroplax system.The second activator, 3-(a-bromomethyl)-3'-[a-(trimethylammonium)methyllazobenzene bromide (QBr) can be covalently attached to the electroplax membrane after reduction of the membrane with dithiothreitol. Activation of the membrane is induced by the covalently linked reagent. Its cis isomer, obtained from the trans by exposure to light of 330 nm, is, like cis-Bis-Q, of very low activity. Both isomers of Bis-Q are equally active as inhibitors of acetylcholinesterase, 50% inhibition occurring at a concentration of 10-5 M. The possibility of using trans-Bis-Q and trans-QBr to characterize and isolate the receptor protein is discussed.Systems in which photoregulation could be studied at the molecular level were described in previous papers. In these systems, photochromic azo derivatives were used as effector molecules to regulate the activities of chymotrypsin (1) and acetylcholinesterase (2, 3) and to photoregulate the potential of the excitable membrane of the monocellular electroplax preparation (4). Photoregulation was achieved by exploiting differences between the biochemical activities of the cis and trans isomers of the photochromic compounds, the relative concentrations of which were influenced by the wavelength of light to which the solution was exposed [or light vs. darkness, in one case (3) ].Light-induced changes in potential of the electroplax membrane may be considered as a Mtodel for the process of vision, in which the cis to trans isomerization of retinal is the first step in the initiation of a neural impulse. In the latter case, however, as well as in the phytochrome system of plants (5), the photochromic substances are located intracellularly, making for a highly efficient process. It thus appeared of interest to prepare a light-sensitive ligand that would form a covalent bond with the receptor protein of the electroplax. A compound with the desired properties was prepared: 3-(a-bromomethyl)-3'-[a-(trimethylammonium)methyl]azobenzene (QBr). Also synthesized was the closely related 3,3'-bis[a-(trimethylammonium)methyl]azobenzene (Bis-Q). the trans isomer of which was found to be a potent receptor activator, one of the most potent thus far described.The high affinity and specificity of Bis-Q may make it a useful reagent for the characterization, isolation, and purification of the receptor protein. Some experiments with the two azo compounds are presented in this paper. METHODSPreparation of 3,3'-bis(a-bromomethyl)azobenz...

After disulphide bonds are reduced with dithiothreitol, trans-3-(a-bromomethyl)-3'-[a-(t rimethylammonium) methyl]azobenzene(trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this "tethered agonist" shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 btM carbachol. Voltage-jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 #M carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-QBr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to "open-channel blockade" by QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3',bis-[a-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis ~ trans photoisomerizations and decreased by trans --* dsphotoisomerizations. As with Bis-Q, light-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered cis-QBr have a channel duration severalfold briefer than with the tethered trans isomer. By comparing the agonist-induced conductance with the cis/trans ratio, we conclude that each channel's activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. J. GEN. PHYSIOL. ~) The Rockefeller University Press

These experiments employ the photoisomerizable compound, 3,3'-bis-[a-(trimethylammonium)methyl]azobenzene (Bis-Q), to study the response to muscarinic agents in frog myocardium . In homogenates from the heart, transBis-Q blocks the binding of [3 H]-N-methylscopolamine to muscarinic receptors .In voltage-clamped atrial trabeculae, trans-Bis-Q blocks the agonist-induced potassium conductance. The equilibrium dose-response curve for carbachol is shifted to the right, suggesting competitive blockade . Both the biochemical and electrophysiological data yield a dissociation constant of 4-5 AM for trans-Bis-Q ; the cis configuration is severalfold less potent as a muscarinic blocker . Voltageclamped preparations were exposed simultaneously to carbachol and Bis-Q and were subjected to appropriately filtered flashes (<1 ms duration) from a xenon flashlamp . Trans --+ cis and cis -> trans photoisomerizations cause small (<20%) increases and decreases, respectively, in the agonist-induced current . The relaxation follows an S-shaped time course, including an initial delay or period of zero slope. The entire waveform is described by [1 -exp(-kt)]" . At 23°C, k is -3 s -' and n is 2 . Neither k nor n is affected when : (a) [Bis-Q] ature ; the Qto is between 2 and 2.5 . In the same range, n does not change significantly . Like other investigators, we conclude that the activation kinetics of the muscarinic K + conductance are not determined by ligand-receptor binding, but rather by a subsequent sequence of two (or more) steps with a high activation energy .

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tran.-3,3'-Bis[a-(trimethylammonio)methyl]azobenzene bromide (BisQ) is a potent agonist of the acetylcholine receptor (AcChoR) ofElectrophorus electrics. BisQ is highly constrained, suggesting that its structure is complementary to the combining site of the AcChoR when the latter is in its activated state. Antibodies produced in rabbits to a conjugate of bovine serum albumin and a derivative ofBisQ mimicked the binding characteristics of the AcChoR with respect to the order of binding of a variety of agonists and to the preferred recognition of decamethonium ion (an agonist) over hexamethonium ion (an antagonist). Immunization of three rabbits with purified anti-BisQ yielded antisera having binding characteristics of anti-AcChoR in that, by complement fixation and enzyme immunoassay, crossreactions with receptor preparations from rat, Torpedo, and eel could be demonstrated in sera ofall three rabbits immunized. Two ofthe three rabbits showed signs ofmuscle weakness similar to that seen after immunization with the AcChoR. One of the rabbits was injected intramuscularly with neostigmine and showed temporary improvement. Another showed post-tetanic exhaustion of hindlimb muscles after stimulation of the sciatic nerve at 50 Hz. Antibodies reactive with the AcChoR, therefore, were elicited by immunization with an antibody to a potent ligand of the AcChoR without the necessity of isolating the receptor itself. A similar mechanism may play a part in the etiology of at least some autoimmune diseases in which antibodies to various other receptors are involved.

