. Juli-l. IIIEOIEI Kahlil-r“, .h . in . .3 . .7 li . - ABSTRACT MEMBRANE-BOUND ATP-ASE ACTIVITY, INTRACELLULAR pH AND MEMBRANE POTENTIAL OF MYCOPLASMAS PART I: LIPID AND TEMPERATURE DEPENDENCE OF MEMBRANE- BOUND ATP-ASE ACTIVITY OF ACHOLEPLASMA LAIDLAWII PART II: INTRACELLULAR pH AND MEMBRANE POTENTIAL OF THERMOPLASMA ACIDOPHILA By Jean-Cheui Hsung The activity of membrane-bound enzymes may depend upon the prey sence of lipids, and protein-lipid interaction may play an important role in enzyme function. Thus, the effects of fatty acyl chain length on the membrane physical states and membrane—bound ATPase activity of Achole- plasma ladlawii were investigated. Three membrane preparations from cells were obtained by supple- menting the growth media with either arachidic (C20:0), oleic (Cl8zl) or lauric (C12:0) acid. The cells grown with arachidic or oleic acid supplementation yielded membrane lipids enriched with arachidoyl or oleoyl groups respectively. Those supplemented with lauric acid yielded membrane lipids enriched with myristoyl and palmitoyl groups. The membrane-bound ATPase activity of membrane preparations were measured in the temperature range from l5 0C to 45 0C. Arrhenius plots of the ATPase activity of membranes enriched with either arachidoyl Ma's} . “a; \ «I vb OJ any .1 .r: a... HUIC Cl V fi‘| d‘er.fi Jean-Cheui Hsung or myristoyl-palmitoyl groups exhibited a pronounced discontinuity in slope around 26 0C - 30 0C. 0n the other hand, the Arrhenius plots of the ATPase activity of membranes enriched with oleoyl groups showed no breaking point in the temperature range from l5 0C to 45 0C. The membrane lipid fluidity was measured with a nitroxyl stearic acid spin label. At the growth temperature (37 0C) the membrane lipid fluidity of all three kinds of enriched membranes was virtually identi- cal. For membranes enriched with saturated acyl chains, a plot of the EPR anisotropy parameter 2Tu , versus the reciprocal of temperature showed a blphaSlC profile with a discontinuity in slope around 26 OC - 30 0C. For oleoyl group enriched membranes this plot yields only a straight line, without a breaking point in the temperature range from 15 0C — 45 0C. Thus, the Arrhenius plots of the ATPase activity and 2T" correlated well. This indicates that the physical state of membrane lipids does play a role in determining the activation energy of the membrane bound ATPase. This enzyme is probably localized in the more fluid regions of the membrane where the fatty acid spin label is also preferentially located. Thermoplasma acidophila, a mycoplasma-like organism, was grown at pH 2, at 56 0C. This is a prokaryotic micro-organism devoid of a cell wall bounded by a single membrane without any intracellular membrane bound organelles. The intracellular pH was measured on the basis of the distribution of a radioactive weak organic acid, 5,5—dimethyl-2,4- oxazolidine-dione, "across" the plasma membrane. It was fOund that the Er “ L"; -:_".’._. "1 I11“ v» ir .. I» l‘! n‘- L l ‘i P's, ~LF- \\ Jean-Cheui Hsung intracellular pH lies in the neutral range from pH 6.4 to 6.9. This agrees with a direct pH measurement of the cavitation fluid and indirect conjecture from enzyme profiles. The cell can maintain a huge pH gradient when subjected to heat or metabolic inhibitors. The menbrene potential of the cell was measured by the distribution of a radioactive anion SCN' which dissociates almost completely at pH 2. The membrane potential is l20 mV positive inside, and is independent of the presence of metabolic inhibitors. The membrane potential decreased linearly with the external pH. The membrane poten- tial virtually diminished when the external pH was raised to 6. The surface charge density and the zeta-potential was estimated by microscopic electrophoresis. The cells moved towards the positive elec- trode. The mobility remained constant from pH 2-5, and increased for ij> 6. The mobility decreased dramatically with increased external Ca++ concentration at pH 6, and only slightly depended on Ca++ ion concentration at pH 2. At pH 2 and an ionic strength similar to that of the growth medium, the zeta-potential was about 8 mV, negative rela- tive to the bulk medium; the surface charge density was -l360 esu/cm2 which corresponds to one elementary charge per 3500 A2. The compatibility of the observed data with the chemiosmotic theory of energy coupling is discussed. MEMBRANE-BOUND ATP-ASE ACTIVITY, INTRACELLULAR pH AND MEMBRANE POTENTIAL OF MYCOPLASMAS PART ONE: LIPID AND TEMPERATURE DEPENDENCE OF MEMBRANE-BOUND ATP—ASE ACTIVITY OF ACHOLEPLASMA LAIDLANII PART TWO: INTRACELLULAR pH AND MEMBRANE POTENTIAL OF THERMOPLASMA ACIDOPHILA By Jean-Cheui Hsung A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biophysics 1977 DEDICATION To my parents, brother and sisters and Hwai-chyi ii FL. AH... ACKNOWLEDGMENT The author wishes to express his sincere appreciation to Dr. Alfred Haug for his guidance, encouragement and criticism , as both major professor and friend, during the course of this study. Special thanks are extended to Drs. H.Ti. Tien, Michael Jost, Estelle McGroarty and Ashraf El-Bayoumi for serving as guidance committee members. Very special thanks go to Hwai-chyi for her love, faith and encouragement during my graduate years. The author also wish to thank Dr. Anton Lang for enabling him to carry out this research in the MSU/ERDA Plant Research Laboratory. This work was supported by United States ERDA contract no. EY—76-C- 02-l338 *ooo TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . vii LIST OF FIGURES . . . . . . . . . . . . . . . . viii ORGANIZATION OF DISSERTATION . . . . . . . . . . . . xi CHAPTER I. GENERAL INTRODUCTION . . . . . . . . . . 1 PART ONE. LIPID AND TEMPERATURE DEPENDENCE OF MEMBRANE BOUND ATP-ASE ACTIVITY OF AEHQLEPLASMAHLAIDANII . . . . 17 CHAPTER II, LIPID AND TEMPERATURE DEPENDENCE OF MEMBRANE-BOUND ATP-ASE ACTIVITY OF ACHOLEPLASMA LAIDANII . . . . 18 Introduction . . . . . . . . . . . . . 18 Material and Methods . . . . . . . . . . 20 Organism and Growth . . . . . . . . . . 20 Plasma Membrane Preparation . . . . . . . 21 ATPase Assay . . . . . . . . . . . 22 Fatty Acyl Analysis . . . . . . . . . 23 Solubilization of Membranes . . . . . . . 23 EPR Spectroscopy . . . . . . . . . . . 23 Results . . . . . . . . . . . . . . 26 Fatty acyl Composition . . . . . . . . . 26 ATPase Activity . . . . . . . . 26 Membrane Solubilization Studies . . . . . . 30 Membrane Fluidity as Measured by the Spin Labelling Method . . . . . . . 31 Discussion . . . . . . . . . . . . . . 36 iv PART THO. CHAPTER III. CHAPTER IV. Page INTRACELLULAR pH AND MEMBRANE POTENTIAL OF TEEBNQREASM Afilflfllfl - - - - . . . 40 INTRACELLULAR pH OF THERMOPLASMA ACIDOPHILA. . 4T Introduction . . . . . . . . . . . 41 Material and Methods . . . . . . . . 44 Organism and Growth . . 44 Procedure for pH Measurement by the Method of DMO Distribution . . . 44 Principle of Using DMO Distribution to Measure Intracellular pH . . . . . . 49 Direct pH Measurement . . . . . . . 53 Malate Dehydrogenase Assay . . . . . . 53 Results and Discussions . . . . . . . 54 Intracellular pH Measured by the Distribution of DMO . . 54 Validity of Using DMO Distribution as a Method to Measure Intracellular pH . . 54 Other Evidence Concerning the Intracellular pH . . 62 Influence of Temperature and External pH on Intracellular pH . . . . . 67 How Is the Hydrogen Ion Concentration Gradient Maintained? . . . . . . . . 67 MEMBRANE POTENTIAL AND SURFACE CHARGE OF THERMOPLASMA ACIDOPHILA . . . . . . . 71 Introduction . . . . . . . . . . . 71 Material and Methods . . . . . . . . . 73 Growth of Cells and Harvest . . 73 Procedure of Measuring Membrane Potential by the Distribution of SCN. . . . . . 