Laboratory Techniques in Membrane Biophysics An Introductory Course By W. McD. Armstrong· K. Baumann· P . C. Caldwell T. W. Clarkson· J. Dudel . B. Frankenhaeuser . E. Fromter . G. Gardos B. Z. Ginzburg· J. F. Hoffman· A. Kepes . R. D. Keynes A. Kleinzeller . P. G. Kostyuk . A. Kotyk· A. A. Lev· B. Lindemann P. }rfueller . H. Muth . W. Nonner . E. Oberhausen . H. Passow A. Rothstein· D. O. Rudin' R. Stampfli· T. Teorell· K. J. Ullrich N. A. Walker' W. Wi/brandt Edited by H. Passow and R. StampI Ii With 66 Figures Springer-Verlag Berlin· Heidelberg· New York 1969 ISBN- I3 978-3-540-04592-2 e-ISBN- I3 978-3-642-87259-4 DOL 10.1007/978-3-642-87259-4 All rights reserved. No part of this book may be translated or reproduced in any form without written permission from Springer-Verlag. © by Springer-Verlag Berlin' Heidel- berg 1969. Library of Congress Catalog Card Number 69-19291. The use of general descriptive names, trade marks, etc. in this publication, even if the former are not especially identified, is not to be taken as a sign that such names, as understood by the Trade Marks and Merchandise Marks Act, may accordingly be used freely by anyone. Title No. 1553 Preface The present manual contains a collection of laboratory instructions used during an international training course on membrane biophysics which was held at Homburg in the fall of 1966. The selection of the topics dealt with in the various chapters depended on the scientific interest of the available teachers and on the availability of the necessary equipment in our laboratories. Thus, the material included in this volume does not add up to a systematic course in membrane biophysics. Instead it represents a more fortuitous collection of laboratory problems. In addition, some authors place more emphasis on teaching the more technical aspects of a method whereas others are primarily concerned with the demonstra- tion of a significant biological phenomenon. Nevertheless, in spite of such differences of emphasis and a somewhat haphazard choice of a few methods and phenomena among many others of similar importance, it was felt that the publication of the material is desirable. Since no other laboratory manual exists so far, the present laboratory problems which were tested in actual practice may serve as a useful basis for the shaping of further training courses or for laboratory courses for graduate students in biophysics and physiology. Our thanks are due to the authors and the publisher who were patient and kind enough to cooperate with the editors during the long period between the end of the course and the appearance of the book. We are particularly grateful to a number of authors who were willing to expand their material beyond the limits of their original manuscripts. Homburg, February 1969 H. PASSOW' R. STAMPFLI Contents GINZBURG, B. Z., and N. A. WALKER: Measurement of Hydraulic Conductivity and Reflexion Coefficients of a Plant Cell Mem- brane by means of Transcellular Osmosis .................. 1 GARDOS, G., J. F. HOFFMAN, and H. PASSOW: Flux Measurements in Erythrocytes ........................................ 9 PASSOW, H.: Ion Permeability of Erythrocyte Ghosts .......... 21 WILBRANDT, W.: Countertransport in Human Red Blood Cells .. 28 ARMSTRONG, W. McD., and A. ROTHSTEIN: Kinetics of Cation Transport in Yeast ..................................... 34 KEPES, A.: Bacterial Permeases ............................. 49 CALDWELL, P. C., and R. D. KEYNES: The Exchange of 22Na be- tween Frog Sartorius Muscle and the Bathing Medium ...... 63 KLEINZELLER, A., P. G. KOSTYUK, A. KOTYK, and A. A. LEV: Determination of Intracellular Ionic Concentrations and Acti- vities ................................................. 69 CLARKSON, T. W., and B. LINDEMANN: Experiments on Na Trans- port of Frog Skin Epithelium ............................ 85 ULLRICH, K. J., E. FROMTER, and K. BAUMANN: Micropuncture and Microanalysis in Kidney Physiology . . . . . . . . . . . . . . . . . . . . . . . 106 TEORELL, T.: Oscillatory Phenomena in a Porous, Fixed Charge Membrane ............................................. 130 MUELLER, P., and D. O. RUDIN: Bimolecular Lipid Membranes: Techniques of Formation, Study of Electrical Properties, and Induction of Ionic Gating Phenomena. . . . . . . . . . . . . . . . . . . .. 141 STAMPFLI, R.: Dissection of Single Nerve Fibres and Measurement of Membrane Potential Changes of Ranvier Nodes by means of the D~)Uble Air Gap Method . . . . . . . . . . . . . . . . . . . . . . . . . . .. 157 FRANKENHAEUSER, B.: Ionic Currents in the Myelinated Nerve Fibre ................................................. 167 NONNER, W., and R. STAMPFLI: A New Voltage Clamp Method.. 171 DUDEL, J.: Electrical Activity of Synapses. The Crayfish Neuro- muscular Junction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 Appendix OBERHAUSEN, E., and H. MUTH: Double Tracer Techniques. . . . . 