BioSystems, 13 (lq81) 243--251 243 © Elsevier/North-Holland Scientific Publishers Ltd.

ELECTRICAL MEMBRANE PHENOMENA IN SPHERULES FROM PROTEINOID AND LECITHIN***

YOSHIO ISHIMA*, ALEKSANDER T. PRZYBYLSKI** and SIDNEY W. FOXt Institute for Molecular and Cellular Evolution, University of Miami, 521 Anastasia Avenue, Coral Gables, Florida 33134, U.S.A. (Received August :[2th, 1980) (Revision received January 20th, 1981)

Spontaneous and induced electrical phenomena resembling membrane and action potentials in natural excitable cells have been observed in artificial cells. These artificial cells were made from thermal proteinoid and lecithin in a solution of potassium acid phosphate with glycerol.

Introduction Other related studies are numerous (e.g. Takahashi and Miyazaki, 1971; Kennedy et One of the most studied attributes of the al., 1977). Studies of synthetic bilayer mem- living state in modern is that of branes indicate that polypeptides can form excitability. Because of an extensive literature ionic channels in excitable bilayers. on excitability in cells, and because of accu- The early examples and the results of mulated knowledge of properties of proteinoid chemical analysis of excitable cells (Condrea (Fox and Dose, 1977; Fox, and Rosenberg, 1968; Camejo et al., 1969; 1980), proteinoid-containing models for excit- Ishima and Waku, 1978) suggested that excit- able cells have been sought. ability might be produced in an artificial In somewhal; related studies, excitability composed of suitable polyamino acid and has been observed in internodal cells of phospholipid. tissue (Hill and Osterhout, 1934; Cole and The fact that proteinoid microparticles Curtis, 1938; F indlay, 1959) and in reassem- possess permselective membranes (Fox et al., bled vesicles. Droplets of protoplasm from an 1969) and are capable of junction formation internodal cell of NiteUa form semipermeable and communication (Hsu et al., 1971) also membranes on the surface under certain raised the possibility of electrical phenomena conditions (Yoneda and Kamiya, 1969) and in these units. This paper describes the results display excitabJ~lities when properly activated of experiments in making electrically active (Inoue et al., 1971; Takenaka et al., 1971). artificial cells from proteinoids and lecithin. Various proteinoids were prepared to contain *Present address: Laboratory of Neurophysiology, large proportions of individual amino acids Division of Neuroscience, Tomobe Hospital Medical Center, 654 .4~ahi-Mach, Tomobe-Cho, Nlshi- in an effort to identify which amino acids Ibaragigun, Ibaragi-Ken 319-03, Japan. most contribute to electrical activity. **Present addrem+: Medical Research Center, Polish Academy of Sciences, Dworkowa 3, 00-874 Warsaw, Materials and methods Poland. ***Some of these results were first presented at the Third Annual Meeting of the Society for Neuroscience Proteinoid in San Diego, Cal~[ornia, November 1973. tTo whom repriint requests should be addressed. The polyamino acids were prepared by 244

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¢JR °~ S 0 245 heating in an oil bath at 190°C for 6 h a mix- the surface of the crusts (Fig. 1). This mem- ture of 50 g of each individual brane formed from the crust in an external treated with 50 g of an equimolar mixture of solution containing mannitol at concentra- 18 amino acids under a nitrogen blanket tions between 150 mM and 300 mM. Mannitol (Vegotsky, 1972,). The products were mech- was used to maintain the osmotic balance of anically stirred with for 2 h and then the spherule. If the mannitol in the external filtered. The dissolved fraction was dialyzed solution was concentrated to more than for 2 days in a cold room with two changes of 300 mM, it failed to produce semipermeable water per day. The non-diffusates were spherules. If the tonicity of the solution was lyophilized. Preliminary experiments showed too low the size of spherules increased rapidly that the dissolved fractions yielded the most until they burst. A concentration of 200 mM "active" particles. The leucine-rich - mannitol was found to prevent bursting of the oid that received most attention contained spherules and yet maintain suitable sized 14.9% leucine. spherules for several hours. The selected spherules used in this experiment were Phospholipid 60--150 ~m in diameter. The compositions of the inside and outside solutions for the Phospholipids were purified from Eastman spherules are shown in Table 1. practical grade vegetable lecithin by extrac- tion with 50% aqueous glycerol solution con- Electrical phenomena taining potassium phosphate (pH 5.8). A simple, standard intracellular microelec- Preparation of spherules trode technique was applied to monitor the electrical phenomena (Watanabe and Ishima, A suspension of 60 mg of proteinoid and 1972). Figure 2 shows a schematic outline of 60 rag of lecithin in 3.0 ml of potassium the equipment. The tip diameter of the glass phosphate solution with aqueous glycerol electrode was about 0.2/~m and the electrical (50:50 v/v) was heated to boiling. This resistance was approximately 20 M~2, with solution contained 80 mmol of phosphate 3 M KC1 inside. The reference electrode was a (PO4), and 50% of glycerol (pH 5.8). The silver-silver chloride-KC1 agar placed in the solution was allowed to cool to room tem- external solution. These are employed for perature, and then allowed to stand for a accurate measurements of the membrane period of several hours up to 2 days. Standing TABLE 1 was empirically observed to be helpful to the experiments. After incubation at room tem- Ionic concentrations within spherules. perature, crusts precipitated from the super- saturated solution. They were carefully Inside Outside removed by use of a pipet. The original K÷ 80 (mM) Na÷ 0.2 (mM) external solution was then replaced com- FF 130 K÷ 0.05 pletely by the e:cperimental external solution Mg ~ + 0.1 by a suction device. This was done by adding Ca 2÷ 0.5 (1.0) from the top and drawing from the bottom, PO~- 70 C1- 0.25 SO:- 0.1 while the level of the solution was kept NO~ 1.0 (2.0) constant. Approximately 30 min after the Glycerol 50% Mannitol 200.0 (mM) external solution was completely replaced, in pH 5.8 pH 7.0 (Na- the case of leucine-rich equimolar proteinoid, phos- phate for example, a very thin, transparent, almost buffer) invisible spherical membrane emerged from 246