73 Microsc0pic Electrophoresis . . . . . . 76 Results . . . . . . . . . . . . . 78 Membrane Potential Calculated by the Distribution of SCN . . . . . . . . 78 Page Validity of Using SCN- Distribution Method to Measure Membrane Potential . . . . . 78 Influence of Metabolic Inhibitors on Membrane Potential . . . . . . . . . 81 Electrophoretic Mobility . . . . . . . . 86 Discussion . . . . . . . . . . . . . 94 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . lOl vi LIST OF TABLES Table Page l. Fatty acyl composition of total membrane lipids of Acholeplasma laidlawii. Cells were grown in lipid ore-extracted tryptose medium supplemented with fatty acid depleted bovine serum albumin and the fatty acid as indicated . . . . . . . . . . 27 2. Determination of the intracellular pH in Thermoplasma acidophila cells by measuring the distribution of radioactive DMO across the plasma membrane . . . . 55 3. Determination of the membrane potential v in Thermoplasma acidophila cells ]by measuring the distribution of radioactive S CN across the plasma membrane . . . . . . . . . 79 4. The electrophoretic mobility u, zeta-potential E , and surface charge density a of Thermoplasma acidophila at various pH, ionic strength, and divalent ion concentration . . . . . . . . . 89 vii LIST OF FIGURES Figure Page I. The fluid mosaic model of biological membrane; schematic three-dimensional and cross-sectional views. The solid bodies with stippled surfaces represent the globular integral proteins, which at long range are randomly distributed in the plane of the membrane. At short range, some may form specific aggregates. Lipids form a matrix . . . . . . . . . . . . . . . . . 5 Chemiosmotic hypothesis: extrusion of protons by the respiratory chain, generation of ADH and AW, and the poising of ATPase by the proton-motive force . . . . . ll The molecular structure of the Spin labels 2-(3-carboxy- propyl)—4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, 5- ~nitroxyl stearate, abbrev1ated as SNS. 2- (l0-carboxydecyl)- 2- ~hexyl- 4, 4- -dimethyl- -3— —oxazolidinyloxyl, lZ-nitroxyl stearate, abbreviated as lZNS . . . . . Arrhenius plot or ATPase activity in membranes isolated from Acholeplasma laidlawii cells grown in a lipid free medium supplemented (6 mg/l) with lauric acid (-------), oleic acid (-o-o-o-), or arachidic acid (~A—A-A-). Arrhenius plot (-A-A-A-) of ATPase activity of solubilized (0.1% Triton X-lOO; l mg protein/ml; lzl, v/v) membrane enriched with arachidoyl groups . . . . . . . . . . . . 29 Typical EPR spectrum of the Spin label SNS incorporated in vitro into Acholeplasma laidlawii membranes enriched with arachidoyl groups. The spectrum was taken from a sample at 25 °C. The magnetic field strength increase from left to right. 2T" is the hyperfine splitting parameter. The vertical arrows indicates signals from unincorporated spin label . . . . . . . . . . . . . . . . . 33 Temperature dependence of the EPR hyperfine splitting parameter 2Tn as defined in Figure 5. The spin label SNS was incorporated into membranes isolated from Acholeplasma laidlawii cells grown in a medium supple- mented'with different fatty acids as explained in Material and Methods. The final concentration of the spin label solution was 0.1-0.2 umole/ml with 10 mg/ml membrane protein . . . . . . . . . . . . . . . . . 3S viii P' _ r1911 O7 ‘10. Figure 7. 10. 11. 12. 13. 14. 15. 16. An electron micrograph from thin sections of Thermoplasma acidophila cells . . . . . . . . . . Measurement of intracellular pH by the distribution of a radioactive weak organic acid C-labelled 5,5-dimethyl- 2,4-oxazolidine-dione (DMD) . . . . . . An experimental scheme of the procedure for measuring intracellular pH by the distribution of DMO . . . The concentration of l4C-DMO inside the cell and in the extracellular pellet space were calculated, and their ratio was related to the incubation time The effect of washing on the accumulation of radioactive 0M0, both supernatant and pellet counts show linear decrease in a semi-109ar1thm1c plot . . . . . The ratio of the intracellular 0M0 concentration to the extracellular 0M0 concentration Effect of pH on cell lysis. o opH change in an aqueous cell suspension (6 mg/ml) upon dropwise addition of 0.04N NaOH. X X Release of protein observed when an aqueous suspension of cells (6 mg/ml) is diluted ten fold by buffers of varying pH. All buffers used were 1 M. Buffer at pH 9,10 and ll were glycine-NaOH, at pH 8 and 4, citrate- NaOH; at pH 5,6, and 7 phosphate- -Na0H . . . . . . . The pH profile of cytoplasmic malate dehydrogenase from Thermoplasma acidophila, assayed in four different buffers at 56 5C. The enzymatic activity was measured by reduction of oxaloacetate and monitoring the absorbance decrease of NADH at 340 nm . . . The effect of external pH on the intracellular pH of Thermoplasma acido hila. For pH2, the suspension medium was composed of O. BZN KCl, 4.2 mM M9504 7H20, 1.7 mM CaCl 2H 20 and 0.01 M glycine buffer. For pH 4 and 6, the gTycine buffer was replaced by citrate buffer . . . . . . . An experimental scneme of the principle and procedure for measuring the membrane potential by the distri- bution Of SC“- 6 0 Q o o a o o o o a o o 0 ix 0.04M sucrose, 1.5 mM (NH 412504, Page 43 46 48 51 58 60 64 66 69 75 :lCL Figure 17. 18. 19. 20. 21. 22. Page Removal of radioactive material by washing Thermoplasma acidophila cells with suspension buffer. X 77173 X, KSCN; ...... Acetate. Acetate included for comparison . . . . . . . . 83 Dependence of intracellular SCN’ concentration on the extracellular one in Thermoplasma acidophila cells . . 85 Dependence of membrane potential on the external pH of Thermoplasma acidophila cells suspended in suspension media composedS of'O. GEN KCl, 0.04M sucrose, solution T (l. 5 mM (NH) 4. 2 mM MgSO4 7H20 and 1.7 mM CaClz- ZHZO), and 3? 61M 4glycine buffer , for pH 2 and 3. 85. For pH 5 and 6, the glycine buffer was replaced by citrate buffer . . . . . . 88 Dependence of electrophoretic mobility on pH. The suspension solution was 0. OlM glycine (pH 2 to 4) or O. OlM glycyl- glycine buffer (pH 4 to l0. 5), 0.02N KCl, and 50 tion T . . . .9] Dependence of electrophoretic mobility on the cation concentrations. For pH 2. O. OlM glycine buffer, for pH 6, 0.01M glycyl-glycine buffer . . . . . . 93 A model for Thermoplasma acidophila; the surface is negatively charged, and the potential difference between the two bulk phases is l20 mv (positive inside), a zeta-potential of about 8 mV exists between the external surface of shear to the bulk phase of the externalinediun, when the external medium is at the same pH, ionic strength and divalent ion concentrations of the growth medium . . 