181 Author Index ............................................. 195 List of Authors ARMSTRONG, W. McD.: Department of Physiology, Indiana University, Indianapolis (USA) BAUMANN, K.: Physiologisches Institut der Freien UniversWit, Berlin (Germany) CALDWELL, P. C.: Department of Zoology, University of Bristol (Great Britain) CLARKSON, T. W.: Department of Radiation Biology and Biophysics, University of Rochester, Rochester (USA) DUDEL, J.: II. Physiologisches Institut der Universitat Heidelberg, Heidelberg (Germany) FRANKENHAEUSER, B.: Nobel Institute for Neurophysiology, Karolinska Institutet, Stockholm (Sweden) FROMTER, E.: Physiologisches Institut der Freien Universitat, Berlin (Germany) GARDOS, G.: Department of Cell Metabolism, Research Institute of the National Blood Center, Budapest (Hungary) GINZBURG, B. Z.: Department of Botany, Hebrew University, Jerusalem (Israel) HOFFMAN, J. F.: Department of Physiology, Yale University, New Haven (USA) KEPES, A.: College de France, Laboratoire de Biologie Moleculaire, Paris (France) KEYNES, R. D.: Institute of Animal Physiology, Agricultural Research Council, Babraham, Cambridge (Great Britain) KLEIN ZELLER, A.: Department of Physiology, University of Pennsyl- vania, Philadelphia (USA) KOSTYUK, P. G.: Institute of Physiology, Kiev (USSR) KOTYK, A.: Institute of Microbiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) LEv, A. A.: Institute of Cytology, Academy of Sciences of USSR, Lenin- grad (USSR) LINDEMANN, B.: II. Physiologisches Institut, Universitat des Saar- landes, Homburg a. d. Saar (Germany) MUELLER, P.: Eastern Pennsylvania Psychiatric Institute, Philadelphia (USA) MUTH, H.: Institut fUr Biophysik, Universitat des Saarlandes, Homburg a. d. Saar (Germany) VIII List of Authors NONNER, W.: I. Physiologisches Institut, Universitat des Saarlandes, Homburg a. d. Saar (Germany) OBERHAUSEN, E.: Institut fUr Biophysik, Universitat des Saarlandes, Homburg a. d. Saar (Germany) PASSOW, H.: II. Physiologisches Institut, Universitat des Saarlandes, Homburg a. d. Saar (Germany) ROTHSTEIN, A.: Department of Radiation Biology and Biophysics, University of Rochester, Rochester (USA) RUDIN, D. 0.: Department of Basic Research, Eastern Pennsylvania Psychiatric Institute, Philadelphia, Pennsylvania (USA) STAMPFLI, R.: I. Physiologisches Institut, Universitat des Saarlandes, Homburg a. d. Saar (Germany) TEORELL, T.: Department of Physiology, University of Uppsala, Uppsala (Sweden) ULLRICH, K. J.: Physiologisches Institut der Freien Universitat, Berlin (Germany) WALKER, N. A.: School of Biological Sciences, University of Sydney, Sydney (Australia) WILBRANDT, W.: Pharmakologisches Institut, Universitat Bern, Bern (Switzerland) Measurement of Hydraulic Conductivity and ReHexion Coefficients of a Plant Cell Membrane by means of Transcellular Osmosis By B. Z. GINZBURG and N. A. WALKER I. Introduction The aim of this experiment is to measure the hydraulic conductivity (Lp) of the "membrane" of the Nitella internodal cell, and its reflexion coefficient (0') for a rapidly penetrating solute. The coefficients Lp and 0' appear in the phenomenological equations for the movement of water and solute across a homogeneous membrane separating solutions of the same nonelectrolyte. It is possible (KEDEM and KATCHALSKY, 1958) to write the dissipation function as: (1) where I v is the flow of volume across the membrane, I D is the relative velocity of solute to solvent in the membrane, L1 P is the hydrostatic pressure difference across the membrane, L1II is the osmotic pressure difference across the membrane. Making the assumption that flows are linear functions of forces, Iv = Lp·L1P + LpD·L1II (2) I D = LDp ·L1P + LD·L1II. (3) And Eq. (2) can be rewritten Iv = Lp·L1P - O'·Lp·L1II (4) where 0' is defined as (-LPD!Lp). At L1II = 0, Lp clearly measures the relation between hydrostatic pressure difference and volume flow. It is a kinetic parameter (DENBIGH 1951) and can only be obtained by a measurement of flow and driving force. At Iv = 0,0' measures the "relative effectiveness" of hydrostatic and osmotic pressure differences: it is a ratio of two similar kinetic parameters - i.e. a thermodynamic parameter. It can be measured by a nul method (at Iv = 0). Its meaning can be grasped from this method 1 Membrane Biophysics 2 B. Z. GINZBURG and N. A. WALKER: of measurement, and from the relation (f = 1 - ~ , where V8 is the velo- v. . city of the solute in the membrane and Vw the velocity of water. Normally then values of (f will range from 1, for impermeant solutes (vs = 0) to 0, for solute permeating as readily as water (vs = vw); though negative values of (f are possible (KATCHALSKY and CURRAN, 1965). In this experiment Lp is measured by volume flow and (f by the nul method. Transcellular osmosis was first observed by OSTERHOUT, and it has been fully treated by KAMIYA and TAZAwA (1956) and by DAINTY and HOPE (1959). The