ION TRANSPORT ACROSS MEMBRANES Incorporating Papers Presented at a Symposium Held at the College of Physicians & Surgeons Columbia University October, 1953 HANS T. CLARKE, Editor DAVID NACHMANSOHN, Associate Editor IQ54 ACADEMIC PRESS INC., Publishers NEW YORK, N. Y. Copyright, 1954, by ACADEMIC PRESS INC. 125 East 23rd Street New York 10, N. Y. All Rights Reserved NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS. Library of Congress Catalog Card Number: 5^-10754 PRINTED IN THE UNITED STATES OF AMERICA Dedicated to the memory of JACQUES LOEB Contributors J. J. BLUM, U. S. Naval Medical Research Institute, Bethesda, Maryland. SHELDON DRAY, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland. P. DEBYE, Department of Chemistry, Cornell University, Ithaca, New York. JOHN T. EDSALL, University Laboratory of Physical Chemistry Related to Medicine and Public Health, Harvard University, Boston, Massachusetts. HENRY EYRING, Department of Chemistry, University of Utah, Salt Lake City, Utah. S. L. FRIESS, U. S. Naval Medical Research Institute, Bethesda, Maryland. EUGENE GRIM, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland. FRANK R. N. GURD, University Laboratory of Physical Chemistry Related to Medi- cine and Public Health, Harvard University, Boston, Massachusetts. TERRELL L. HILL, U. S. Naval Medical Research Institute, Bethesda, Maryland. J. F. HOFFMAN, Department of Biology, Princeton University, Princeton, New Jersey. A. F. HUXLEY, Physiological Laboratory, University of Cambridge, England. JOHN G. KIRKWOOD, Sterling Chemistry Laboratory, Yale University, New Haven, Connecticut. M. F. MORALES, U. S. Naval Medical Research Institute, Bethesda, Maryland. GILBERT H. MUDGE, Department of Medicine, College of Physicians & Surgeons, Columbia University, New York. DAVID NACHMANSOHN, Department of Neurology, College of Physicians & Sur- geons, Columbia University, New York. REX NEIHOF, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland. W. J. V. OSTERHOUT, Rockefeller Institute for Medical Research, New York. RANSOM B. PARLIN, Department of Chemistry, University of Utah, Salt Lake City, Utah. A. K. PARPART, Department of Biology, Princeton University, Princeton, New Jer- sey. GEORGE SCATCHARD, Department of Chemistry, Massachusetts Institute of Technol- ogy, Cambridge, Massachusetts. KARL SOLLNER, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland. HANS H. USSING, Zoophysiological Laboratory, University of Copenhagen, Copen- hagen, Denmark. IRWIN B. WILSON, Department of Neurology, College of Physicians <fc Surgeons, Columbia University, New York. Preface The collection of papers in this volume is based upon a Symposium on The Role of Proteins in Ion Transport across Membranes, sponsored by the National Science Foundation and held at the College of Physi- cians & Surgeons, Columbia University, on October 2 and 3, 1953. The problem of ion transport across cell membranes has long at- tracted the attention of biologists in various fields. It is of prime impor- tance for an understanding of the mechanism of bioelectric currents and the conduction of the nerve impulses; it has also played an important role in other studies such as the functions of the kidney and of red blood cells. The recent advances of biochemistry, especially in protein and enzyme chemistry, and the availability of isotopic tracers have made possible rapid progress in the area here under consideration. The prob- lem is reaching a stage where the molecular forces involved have be- come a subject of more than speculative interest. Now that experimental biology is approaching the molecular level, active cooperation between biologists, physical chemists, and protein chemists should lead to significant developments in basic theory. His- tory records many such cases of cross-fertilization of ideas. For example, the membrane theory which is still the basis of all modern concepts of nerve impulse conduction was built upon the fundamental work of Ostwald, Nernst, Planck, and other physical chemists and physicists. The plan of this Symposium was not, as is the usual procedure, to bring together investigators working in similar areas for the discussion of limited, specific problems. It was planned, rather, to bring together a small group of physicochemically minded biologists working on ion transport and the role of proteins in this process, with a small group of distinguished physical chemists and protein chemists who are or may become interested in this borderline problem. The study of ion trans- port across membranes and the interaction between ions and proteins transcends the field of biology and has always been a domain in which physical chemists were interested. At