95 ORGANIZATION OF DISSERTATION A General Introduction is in Chapter I. The main body of this dissertation con51sts of two parts. Part One, (Chapter 11) deals with Lipid and Temperature Dependence of Membrane-Bound ATPase Activity of Acholeplasma laidlawii. Part Two, (Chapter III and IV) deals with Intracellular pH and Membrane Potential of Thermoplasma acidophila. References are combined at the end of the dissertation. xi they a' differ. aaeric CIYSIB inform BAG t1 mEch Slnt CHAPTER I GENERAL INTRODUCTION Living systems and non—living systems have one thing in common, they all consist of matter. But their structures and functions are different, the living systems consist of what Schrbdinger called the aperiodic crystal, and the non-living systems consist of the periodic crystal if there is a crystal structure at all. According to their information content living systems tend to maintain an organization, and to keep the local entropy low within the biological systems; on the other hand, non-living systems tend to increase their entropy as thermodynamic equilibrium approaches (1). Living cells have to keep themselves separated from their surroundings; at the same time they must be in dynamic interaction with their environment. At the interface of the cell and its surround- ing, there is the cell membrane. Biological membranes not only play an active role in regulating the internal environment of the cell, selecting substrates through the mechanism of facilitated diffusion or active transport (for review 2-4); but are also important in energy transduction (5,6) and macromolecular synthesis (7). They may also play an important role in protecting the organisn1when under environmental stress (8). ~A-O- 9" ‘vUb 9-465 Patl Out: Evi Biological membranes consist mainly of lipids and proteins in various ratios, and some polysaccharides. The exact molecular arrange- ment of these components in three-dimensional structure is still unknown, but several membrane models have been proposed. Two important models will be reviewed. The classical lipid bilayer model proposed by Davson-Danielli (9,10) was based on thermodynamic considerations and experiments of Gorter and Grendel (11). They prepared a lipid extract from erythro- cytes, and Spread it as a monomolecular film on a Langmuir trough, and found that the film area was approximately twice the total surface area assumed for the total red cells used. In this model, lipids are arranged in two layers, the nonpolar sidechains of the lipids are sequestered together and shielded from water, whereas the polar head groups of the lipids are in direct contact with the aqueous phase on either side of the membrane, and therefore, maximize both the hydro- phobic and hydrophilic interactions. Although the original Gorter- Grendel experiment may have several mutual "offsetting errors" (for modern review, see 12), the basic concept of the lipid bilayer has survived for all these years. It has induced the development of several artificial systems such as that of the Black Lipid Membranes, as model systems for membrane research (13,14). The main shortcoming of the Davson-Danielli model is not the question of whether or not the lipids are arranged in a bilayer, but rather the position of the proteins. Originally the proteins were put outside the bilayer, in globular or extended B-form. Based on thermodynamic considerations and some experimental evidences, Singer—Nicolson proposed the fluid-mosaic model (15,16). A sketch of the model is presented in Figure 1 (16). “In this model, the proteins that are integral to the membrane are a heterogeneous set of globular molecules, each arranged in an amphipathic structure, that is, with the ionic and highly polar groups protruding from the membrane into the aqueous phase, and the nonpolar groups largely buried in the hydrophobic interior of the membrane. These globular molecules are partially embedded in a matrix of phospholipids. The bulk of the phos- pholipids is organized as a discontinuous, fluid bilayer, although a small fraction of the lipid may interact specifically with the membrane proteins. The fluid mosaic structure is therefore formally analogous to a two-dimensional oriented solution of integral proteins (or lipo- proteins) in the viscous phospholipid bilayer solvent.‘I This model is strongly supported by the following experimental evidence. In freeze-etch electron microscopic studies, the proteins can be visualized as intercalated particles (17,18). Enzymatic iodi- nation of biological membranes by lactoperoxidase has also shown that some proteins can be labelled from both sides of the membrane, which indicates that these kinds of protein span the membrane (19,20). One feature of the fluid mosaic model is the dynamic character of the proteins and lipids in the membrane. The model suggests that at physiological conditions, the membrane is in a liquid-crystal fluid state and the proteins can laterally diffuse in the two dimen51onal lipid solvent. To investigate the physical states of the lipids in the biological menbrane, several physical techniques have been used (for recent review, see reference 21,22). They can be broadly divided into two categories. x -NVNVNNVVAYHVNTVAV (\ i .chums m Eco» mvwawb .mmummmcmmm owmmumam Ecow >85 wEom .wmcmg ugocm u< .mcmcnsms ms» mo mcmpa mgu c? uwpanwcumwu xFEovcmL men mace; acop um cows: .mcwmpoca Pmcmmpcw cmpznoFm mg» pcmmmcamg mmumegzm umpaqwum cue; mmwuon uwpom mgp .mzmw> —w:owuumm immoeu vcm chowmcmewu-mmccu uwumsmgum mmcmngms Pmowmopown mo Pmnos owemoe uw:_e wsp .~ mczmwm are The techniques in the first category, such as X-ray diffraction and differential scanning calorimetry, measure some intrinsic characters of the membrane. X-ray experiments measure the change of diffraction patterns in dependence of physical states of the membrane lipids, for example, at a transition from a thermotropic gel (order) to liquid crystalline (disorder) (23). Differential scanning calorimetry measures an enthalpy change when an endothermic phase transition of the membrane occurs (24). Those in the second category involve sending “reporter" molecules into the membrane. The reporter molecules are sensitive to their micro- environment, so they will report any change in the physical states as they perceive. The commonly used "reporters" include fluorescent probes (25) and spin labels (26,27). The advantage of this kind of probing technique is that it can provide information of the micro- environment such as microviscosity or fluidity very sensitively, in contrast, differential scanning calorimetry can only measure a gross average property of the membrane. The disadvantage of probing studies is that the news breaker may become news maker, i.e., the probing mole- cules may disturb the system which they intend to measure. In Part One of this dissertation, the influence of thermotropic phase transitions on membrane-bound ATPase activity of Acholeplasma laidlawii has been investigated by the spin labelling method. Since detailed reviews on this organism have been published in several articles and books (28-30), only a simple summary is presented here. Acholeplasma laidlawii belongs to the order Mycoplasmatales which are a group of procaryotic micro-organisms. They are generally small {10 C011 01‘ in size (0.2 to few pm in diameter), devoid of a cell wall, bounded by a simple membrane without any intracellular membrane bound organelles. Acholeplasma laidlawii differs from other mycoplasmas by requiring no sterol for growth. Acholeplasma laidlawii has been the subject of many membrane studies in recent years. Electron microscopy reveals a "unit membrane" structure both in cells and in isolated membranes with a thickness of 75-100 A. The lipids are mainly phospholipids, glycolipids and varing amounts of carotenoids. The lipids can be highly enriched with a particular fatty acyl group when the corresponding fatty acid is supplied exogeneously. Polyacrylamide gel electrophoresis of the solubilized membrane exhibits at least 20 to 30 bands (31,32). It is possible to solubilize the membrane into small subunits by detergents, and form a reaggregated membrane after removing the detergents. Lipid bilayer structure resumes in the "reaggregated membrane". However, the proteins are incorrectly reassembled in these membranes (33,34). Phase transi- tions of the hydrocarbon chains in the bilayer have been detected with differential thermal calorimetry, EPR spin labelling, and X—ray diffrac- tion techniques. One of the most important questions in biology is how energy is converted from one form into another one. How energy, gained from light or food stuff, can be employed to carry out biological functions. For mitochondria or