proteinoids, four types proved suitable for experiment. These were leucine-rich equimolar proteinoid (AMX-9, dfl), threonine-rich equi- molar proteinoid (AMX-105, dfl), proline- rich equimolar proteinoid (AMX-105, dfl), and leucine-rich equimolar proteinoid (T-83, dfI). These water-soluble proteinoids formed semipermeable membranes with lecithin. Among these, the leucine-rich equimolar proteinoid alone produced pronounced elec- trical activities. While a proline-rich protein- 1 M ohm oid (49.8% proline) and a threonine-rich one (19.4% threonine) also formed mem- branes with the same lecithin, they showed Fig. 2. ;chematic diagram of the experimental setup lesser electrical activities. (see text). The individual amino acids included in proteinoids in large proportion represented potential with elimination of any possible quite fully the roster of amino acids common boundary potentials. For DC

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Fig. 4. Flip-flop phenomenon of the spherule. Miniature potential activities were observed on the flopped phase of the potential.

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Fig. 5. Miniature potential activities. A high magnification measurement of the potential activities on the flopped phase of the potential.

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1 Sec Fig. 6. Two different patterns of potential burst (a and b) observed on different spherules. 249

2 mV 1 2xlo-~A 1 10 mSec Fig. 7. Potential jumps induced by the current injection of the spherule.

Fig. 8. A photomicrograph of typical spherule which produced the electrical activity by current injection. Diameter of the spherule was 110 pm with an 80-pro core of the proteinoid crust. 250

Artificial pond water is a physiological on the contrary a selective advantage to a solution for Nitella, an excitable water plant. that thereby permitted unimpeded This is believed to be a minimum requirement inward diffusion of metabolic intermediates for the excitation of the membrane. from the exterior soup. Various patterns of spontaneous potential The results of the experiments with com- activities comparable to those of modern plexes of thermal proteinoid and lecithin living excitable cells were observed notwith- indicate channels for passive transport of standing any current leak through equipment, ions. The ability to generate potential pulses as proved with the current monitor. These is not specifically a biological phenomenon might be related with expanding and re- since it is simulated in the non-biological arranging of the membrane of the spherules systems that have been studied. in a very small area when there were consider- able potential differences between the inside and outside of the membrane in the course of Acknowledgment the development of the spherule. It was rather easy to elicit the activities by electrical This research has been supported by the stimulation in the stable state in the experi- Robert Sterling Clark Research Foundation mental solution containing 1 mM calcium ions and Grant No. NGR 10-007-008 of the if there were spontaneous activities in the National Aeronautics and Space Administra- standard external solution (Ca 2÷ 0.5 mM). tion. Contribution No. 244 of the Institute Contribution of Ca 2÷ to compartmentation for Molecular and Cellular Evolution. {Brooke and Fox, 1977) may explain effects on electrical activity in vesicles. The finding of excitability in a cell-like References microstructure permits investigation of coup- ling of biochemical and other phenomena in Brook, S. and S.W. Fox, 1977, Compartmentalization ways that cannot be accomplished with other in proteinoid microspheres. BioSystems 9, 1--22. models such as bilayer membranes. Although Camejo, G., G.M. Villegas, F.V. Barnola and R. the proteinoid-lecithin vesicles are to a degree Villegas, 1969, Characterization of two different a departure from the proteinoid microspheres membrane fractions isolated from the first stellar nerves of the squid Dosidicus gigas. Biochim. that have been placed conceptually on a Biophys. Acta 193,247--259. primary evolutionary pathway (Fox and Dose, Cole, K.S. and J.H. Curtis, 1938, Electric impedance 1977), evolution itself occurred in a cell-like of NiteUa during activity. J. Gen. Physiol. 22, 37-- unit composed of both and lipid, 64. Condrea, E. and P. Rosenberg, 1968, Demonstration not in a simple lipid bilayer membrane (cf. of phospholipid splitting as the factor responsible Nachmansohn, 1970). for increased permeability and block of axonal The difficulty of inserting microelectrodes conduction induced by snake venom. II. Squid in the usual size proteinoid microsphere axons. Biochim. Biophys. Acta 150, 271--284. (1--5 •m) has precluded testing the hypo- Findlay, G.P., 1959, Studies of action potentials in thesis that they are capable of electrical the vacuole and cytoplasm of Nitella. Aust. J. Biol, Sci. 12, 412--426. activity. In general, the most results have, Fox, S.W., 1980, The origins of behavior in macro- however, been obtained in these experiments molecules and protoceUs. Comp. Biochem. Physiol. in microsystems rich in apolar amino acids (cf. 67B, 423--436. Lehninger, 1975). No reason for the need of Fox, S.W. and K. Dose, 1977, an efficient phospholipid barrier and bio- and the Origin of , revised edn., (Marcel Dekker, New York). electric activity in a Pr0tocell is perceived. Fox, S.W., R.J. McCauley, P.O'B. Montgomery, Indeed, Kuhn (1976) has pointed out that T. Fukushima, K. Harada and C.R. Windsor, 1969, leakiness in a membrane would have provided, Membrane-like properties in microsystems as- 251