Abstract.-The enzymic activity of acetylcholinesterase can be photoregulated through the mediation of photochromic inhibitors of the enzyme. N-pphenylazophenyl-N-phenylcarbamyl fluoride, an irreversible inhibitor of acetylcholinesterase, exists as two geometric isomers which are interconvertible through the action of light. The cis isomer, which predominates after exposure to light of 320 nm, is more active than the trans isomer, which results from exposure to light of 420 nm. It was possible, therefore, to use light energy to regulate the inactivation of the enzyme. Similarly, levels of acetylcholinesterase activity could be photo-regulated in a completely reversible manner by means of the photochromic reversible inhibitor p-phenylazophenyltrimethylammonium chloride. These experiments can serve as models for similar phenomena observed in nature, particularly in photoperiodic rhythms of higher animals.A system was recently described in which an enzymic process, in itself insensitive to light, could be made subject to photoregulation through the mediation of a light-sensitive effector molecule.1 The photosensitive compound, N-p-phenylazophenyl-N-phenylcarbamyl chloride2 (PAPC), is a specific inactivator of chymotrypsin.3 PAPC is a photochromic (or phototropic) molecule4 which, under the influence of light, can undergo a reversible configurational change involving the N = N bond, to yield either a cis or a trans isomer. The change in structure is influenced by the wavelength of light as follows: 320 nm trans = cis 420 nm Although both isomers could inactivate chymotrypsin, the cis isomer was found to be about five times more active. Conditions were found in which the rate of inactivation by trans PAPC was very slow. Thus, it was possible to "turn off" (i.e., inactivate) the enzyme by exposing a solution of enzyme in the presence of trans PAPC to light of 320 nm. Similarly, experiments in which the inactivation process could be halted by light were also possible by starting with the cis isomer. It was suggested that these experiments could serve as models for certain photosensitive processes found in nature, e.g., phototaxis.5Our investigations have now been extended to the enzyme acetylcholinesterase (AcCh-esterase). Its activity can be regulated in the same way as the activity of chymotrypsin. Moreover, by using a photochromic reversible inhibitor, it was possible to regulate the level of AcCh-esterase activity reversibly, by the action of light.

Recently we described a procedure for preparing antibodies to the acetylcholine receptor (AChR) based on immunoglobulin idiotypes and on the hypothesis that, regardless of functional differences, macromolecules of the same specificity will show structural homologies in their binding sites. Antibodies were prepared in rabbits to a structurally constrained agonist of AChR, trans-3,3'-bis[alpha-(trimethylammonio)methyl]azobenzene bromide (BisQ). These antibodies mimicked the binding specificity of AChR in its activated state--agonists were bound with affinities that were in accord with their biological activities and antagonists were bound poorly. Rabbits were then immunized with a specifically purified preparation of anti-BisQ to elicit a population of antibodies specific for the binding sites of anti-BisQ. A portion of the anti-idiotypic antibodies produced in the second set of rabbits cross-reacted with determinants on AChR preparations from Torpedo californica, Electrophorus electricus and rat muscle. Moreover, several of the rabbits showed signs of experimental myasthenia gravis, in which circulating AChR antibodies are typically found. To devise a more direct route to monoclonal anti-receptor antibodies we based our strategy on acceptance of the concept of the anti-idiotypic network theory of Jerne. According to this theory, injection of an antigen elicits, in addition to antibodies to the antigen, other populations that include anti-idiotypic antibodies directed at the combining sites of the antigen-specific antibodies. If the antigen-specific antibodies recognize a ligand of a receptor, then the anti-idiotypic antibodies should bind receptor. Thus, when a mouse is immunized with a bovine serum albumin conjugate of BisQ (BisQ-BSA), it should be possible to expand populations of spleen cells that secrete antibodies which bind anti-BisQ and AChR, in addition to populations specific for BisQ. Fusion of the spleen cells with an appropriate myeloma line should yield monoclonal anti-AChR antibodies. Here we report the success of this approach and its implications.

Abstract. Levels of acetyicholinesterase activity can be made to vary in response to the presence or absence of sunlight in a system that can be considered as a model for photoperiodic processes found in nature. The enzyme is rendered photosensitive by the presence of a photochromic inhibitor, N-p-phenylazophenylcarbamyl choline, which changes from a trans to a cis isomer under the influence of the light of the sun and reverts back to the trans isomer in the dark. The two isomers differ in their ability to inhibit acetylcholinesterase, thus rendering the enzyme system responsive to sunlight. The relationship of this system to photoresponsive processes in nature is discussed, and a possible role in photoregulation is suggested for naturally occurring carotenoids.We have recently shown how systems normally insensitive to light (the enzymes chymotrypsin' and acetylcholinesterase2 and the electroplax of the electric eel)3 can be photoregulated by means of photochromic effector molecules. Those molecules share a common p-phenylazophenyl group which, under the influence of light, undergoes a reversible configurational change to yield a cis or trans isomer (Fig. 1). Their ability to induce photoregulation derives from differences in the biochemical activities of the two isomers. For example, carbamylcholine-induced depolarization of the excitable membrane of the electroplax was inhibited unequally by the cis and trans isomers of p-phenylazophenyltrimethylammonium chloride (in Fig. 1, substitute N(CH3)3+Cl-for R1-N-R2).