micro-organisms, this question would be: how does the energy from the respiratory electron transport chain couple to lies . {Yen 1 let be agree< the ATP synthesis and other biological functions, such as active trans- port of cations? Currently, there are three theories of energy coupling: (1) Chemical intermediate theory (35,36), (2) Chemiosmotic theory (37-40) and (3) Mechanoconformational coupling theory (41-43). The chemical intermediate theory is the oldest of the three. A modified version of the original form proposed by E. C. Slater (35) put the scheme of this hypothesis as follows (44): Aired + Biox 7:: Aiox~I + Bired (1) A10X~I + X === A1.0x + X~I (2) X~I + Pi ==e X~P + I (3) X~P + ADP # x +ATP (4) Here A1, Bi are adjacent respiratory carriers at a given coupling site and X and I are energy—transfer carriers common to all three sites. Equation (1) shows the "energy-coupling reaction" and the other equa- tions represent "energy-transfer reactions". The chemical hypothesis has many virtues, but it is somewhat unfortunate that no high energy intermediate, phosphorylated or non- phosphorylated, of mitochondrial respiratory chain phOSphorylation has ever been discovered (45,46). A merit of the chemiosmotic theory as a scientific hypothesis lies in the fact that it can be subjected to crucial experimental tests. Even the validity of the chemiosmotic theory of energy coupling has not yet been accepted jg_tgtgl_by the scientific community, it is universally agreed that it is the most influential theory in energy coupling. Since Mitchell proposed the hypothesis in 1961 (37), and in more elaborated thrr the L0 1 1.1112: trai Dl‘: .I- F) (D 3‘3 (‘3 a) form afterwards (38-40), it has triggered extensive research trying to diSprove or verify this hypothesis. The mechanoconformational coupling theory proposed that the primary energy-conservation process is a conformational change in a respiratory protein - a flavoprotein, an iron-sulfur protein or a cytochrome - and this energy may be utilized to make ATP. This theory has often been critisized for its vagueness and difficulty in putting forward for experimental test (53). The basic feature of the chemiosmotic theory of energy coupling is as follows (A sketch is presented in Figure 2): (1) The coupling membrane of the inner mitochondrion or plasma membrane of micro-organisms is permeable to water and virtually imperme- able to protons and most other ions, and therefore is of very low electrical conductivity. (2) The oxidoreductions, causing hydrogen and electron transport through the respiratory chain in the forward direction, result also in the translocation of protons from the inner phase of the mitochondrion to the outer phase. The respiratory chain is supposed to be folded into "oxidoreduction loops'I corresponding to the three coupling sites. (3) The respiration-driven proton translocation generates a transmembrane electric potential and/or pH difference, together called "proton motive force". In electric units, a 270 mV* proton motive * Based on the high (ATP)/(ADP)x(Pi) ratio observed in state 4 mito- chondria, the opponents of Mitchell's hypothesis have put the proton motive force required for ATP synthesis in state 4 mitochondria as high as 370 mV, if the P:2e ratio is assumed to be equal to 2 for each coupling site of respiratory chain. This statement has been considered by some as not to be rigorously substantiated because of the complication of the existence of ATP, ADP porters in the inner mitochondria membrane (54,55,66) Figure 2. 1O Chemiosmotic hypothesis: extrusion of protons by the respiratory chain, generation of ApH and Alp, and the poising of ATPase by the proton-motive force. (From reference 53). / 11 ‘v‘v‘ + NADH NAD 02 H20 ? ATPase RQSpiralovy chain Aim and AV’ Reversible ATPase poised by ApH and A“! Figure 2 ‘0 rt 12 force is needed for ATP synthesis, if H+/P ratio is 2. (4) There exists a membrane-bound reversible ATPase which can transport protons from the inside of the mitochondrion to the outside. Protons flow back from the outside to the inside of the mitochondrion, i.e., down their electrochemical gradient, trigger ATP synthesis. (5) For this coupling mechanism to function, two corollaries must hold: (i) A closed membrane is necessarily required for energy coupling. (ii) To compensate for what Mitchell called "backlash", exchange transport systems should exist, in which cations may antiport and anions may symport with protons, or with each other. The fundamental difference between the chemiosmotic theory and the conventional ones is that in Mitchell's hypothesis, H+ ions sepa- rated by an osmotically intact membrane are the coupling agents. Both the membrane potential, and the H+ ion chemical potential difference between two aqueous phases separated by the coupling membrane, are properties belonging to the whole membrane phase. It does not require "direct microscopic contact" of the redox chain with the reversible ATPase in physical space. In contrast, the chemical, conformational, or the modified "electromechanochemical" coupling developed by Green et. a1 (47-49) all require a direct contact of the two. Thus, it would be expected that normal biochemical fractionation methods should provide evidence for well defined structural associations and func- tional interrelationships between redox and the ATPase components. This is contrary to the experimental finding of biochemists despite forty years of diligent searching (51). 13 Although some controversial points.still remain, (for unsympa- thetic reviews 36, 47-50), a vast amount of evidence has been accumu- lated in recent years in support of the main feature of the chemiosmotic theory (for review, 44, 51-55). Some of them are summarized below: (1) In both mitochondria and submitochondrial particles, oxygen pulse and ATP hydrolysis do cause vectorial proton translocations in the right stoichiometry (56—59). Two proton pumps have been isolated and reconstituted in liposome systems and are found to be intrinsic expressions of the redox chains and the reversible ATPase systens. The two pumps are apparently separate. They do not share any chemical component in the membrane but can be shown to be energetically coupled one to another through cyclic proton current (60-63). Kinetically, they are fast enough to mediate between respiration and phosphorylation (59, 64). (2) There is no doubt that there exist energy-linked exchange diffusion systems of cations and anions with proton translocation (65, 66). But which one is directly linked to metabolism is a controversial point. Evidence accumulated in recent years seems to indicate that it is the proton current which is primarily linked to metabolism (54,55). The inhibitors of cation transport have no direct effect on the proton pump (59,67). Kinetic analysis shows that both the redox and the hydrolytic proton translocations are immediate expressions of the oxidation and reduction reactions of the respiratory chain and the hydrolytic reaction of ATPase system reSpectively. They do not depend upon the build-up of labile energy-rich chemical intermediates (59,68, 69). The transmembrane electric potential thus generated causes as {a it“ 1111 Dr 55 31 of de Ac Cu 14 electrophoretic migration across the membrane of any permeant cations or anions aspecifically (52,67,68). (3) The coupling membranes are not readily permeable to protons (70,71). The classical uncouplers of oxidative phosphorylation such as 2,4-nitrophenol function as proton conducting agents specifically equilibrating the electrochemical potential of H+ ions across the coupl- ing membrane (72). The study of a number of structurally dissimilar compounds has revealed a linear relationship