sembled from synthetic protein-like polymer, in: Lehninger, A.L., 1975, Biochemistry, 2nd edn. (Worth Physical PrincJ~ples of Biological Membranes, and Co., New York) p. 1047. F. Snell, J. Wolken, G.J. Iversen and J. Lain (eds.) Mueller, P. and D.O. Rudin, 1969, Biomolecular lipid Gordon and Breach, New York). membranes: techniques of formation, study of Grote, J.R., R.M. Syren and S.W. Fox, 1979, Effect electrical properties, and induction of ionic gating of product from heated amino acids on conduct- phenomena, in: Laboratory Techniques in Mem- ance in lipid bilayer membranes and non-aqueous brane Biophysics, H. Passow and R. Saempfli (eds.) solvents. BioSy.,,~ems 10,287--292. (Springer-Verlag, Berlin) p~ 140. Hill, S.E. and W.SV. Osterhout, 1934, Nature of the Nachmansohn, D., 1970, Proteins in excitable mem- action current in Nitella; II. Special cases. J. Gen. branes. Science 168, 1059--1066. Physiol. 18,377--383. Takahashi, K. and Miyazaki, S., (1971)Development Hsu, L.L., S. Brooke and S.W. Fox, 1971, Conjugation of excitability in embryonic muscle cell membranes of proteinoid microspheres: a model of primordial in certain tunicates. Science 171, 415--418. communication. Curr. Mod. Biol. (now Bio- Takenaka, T., I. Inoue, Y. Ishima and H. Horie, 1971, Systems) 4, 12--25. Excitability of the surface membrane of a proto- Inoue, I., Y. Ishima, H. Horie and T. Takenaka, 1971, plasmic drop produced from protoplasm in Nitella. Properties of an excitable membrane produced on Proc. Jpn. Acad. 47,554--557. the surface of a protoplasmic drop in NiteUa. Proc. Tasaki, I., 1968, Nerve Excitation (Charles C Thomas, Jpn. Acad. 47,549--553. Springfield, IL). Ishima, Y. and K. Waku, 1978, Phospholipid analysis Vegotsky, A., 1972, The place of the origin of life in of the chick ventricles in the early stages of the undergraduate curriculum, in: Molecular development when their excitability changes in Evolution, D.L. Rohlfing and A.I. Oparin (eds.) tetrodotoxin or in sodium substitute media. Comp. (Plenum Press, New York). Biochem. Physiol. 61C, 283--286. Watanabe, A. and Y. Ishima, 1972, The preparation Kennedy, S.J., R.W. Roeske, A.R. Freeman, A.M. and properties of cells in the heart ganglion of Watanabe and A.R. Besch, Jr., 1977, Synthetic Squilla. Exp. Physiol. Biochem. 5, 91--125. form :ion channels in artificial lipid bilayer Yoneda, M. and N. Kamiya, 1969, Osmotic volume membranes. Science 196, 1341--1342. change of cytoplasmic drops isolated in vitro from Kishimoto, U. and M, Tazawa, 1965, Ionic composi- internodal cells of Characeae. Plant Cell Physiol. tion of the cytoplasm of Nitella fexilis. Plant Cell 10,821--826. Physiol. 6,507--518. Yoshida, M., N. Kamo and Y. Kobatake, 1972, Kuhn, H., 1976, Model consideration for the origin Transport phenomena in a model membrane of life, Environmental structure as stimulus for the accompanying a conformational change :membrane evolution of chemical systems. Naturwissen- potential and ion permeability. J. Membr. Biol. 8, schaften 63, 6~--80. 389--402.