the suggestion of Dean Willard C. Rappleye a committee was or- ganized, con^sting of Robert F. Loeb, H. Houston Merritt, and David Rittenberg, with David Nachmansohn and Hans T. Clarke as organiz- ing secretary and chairman, respectively. This committee decided to limit the number of participants to about fifty in order to ensure an ix X PREFACE atmosphere favorable to the exchange of ideas, by informal discussion and personal contacts, between the two groups. As the Symposium was held during only two days, the program was limited to eight papers, given by Drs. Ussing, Wilson, Eyring, Kirkwood, Scatchard, Debye, Sollner and Edsall. Drs. Rittenberg, Louis P. Hammett, I. I. Rabi, and Raymond M. Fuoss served as the chairman at each of the four sessions. The Symposium was held on the eve of the 25th anniversary of the Columbia-Presbyterian Medical Center. As stated by Dean Rappleye in his opening address, Columbia University is proud to have been host to so many distinguished scientists. The Symposium was an appropriate expression of the spirit of research prevailing at the Center. The present volume contains the substance of the various addresses, together with six invited articles on allied topics, contributed by various investigators who could not be represented on the program. The organ- izing committee expresses its gratitude to all the authors of the chapters here assembled. The book is dedicated to the memory of Jacques Loeb, pioneer in the application of the principles of physical chemistry to biological problems, whose intensive studies of ion transport across cell membranes are still bearing fruit, as pointed out by Dr. Osterhout, his associate through many years. # Note on the Work of Jacques Loeb W. J. V. OSTERHOUT It is a pleasure to respond to the suggestion of the Editorial Com- mittee to say something about the pioneer work of Jacques Loeba in applying physical chemistry to biology. Loeb was one of the first to see the importance to biology of the dissociation theory of Arrhenius, and he applied it with conspicuous success. A few examples will suffice to show the nature of his work. Loeb's point of view may be illustrated by his experiments with eggs of the fish Fundulus, which develop in distilled water as well as in sea water. In a solution of sodium chloride isotonic with sea water, the eggs soon die. This also happens in an isotonic solution of zinc sulfate, but when these two solutions are mixed in suitable proportions normal embryos are produced. From this he concluded that sodium and zinc ions antagonize each other. He found that, in general, the toxic effects of univalent cations could be counteracted by the addition of small amounts of bivalent cations and by still smaller amounts of tervalent cations. It was a novel and important idea that the physio- logical effects of ions could be predicted by knowing their electrical charges, and it gave a great stimulus to the application of physical chemistry to biology. Loeb suggested that these effects were due to the action of ions on the permeability of the membrane surrounding the egg. The antagonistic action is quite different as soon as the embryo escapes from the covering. He thus directed attention to the importance of the study of permea- bility. A deep interest in the changes which occur in the egg after union with the sperm led him to attempt to analyze and control these by using the techniques of physical chemistry. His first task was to induce development in the absence of sperm (artificial parthenogenesis). This he soon accomplished by varying osmotic pressure, pH, surface tension, and other factors. These experiments aroused great interest and stimulated extensive research which still continues. Loeb concluded a For a fuller account, see the Memorial Volume, J. Gen. Physiol. 8, (1925- 1928). 1 2 W. J. V. OSTERHOUT that the first step is a change at the surface of the egg whereby the normal structure begins to break down (cytolysis). If this continues too long, death occurs, but, if stopped at the right time, it is followed by cell division and development. In the course of Loeb's experiments it became clear that in many cases a knowledge of the properties of colloids is important. The whole subject was at that time in a state of confusion and he sought to clarify it by using the methods of physical chemistry. He therefore began experiments on the colloidal properties of gelatin. An important aspect of this work was his study of the effects of pH. McBainb speaks of the invaluable services of Loeb in pointing out the predominant role of pH on amphoteric basic and acidic colloids. In this field his scientific imagination and insight had a clarifying effect, as is evident