between their effectiveness as protonphores in artificial phospholipid membranes, and uncouplers in mitochondria (73). Uncouplers do not depress the H+/0 ratio in both intact mitochondria and submitochondrial particles obtained by cavitav tion (56,74). (4) Energization of coupling membranes either by redox chain or by ATP hydrolysis can generate a transmembrane electric potential and/or a pH difference irrespective of the formation of high-energy intermediates (54). In state 4 mitochondria, a membrane potential of 230 mV (negative inside), has been observed by measuring the distribution of K+ in the presence of valinomycin (75) and synthetic organic Skulachev ions, such as phenyl dicarbaundecaborane (PCB—), and N,N-dibenzyl N,N-dimethyl ammoninum (DDA+) (76,77). A pH gradient of 0.4 to 0.8 pH units has also been obtained by titrimetric methods or by measuring the distribution of a weak acid (78). The total proton motive force of 270 mV is consi- dered by some as thermodynamically adequate for ATP synthesis (see foot note on page 9). (5) Energy accumulated in the form of transmembrane electric and osmotic gradients can be utilized for ATP synthesis. In chloroplasts, an artificially imposed pH gradient can induce ATP formation in the dark with efflt ritoc 311d 1 cert it) my 15 with electron flow prevented by inhibitors (79). An artificial K+ efflux, down their electrochemical gradient can cause ATP synthesis in mitochondria (80). (6) The three-dimensional arrangement of the respiratory chain and ATPase system in the mitochondrial membrane is still far from certain. Some evidence indicates that the inner mitochondrial membrane is both structurally ahd functionally asymmetric. ATPase (F1) is attached to the matrix side of the inner membrane by the stalk component (oligomycin sensitivity conferring factor) of the ATPase complex (81,82). The active sites of NADH dehydrogenase and succinate dehydrogenase are also located on the matrix side of the membrane. Cytochrome c is located on the outer cytoplasmic side of the membrane (81,83). Cyto- chrome oxidase appears to be a transmembranous molecule with its oxygen- reaction site located at the matrix side of the inner mitochondrial membrane (84,85). All these aspects are basically consistent with the chemiosmotic hypothesis. The chemiosmotic hypothesis put the proton in a unique position for biological function. In the second part of this dissertation, a mycoplasma-like organism Thermoplasma acidophila was investigated. It is a procaryotic micro-organism with a single compartment enclosed by a simple plasma membrane. It was first isolated from a coal refuse pile and has an optimal growth at pH 2 and 59 0C (86). It is interest- ing to know how this organism can live in such an environment without the protection of a cell wall. Thermoplasma acidophila can not grow at a temperature higher than 62 0C, which is very close to the high tem— perature limit found empirically for acidophiles (87). No organism which requires a pH less than 3 and yet can grow at 70 0C has been 16 found. Thus, Thermoplasma acidophila represents the edge of the acido- philic, thermophilic frontier at which life may exist. It becomes both important and interesting to know what is the intracellular pH, the membrane potential of this thermophilic, acido- philic organism. The compatibility of the experimental results from this organisms in extreme environment, will be discussed in terms of the chemiosmotic hypothesis. PART ONE LIPID AND TEMPERATURE DEPENDENCE OF MEMBRANE-BOUND ATPvASE ACTIVITY OF ACHOLEPLASMA LAIDLAWII 17 CHAPTER II LIPID AND TEMPERATURE DEPENDENCE OF MEMBRANE-BOUND ATP—ASE ACTIVITY OF ACHOLEPLASMA LAIDLAWII Introduction The activity of membrane bound enzymes may depend upon the presence of membrane lipids (88,89). Alteration of the membrane lipid composition can be achieved by nutritional supplementation of selected fatty acids for organisms with limited fatty acid biosynthetic capa- bilities (90,91) or by adjusting the growth temperature of poikilotherms (92). Acholeplasma laidlawii cells have the property that the membrane lipids can be enriched with respect to a selected fatty acid group via proper supplementation of the growth medium (93). Therefore, this organism has been the object of numerous studies to correlate various membrane properties with the acyl group composition. Previous investigations have demonstrated that Acholeplasma laidlawii cells have a tightly bound membrane ATPase which is Mg++ dependent and independent of Na+ and K+ ions (94). Alteration of the lipid acyl groups by nutritional supplementation and measurement of the ATPase activity from membrane suspensions at various temperatures can determine lipid dependence while avoiding the difficult experimental problem of membrane solubilization, enzyme purification, enzyme stability, and lipid reconstitution. These harsh treatments might 18 19 possibly introduce artifacts. A careful approach was also carried out for Escherichia coli membrane bound enzymes (95,96). In an unsaturated fatty acid auxotroph of Escherichia coli, membrane fatty acyl composition was changed by supplementing the nutrient medium with either cis-vaccenic, oleic, linoleic, linolenic acids or their corresponding trans~unsaturated fatty acids as the sole unsaturated fatty acid. One class of enzymes, such as acyl-CoAzglycerol 3—phosphate acyltransferase, were inactivated at a rate similar to the rate of phospholipase C hydrolysis of total membrane lipids, and were also characterized by an Arrhenius plot depending on whether the membrane contains cis-unsaturated or trans-unsaturated fatty acids. The other class of enzymes, such as glycerol 3-phosphate dehydrogenase, remained completely active after 95% of their membrane phospholipids had been hydrolyzed by phospholipase C, and their Arrhenius plots were charac- terized by a linear curve without a breaking point, and identical in slope, independent of membrane fatty acyl composition. In the case of Acholeplasma laidlawii, strain 8, discontinuities in the Arrhenius plots of the ATPase activity occurred at the lower end of the lipid phase transitions, as measured by differential scanning calorimetry. The organism was enriched with saturated or unsaturated fatty acids varying in chain length from 16 to 18 carbon atoms (97). With respect to the membrane of Acholeplasma cells, structural changes associated with phase transitions in the lipid bilayer portion have been studied with X-ray diffraction (98), electron paramagnetic resonance (99,100), and differential scanning calorimetry (24,97). Recently, freeze fracture studies of membranes demonstrated that the or oi ply 3e 66 DD If?