treatment below is somewhat simplified. n Fig. 1. A turgid plant cell is shown, sealed between two compartments x, n. Com- partment x is open to the atmosphere, while n is also open via the capillary. Changes in volume of the contents of compartment n may be measured by the movement of the meniscus in the capillary. Meaning of symbols: lID osmotic pressure of solution in compartment -x. II."n osmotic pressure of solution in cell, at end in compartment x, n. P hydrostatic pressure of cell contents. A.,.n area of cell surface in compart- ment x, n. V."n volume of cell in compartment x, n. R."fI rate of volume flow out ofl into cell in compartment x, n. Rm net rate of change of volume in compartment n A turgid plant cell (Fig. 1) is sealed between two compartments (here labelled x and n) so that the areas and volumes of the cell exposed in the two compartments are Az, Vz ; An, Vn respectively. The hydro- static pressure in the cell interior is P, while the osmotic pressures of the solutes in the cell interior are lIz, lIn respectively. The exposed cell sur- faces have values of Lp denoted by Lpz and Lpn respectively. We imagine an experiment in which initially there is water on both sides, and no net water movement, IIo = 0, lIz = lIn: at zero time the water on side x is replaced by sucrose solution of osmotic pressure IIo, and the rate of volume change Rm in compartment n is measured. It will be assumed that for sucrose (f = 1. At end x of the cell water will move from cell interior to solution at a rate: Rz = Lpz Az(IIo - lIz + P) (5) Measurement of Hydraulic Conductivity and Reflexion Coefficients 3 and since the exit of water will reduce P below its initial value there will be a flow of water into end n of the cell from compartment n: Rn = Lpn An(IIn - P) . (6) Water will move along the cell interior from n to x, sweeping internal solutes towards end x, and for this reason we write the osmotic pressures of internal solutes as IIn and IIx. If Rn =1= Rx the cell will change in volume at a rate (Rn - Rx). Now in this experiment one measures the rate of net volume change (Rm) in compartment n, and this rate is composed of Rn and the change in volume of that part of the cell in compartment n, viz: Rm = Rn - (Rn - Rx)' ( v" ) (7) v" + V:. and if the cell is cylindrical V" V = A n A ,so V .. + '" An + '" Rm = Rn - (Rn - Rx)' ( A" ). (8) A .. + A., Inserting values from Eqs. (5) and (6): A",A" J1 A",A.. II II Rm = A A Lpx ° - A A [Lpx( x - P) - Lpn( n - P)] , (9) ., + .. '" + " Calculation from actual rates of flow and cell dimensions shows that during the first min of transcellular osmosis the volume of water flowing through the cell is about 2% of its volume, so that if we restrict measure- ments to this period we can assume IIx " IIn , and if II~ = IIx = IIn then A",An II A.,A n II Rm " Lpx ° - ----- [(Lpx - Lpn) ( j - P)], (10) A", + A" A", + An Further simplification, which is not strictly justified, may be introduced for the purposes of this one-day experiment: we assume Lpx = Lpn and obtain: ,A",An J1 Rm=---Lp o· (11) , A", + An This will give the rate at any moment after zero time; whether due to cell shrinkage or to water flow from one compartment to the other, until IIx is significantly different from IIn, From this equation, having measured Rm, Ax, An and IIo, we can obtain Lp, If the solute used (say e) has a value of (J not equal to 1, a similar argument will show that , A",A" C Rm--: Lp'(Je'RT e (12) A.,+A" where Ce is the concentration of the solute at side x. If Rm, Ax, An, C, and Lp are known, (Je may be calculated. 1* 4 B. Z. GINZBURG and N. A. WALKER: Alternatively if we have sucrose at concentration Cs on side nand find a concentration of solute Ce at side x for which Rm = 0 initially, we can show (again assuming Lpz = Lpn = Lp and as = 1) Rm=O=Rn-(Rz-Rn).( v .. ) [Eq.(7)] v .. + v" A"A .. = A A RTLp(ae Ce - Cs) "'+ .. whence ae = CslCe • (13) ll. Apparatus and Solutions 1 Transcellular osmosis apparatus (Fig. 2). 1 Large stirred waterbath to accommodate apparatus and twelve 100 ml bottles of solution. 12 100 ml bottles. 1 Stopwatch. 1 Mirror scale (mm). 1 Microscope with 10 x objective and 10 x micrometer exepiece. 320 ml syringes with flexible plastic tubing attached. "Kleenex" . Solutions of sucrose: 0.1, 0.2, 0.3, 0.4, 0.6, 0.8 molal and { isopropanol: 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 molal or ethanol: same concentrations. "Vaseline". c '~ a b d --------;>4--J--.......:::::--....r-----------==r--' perspex rubber Fig. 2. Practical transcellular osmosis apparatus. a, b two halves of apparatus containing solutions. c capillary, of 0.2 mm i.d. if mirror scale is used or 1.0 mm if travelling microscope is used. d rubber bung with nylon screw insert for adjusting zero position of capillary. e internodal cell of Nitella or ehara. f split bung of "perspex" or rubber, with groove just larger than cell