in his well-known book on proteins and the theory of colloidal behavior. These examples may suffice to illustrate the character of his work, in all of which he sought, with notable success, to apply the methods of the exact sciences. In many cases Loeb was able to replace obscure biological ideas by clear-cut, mechanistic conceptions and thereby opened up new fields of research of fundamental importance. His example was inspiring and his influence was great. For this we "owe him a debt of gratitude. b J. W. McBain, "Colloid Science," p. 215. D. C. Heath & Co., Boston, 1950. Ion Transport Across Biological Membranes HANS H. USSING DIFFUSION THROUGH BIOLOGICAL MEMBRANES At the outset it might be worth while to define briefly what we ex- perimental biologists mean by a biological membrane. I think most of us can agree upon a formulation like this: Whenever we meet, in a liv- ing organism or part thereof, a boundary that presents a diffusion re- sistance to solutes higher than that of the phases separated by the boundary, it is called a membrane. The membrane is often, but not always, anatomically discernible. The objects we study under the name of biological membranes are extremely diverse. Thus.we have membranes on the multicellular level like the gastric mucosa or the frog skin epithelium. Then there are the cell membranes like, for instance, the membranes of the nerve fiber. Finally, the work of the last few years tends to show that even membranes on the subcellular level are highly important. Notably, the surface of the mitochondria shows membrane-like properties, such as the ability to maintain, and under certain circumstances to create, within the mitochondrium concentrations of a number of substances which differ from those of the surroundings. As an example we may take a table from a recent paper by Bartley and Davies (1952) (Table I). It is seen that the Na ion undergoes a conspicuous concentration in the mitochondria as compared to the surrounding medium. At first sight there seems very little in common between the nerve fiber membrane and the skin of a frog, or between the tip of a plant root and the gill of a crab. Nevertheless, these different structures show many similarities in the way they handle inorganic ions. Formerly it was generally assumed that the similarities stemmed from the fact that the element determining the behavior of ions was in all cases a cell membrane, or possibly a number of cell membranes placed in series. This may still be true. With the increasing knowledge concerning the ability of mitochondria to concentrate and exclude certain ion species, one may, however, speculate as to whether some of the phenomena involving the transfer of ions across cell boundaries are actually the result of the activity of mitochondria. Discussing this problem at the 3 4 HANS H. USSING TABLE I INTERNAL-TO-EXTERNAL CONCENTRATION RATIOS FOUND FOR METABOLIZING SHEEP-KIDNEY CORTEX IN MITOCHONDRIA AT 20°C Concentration in medium after Substance Ratio separation (M) H+ 2.5 1.6 X 10~7 Na+ 26 5.9 X 10~4 K+ 2.0 9.0 X 10"2 Mg++ 4.5 2.0 X 10~4 Orthophosphate 6.0 1.9 X 10~4 Adenosine polyphosphates 0.7 4.3 X 10"4 Pyruvate 0.8 3.5 X 10~3 Fumarate 8.0 2.8 X 10"4 Oxaloacetate 0.1 1.8 X 10"3 a-Ketoglutarate 1.0 6.3 X 10-2 Citrate 0.8 1.4 X 10-2 Water content of metabolizing mitochondria = 80%. Water content of nonmetabolizing mitochondria = 91%. (After Bartley and Davies, 1952) conference on active transport at Bangor this summer, Davies pointed out that in secreting kidney cells the mitochondria are arranged longi- tudinally from the cell wall bordering the lumen of the tubules. During the discussion following Dr. Davies' paper, Professor Wigglesworth then mentioned that, in the salt-reabsorbing part of the Malpighian tubule in insects, the giant mitochondria actually pierce the luminal cell boundary, waving with one end in the pre-urine while having the base well within the cell body. Alternatively one may imagine that the surface of the mitochondrium and that of the cell have in common certain properties which enable them to handle ions in a characteristic manner, and resist their free diffusion. This brings us to the point of asking: What is the nature of the cell membrane? Or, to be more cautious, how do the biologists picture the cell membrane? Without having made use of Mr. Gallup's methods, it is probably fair to assume that the concept most widely accepted is that of the lipoid-pore membrane, as proposed by Collander (1937). It implies that the less polar substances penetrate by dissolving in the membrane phase, whereas the polar substances, notably the inorganic ions, penetrate only in so far as the ionic diameter is smaller than the