“ 20 lipid bilayer continuum becomes interrupted by protein particles which aggregate or disperse dependent on fatty acyl composition and tempera- ture (101,102). The possibility of interpreting the change in activation energy in biological system, revealed in the presence of breaking points in Arrhenius plots, as a phase transition or at least as an indication of the beginning of the fluid-order transition has been discussed (103-105). In the following studies, electron paramagnetic resonance (EPR) spin labelling methods are used to monitor the membrane lipid fluidity and to detect any possible phase transition. The Acholeplasma laidlawii cells are grown on a nutrient medium supplemented with either oleic acid or saturated fatty acids containing 12 or 20 carbon atoms (106). Because of such a difference in acyl chain length, it is expected that the physico-chemical membrane properties vary distinctively. Attempts will be made to correlate the dependence of the membrane bound ATPase activity on fatty acyl composition and temperature with information obtained from EPR spin labelling experiments. Material and Methods Organism and Growth Acholeplasma laidlawii (oral strain) was a gift from Dr. S. Rottem (The Hebrew University, Jerusalem, Israel). The basal growth medium consisted of 20g Bacto-tryptose (Difco, Detroit, Michigan) which had been lipid extracted (107), 59 D-glucose, 59 sodium acetate, and 59 tris (hydroxymethyl) aminomethane per liter. The pH of the medium was 8.2 to 8.4 without adjustment. Four g/l of bovine plasma albumin fraction V (Armour Pharmaceutical Co., Chicago, Illinois) was charcoal treated to remove fatty acids (108) and then sterilized by Millipore 21 filtration together with 500 units/ml of penicillin G (Sigma, St. Louis, Missouri). The filtrate was then added to the basal medium. Either arachidic, oleic or lauric acid (5mg/l) was introduced into the growth medium as a 70% aqueous ethanol solution. The final ethanol concen- tration in the medium never exceeded 0.5%. The cells were originally grown in oleic acid supplemented medium. They could be adapted to supplementation with arachidic or lauric acid after 6-10 daily transfers. The medium was inoculated (1% V/v) with a 24 hour-old cell culture. The cells were grown statically at 37°C. and harvested after 20-24 hours for oleic or arachidic acid supplemented cells and 96 hours for lauric acid supplemented cells (106). Plasma Membrane Preparation Cells were harvested at late log phase of growth by centrifugation at 10,000 x g and were washed once in B-buffer, which contains: 1% NaCl, 0.6% tris(hydroxymethyl)aminomethane, and 0.07% (V/V) 2-mercaptoethanol prepared in deionized distilled water and adjusted to pH 7.4 with HCl. (109). To isolate membranes, cells were lysed by squirting washed cells into 1:20 diluted B-buffer, then incubated and stirred gently at room temperature for one hour. After incubation, unlysed cells were removed from the nenbrene preparation by centrifuging at 3,000 x g for 5 minutes, the supernatant was centrifuged for 30 minutes at 4°C at 37,000 x 9, then washed once with 1:20 B-buffer. The pellet was then resuspended in 1:20 B—buffer and adjusted to 1 mg/ml protein concentration and stored at 4°C. The same batch of membrane preparation was divided into three parts, one for ATPase assay (which was always completed within 48 hours), one for lipid fatty acyl composition analysis, and one for spin labelling 22 experiments. ATPase Assay The ATPase assay followed the method of Ruthbun and Betlach (110). Although the ATPase activity of Acholeplasma laidlawii was not dependent on K+, Na+, the assay solution always contained 0.1 ml of 10 mM KCl, 0.1 ml of 10 mM NaCl, 0.1 ml of MgClZu and 0.1 ml of 0.5 mM tris- (hydroxymethyl)aminomethane, HCl (pH7.6), 0.05 ml 20 mM ATP (Sigma, St. Louis, Missouri) plus appropriate amounts of membrane preparation and adjusted with deionized distilled water to a total volume of 1 ml. After incubation for 15 or 45 minutes (dependent on temperature), the reaction was stopped with cold 10% trichloroacetic acid (TCA), and centrifuged at 10,000 rpm (Sorvall GS 34 rotor) for 5 minutes. The supernatant was added to 1 ml 3M sodium acetate-acetic acid (1:1 V/V), the final pH was 4.2. To develop color, 0.1 ml 2% ammonimn "mlybdate and 0.2 m1 stannous chloride 1.52 mg/ml was added. The last two reagents were freshly prepared on the day of the experiment. After 15 minutes of incubation, the absorbance at 730 nm was recorded with a Gilford spectrophotometer, model 2400. A blank with ATP and a blank with the membrane preparation were always run in conjunction with the rest of the assays. All experiments were done in a cold room (4°C), in water baths maintained at temperatures from 6°C to 45°C. The duration of one set of experiments usually lasted from 12 to 18 hours. The enzyme has a half life of 10 days when stored at 4°C. Therefore, the enzyme will change its activity less than 2% during the period for one set of experiments. The protein concentration was determined by the method of Lowry et. al. (111). 23 Fatty Acyl Analysis Membrane lipids were extracted with chloroform-emthanol (2:1, v/v), washed with salt solution (0.02% CaClZ, 0.017% MgClZ, 0.29% NaCl and 0.37% KCl), and dried under nitrogen according to Folch et. al.(112). Methyl esters of fatty acids were prepared by reacting about 4 mg of lipids in 4 m1 of 2% (v/v) concentrated H2S04 in methanol at 40°C for 24 hours. The methyl esters were extracted with hexane and quantitative- ly analyzed by chromatography on a diethylene glycol succinate column at 190°C. A Hewlett-Packard gas chromatograph. model 402, equipped with a flame ionization detector was employed. The esters were identified by comparison with standards obtained from Applied Science Lab., Inc., State College, Pennsylvania. Relative quantities of fatty acyl composi- tion were measured from the areas under the peaks of the chromatogram. Solubilization of Membranes Membrane suspensions (1 mg protein/ml) were treated for one hour at 37°C with an equal volume of 0.1% Triton X-100. The clear solutions were centrifuged for one hour with a Beckman L-65 ultracentrifuge, SN 40 rotor at 38,000 rpm (180,000 x 9). ATPase activity was present in the supernatant. EPR Spectroscopy Isolated Acholeplasma laidlawii membranes were labelled jn_vitro with a fatty acid spin label. We used the label 2-(3-carboxypropyl)- 4, 4-dimethyl-2-tridecyl-3-oxazolidinyloxyl (abbreviated as: 5NS), or the spin label 2-(10—carboxydecyl)—2-hexyl-4,4-dimethyl-3-oxazolidinyloxyl (abbreviated as: 12NS), (for molecular structure see Figure 3), obtained from Synvar Corporation, Palo Alto, California. Figure 3. 24 The molecular structure of the spin labels 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl—3- oxazolidinyloxyl, 5-nitroxyl stearate, abbreviated as 5NS. 2-(10-carboxydecyl)-2-hexy1-4,4-dimethyl-3- oxazolidinyloxyl, 12-nitroxyl stearate, abbreviated as 12NS. 25 H30 /c\ /c\ /c\ /c\ /c\ /C\c /c\/ c \CCCCCC WCCOH oC/\~ ll N-O o 5-niiroxyl siedrdie: 5 NS C C C C C C C C 0 /\/\/\/\/\/\/\/\/<">-0H C C C C C C C C C 3 0 N-O \—i< l2-nilroxyl sieordie: l2 NS Figure 3 26 O.2-0.4 pmole of the spin label was dispersed in 0.6 ml of distilled water by Vortex agitation, followed by brief sonication, and then 0.3 ml of that dispersion was mixed with 5 mg protein of the membrane pellet (106). The spin labelled membrane suspension contained about 10 mg/ml protein and 0.1-0.2 umole Spin label / ml. EPR spectra were recorded with a Varian EPR spectrometer, model 4502 -15, equipped with a variable temperature controller, model 4540. Results Fatty Agyl Composition Table 1 lists the total fatty acyl composition of the membranes. For arachidic acid supplemented cells, the arachidoyl group comprised about 53% of all acyl groups found in cell membrane lipids. For lauric acid supplemented cells, the sum of myristoyl and palmitoyl groups amounts to 85% of the total fatty acyl groups. For cell grown in an oleic acid supplemented medium, the oleoyl group comprised about 60% of fatty acyl groups found in the cell membrane. ATPase Activity ATPase activity of the three types of membranes, enriched as described above, was assayed at different temperatures (Figure 4). At 37°C, the membranes enriched with saturated acyl groups, either short or long ones, have practically the same specific activity. For temperatures below 25°C, the ATPase activity of oleoyl group enriched membranes was higher than that from the other two types of membrane which have virtually the same specific activity. For the ATPase activity of arachidoyl group or short chain enriched membranes, an 27 .»_co ouooFo fixcoos mo mocomosa one omooowocw msopmm Fxspme owoo xupoe Pogo» 05p to mwmeoco owooomocuomom no; ostcw .Lm>ozoI .oosmwomcwumeo on po: o—ooo mcosomw owsmeomm >-oowcoocmoposoggo moon .mocoo m_o:oo any to Logan: asp op msmmwg acme; mgp op Longs: mg» mmeopo coocoo to Logan: on» op mcmwme coFou on» to uth mgp op consozo m.NH 1 w.mm N.H m.v m.H v.0H ¢.H N.om m.H uwuwsoog< m.o H.m i i m.mm N.N H.0N m.o o.¢ m.H uwmpo m.m i 1 i m.m ¢.N n.mv o.o o.N¢ m.m uwgsmo AmFoE\m_oEv mooosm H: onom Numfi Huwfl onma ouoH Huvfi onvfl moumfl Fxoo xppoe omuog o ooucmempaozm iopomco\ooposouom fie oFoEv cowpwmoosoo fixoo synod ovum xuuom .oopoowocw mo ovum zoom; mg“ oco seasoFo Eosmm ocw>oo ooquooo owoo xupoe sue: oopcosofiooom Eowoms mmopoxgu ompuospxmlmeo owowp cw czogo mew; mppmu .AAZoPowoF osmmmmopoco< to owowp ococoEmE Pouop mo co_uwmoosoo F>Uo xuuod H anoH Figure 4. 28 Arrhenius plot of ATPase activity in membranes isolated from Acholeplasma laidlawii cells grown in a lipid free medium supplemented (6 mg/l) with lauric acid (-I-----), oleic acid (-o-o-o-), or arachidic acid (-o-.-.-), Arrhenius plot (-4-4-A-) of ATPase activity of solubilized (0.1% Triton X-100; 1 mg protein/ml; 1:1, v/v) membranes enriched with arachidoyl groups. 29 20 25 3O 37 so .P L 3.3 31.4 I/Txlo3'K' :iz 3N b M“ M“ MW 25.52.85 ovaoSfiaocd 3.061 3.284 2.8on .4 <5 01» no .o nu Figure 4 3O Arrhenius plot revealed a biphasic character (Figure 4). The exact temperature where the discontinuity in slope occurred could not be determined; however, it was around 25 - 30°C. For all batches employed, the profile of the Arrhenius plot was always reproducible. The typical plot in Figure 4 shows a slope discontinuity at 26°C for arachidoyl and at 28°C for short chain enriched membranes. From the slope of the Arrhenius plot, the apparent activation energies are about 7-9 kcal/mole and about 25 kcal/mole for saturated acyl group enriched membranes. For oleoyl group enriched membranes, the Arrhenius plot was essentially a straight line; the activation energy was 8:1 kcal/mole. Membrane Solubilization Studies Membranes solubilization studies were only carried out with arachidoyl group enriched membranes. The ATPase activity was found in the supernatant. Passing the supernatant through a Sephadex G 200 column, the ATPase activity was associated with the protein which came out from the void volume. This finding is consistent with that described by Ne'eman et_al, (113). At any temperature studied, the specific ATPase activity was only 20% that of the non-solubilized material. Apart from the absolute value of the specific enzyme activity, the Arrhenius plots of solubilized and non-solubilized membranes are the same (Figure 4). This may suggest that the solubilized material still contained minute amounts of lipids such that the activation energy remained practically unchanged. This is in agreement with the results reported by Ne'eman et al. (113). 31 Membrane Fluidity as Measured By the Spin Labelling Method Membrane lipid fluidities of the three types of membrane were monitored by the spin labelling method. The fatty acid spin labels were introduced into the isolated membrane jg_yitrg, A representative EPR spectrum is shown in Figure 5. The hyperfine splitting 2T" in such a spectrum is related to the molecular order and the rotational mobility of the spin label and therefore reports the local fluidity of the membrane lipids (114,115). A high value of 2Tu reflects a low local fluidity. Over the entire temperature range (Figure 6), the parameter ZTu could be easily determined from spectra taken from 5NS labelled membranes. If membranes were labelled with a 12NS spin label, the high field "dip" in the EPR spectrum became so shallow that an accurate measurement of 2Tu was impossible. In such a case, the rotational correlation time TC of the spin label could be calculated by the follow- ing formula (115). h _ -10 o _ Tc - 6.5 x 10 x wo x ( i_h-1 1 ) sec. (5) where NO is the peak-to-peak width (gauss) of the central resonance peak; ho and h_.1 are the peak heights of the central and high-field peaks respectively. Around the growth temperature (37°C), the three types of membrane tested have approximately the same fluidity (Figure 6). This result is supported by data obtained from 12NS spin labelled membranes. At 37°C, the correlation time 1c is 3.16:0.33 nsec for membranes enriched with arachidoyl groups, Tc = 2.78 i 0.18 nsec for membranes enriched with short chain saturated acyl chains, and TC = 2.80 i 0.26 nsec for oleoyl 32 .Fmoop swam oouocoocoocwc: Eoct mFocm_m mmooowocw mzocco Poomusm> on» .LopoEoLoo mcwuaw_om mcwmcoox; mg» m? :FN .usowc 0» atop acct momoococw somcospm oymwm ovumcmoe one .uo mm no opoEom o soc» :oxop mo; Eocpomom mge .mooocm onowsooco gum; omgowcco mucoLoEmE wwzofiowoF oEmoPooPogo< ops? ocuw>.mfl oopocoocoocw mzm FmooF :wom mcp to Eosuooom mom Foowoxw .m mcomwu 33 ’i\ m m Lam r .m Figure 6. 34 Temperature dependence of the EPR hyperfine Splitting parameter 2Tn as defined in Figure 5. The spin label 5N5 was incorporated into membranes isolated from Acholeplasma laidlawii cells grown in a medium supplemented with different fatty acids as explained in Material and Methods. The final concentration of the spin label solution was 0.1-0.2 pm/ml with 10 mg/ml membrane protein. Hyperfine Splitting 2T1: (gauss) (B) so . . 1- . O "‘ i- .. C 55 - ' - C l- . " . d ’ / so - l r- '1 + I! r . r . 45 3‘1 3.? ‘53 3.14 3T5 l/T x 103 'k" 35 c. so 45 4933 30 as 2915 Figure 6 60 55 45 36 group enriched membranes, respectively. At temperatures lower than the growth temperature, oleoyl group enriched membranes were appreciably more fluid than the other two types of membrane. For arachidoyl group and short chain (C14:0-C16:0) enriched membranes, a plot (Figure 6) showed a discontinuity in slope around 26-280C, whereas in the case of oleoyl group enriched membranes such a discontinuity was absent. The exact point where that discontinuity occurs can not be measured. Such a discontinuity can be interpreted as a liquid/gel like membrane lipid phase transition (116). Discussion Our experiments demonstrate that the membrane lipid fluidity and ATPase activity behave similarly with respect to the absence or presence of a discontinuity in slope and also where that discontinuity occured in the Arrihenius plots. A low membrane lipid fluidity is accompanied by a high activation energy for ATPase. This is demonstrated by the following facts: (a) compared to the liquid phase, the activation energy is higher below the discontinuity for ATPase from membranes enriched with saturated acyl chains, and (b) below 25°C both fluidity and enzyma- tic activity are consistently higher for oleoyl group enriched membranes compared with those enriched with saturated acyl chains. For the three kinds of membrane studied, the lipid fluidity is practically the same at the growth temperature (37°C), in contrast to the specific enzyme activities which are the same around 25°C. The discrepancy can perhaps be explained by protein aggregation beginning well above the discontinuity in slope (102). 37 In our experiments we Show that the fatty acyl composition does play a role in determining the activation energy for the membrane-bound ATPase. This finding agrees with that reported recently (97), where cholesterol was used to shift the breaking point in the Arrhenius plot. The exogenously administered cholesterol to the non-sterol requiring Acholeplasma represents obviously a nonphysiological perturbation of the membrane. Our system, however, allows one to shift the breaking point (Figure 6) by simply supplying the physiological growth medium with the appropriate fatty acid. Previously it was reported that Acholeplasma membranes enriched with oleoyl groups have a lower fluidity (37°C) as compared to that measured for membranes enriched with saturated aqyl chains (100,102). These findings do not agree with ours. The reasons for that discrepancy may be the following ones: Our Acholeplasma laidlawii(oral strain) grows excellently (109 cells/ml within one day) on a nutrient medium supple- mented exclusively with arachidic acid (105). Usually cells show a poor growth when cultured in a medium containing long chain fatty acids (C16:0’°18:0) (28,117). Thus, it is conceivable that those poorly grown cells lack a regulatory mechanism to maintain the membrane lipid fluidity within a narrow range. Upon increase or decrease of the carotenoid content in the Acholeplasma membrane, we have Shown that our organism maintains the membrane lipid fluidity within a narrow range by modifying the fatty acyl composition of the lipids (118). Our results show that all three types of membrane have a practi- cally constant fluidity at the growth temperature, regardless of fatty acid supplementation to the nutrient medium. From a physiological point of view, the cells are able to maintain a constant lipid fluidity within a narrow range. This capability has been called "homeoviscous adapta- tion" (119), and was found in Escherichia coli (119) and in Achole- plasma laidlawii (118,120). The crucial question in interpreting EPR spin labelling data is as to what extent the spin label really reports the microenvironment. Objections regarding the interpretation of spin label results are (a) the perturbation introduced by the spin label and (b) possible non-random distribution of the spin label within the membrane lipids (97). The question of perturbation is as yet not fully answered; there exists evidence for and against this argument (114). Concerning the non-random distribution of the spin label, experiments indicate that fatty acid spin labels prefer the more fluid regions (114,121,122). This criticism may turn out to be advantageous, if the spin label is able to monitor reproducibly those fluid regions where important membrane functions take place. Because of such a preferential localisa- tion, membrane phase transitions are expected to be sharp ones when reported by the spin label, in contrast to broad transitions when monitored by microcalorimetry (97). Microcalorimetry measures the enthalpy which is an average membrane bulk property. The growth temperature lies generally within the broad lipid phase transition determined by calorimetry (97,121). With the spin labelling method, the relatively sharp transition lies lower than the growth temperature (Figure 6). This latter transition temperature lies approximately around the lower end of the broad lipid phase transition when calorimetric techniques are used (97). This behavior suggests that the spin label provides information about the more fluid membrane lipid regions. Trevor. 11611 a $111. acyl 39 Our results demonstrate that the breaking points (Figure 4,6) of membrane lipid fluidity and ATPase activity correlate well. This may well indicate that the membrane-bound enzyme and the spin probe are in a similar fluid microenvironment. which depends on the type of fatty acyl chain enrichment of the membrane. PART TWO INTRACELLULAR pH AND MEMBRANE POTENTIAL OF THERMOPLASMA ACIDOPHILA 40 CHAPTER III INTRACELLULAR pH OF THERMOPLASMA ACIDOPHILA Introduction Thermoplasma acidophila is a mycoplasma-like organism which grows optimally at 59°C and pH 2 (86). We are interested in the problem of how these cells can live in such a hostile environment without the protection of a cell wall. An electron micrograph from thin sections of Thermoplasma acidophila cells is shown in Figure 7 (123). Since the organism grows under acidic conditions, the question of the intracellular pH becomes significant. If the intracellular pH value should lie in the acidic range, how does the metabolic machinery function? 0n the other hand, if the intracellular pH lies in the neutral region, how can the cell maintain such a huge hydrogen ion concentration gradient across the membrane? The intracellular pH was measured by the distribution of a radioac- tive weak organic acid 14C-labelled 5,5-dimethyl-2,4-oxazolidine-dione (0M0) (Figure 8). This method was first developed for determining the intracellular pH of muscle cells (124), and since then was also applied to the study of pH changes in mitochondria (125), and a few micro- organisms (126-128). 41 42 Figure 7. An electron micrograph from thin sections of Thermoplasma acidophila cells. (From reference 123) 43 Figure 7 44 Material and Methods Organism and Growth Thermoplasma acidophila was grown in a medium containing 1.5 mM (NH4)2SO4, 4.2 mM M9504.7H20, 1.7 mM CaC12.2H20, 0.03% KH2P04 1% glucose, and 0.1% yeast extract (Difco, Detroit, Michigan). The pH was adjust to 2 with concentrated H2504 and the medium then autoclaved. A 10% (v/v) inoculum from a 22-hr old culture into the same medium gave the best growth. Each culture was continuously aerated for 22 hours with air, sterilized by filtration. Cultures (18 1) were incubated at 56°C in 20 1 flasks (129). Cells were harvested at late log phase, after 22 hours of growth, by centrifugation at 15°C, at 9,000 x g for 5 minutes using a Sorval RC 28 centrifuge with a GS 3 rotor. Then, the cells were washed once and resuspended in TG-buffer. The TG-buffer'was composed of 0.02M KC1,0.04 M sucrose, 1.5 mM (NH4)2504, 4.2 mM M9504.7 H20, 1.7 mM CaC12.2 H20 and 0.01 N glycine buffer at pH This TG-buffer had the 2. same osmolarity, ionic strength and divalent cation concentrations as the growth medium. An 18 1 culture usually yielded 35-40 ml of a cellular suspension containing 20-30 mg/ml protein. Procedure forpH_Measurement by the Method of DMO Distribution In aliquots of 1 ml, that cellular suspension was distributed into a set of centrifugation tubes (size: 4 m1), followed by addition of 1 ml of TG—buffer. A scheme of the procedure is shown in Figure 9. In a typical run, 10 tubes were used to determine gravimetrically the total P611€t water, Vt' Another 12 tubes were incubated with 0.1 uCi ‘4 C- dextran (molecular'weight 60,000 - 90,000, Specific activity 1.31 mCi/mM. New England Nuclear, Boston, Massachusetts) which will not penetrate into 45 .AOZQV mcowuimcwowFOme01¢.Nupxsumecim.m twppmnmpiu ovum o_ orcomeo xooz m>_uooowooc o to cowpooweumwo ozu an In co—zppmoosucw to ucoemesmomz .m mesa?“ 46 m weaned :25: no. + :63 «51.. 531 525.521. Eo:aE=3< .58 32:25: a. :0 02o N .1. In oooo Lo _.o 1 v... 0.3.1.3: {ooze oeo_o-v.~ -oe_o__o~oxo_:=oe_oio.o 47 .029 mo cowuznwgumwo we» mo Io co_:~_ooosp:w mcwcomoos so; ocoomooco on» to osmcom Poucmewcmoxm :< .m mcomwm m msomwu 7i 7; oo_+xon:a .883 33:39:...» _> .02 _>no -o>no-ao a 57:2 828; _> .mOu c__HE._oz— Dada—2:9; .203 «0:01 ._O_a__000..—xw no) .22., SEE _2oe n o> 3:8 5 $.58 oo EBoanom 5 £5.50 mo uo> 0+. .4. 4. 4. or 4. 49 the cell, to measure the extracellular pellet water, Ve, An additional MC-labelled DMO (specific 12 tubes were incubated with 0.1 pCi of activity 11 mCi/mM, New England Nuclear, Boston, Massachusetts) to monitor the intracellular pH. After incubation for one hour at 56°C, or at room temperature, all 34 tubes were chilled to 4°C, then centri- fuged simultaneously at 9,000 x g for 10 minutes. Then, the pellets from the ten tubes were cut for gravimetrical determination of total pellet water, weighed immediately, and then dried in an oven for 72 to 96 hours to constant weight. The difference in gram represents Vt in m1. For the rest of the tubes, the supernatant was transferred to test tubes, each pellet was resuspended in 2 ml of TG-buffer in a separate test tube. Then 1 m1 from each test tube was dried separately in a scintillation counting vial (Figure 9). After drying, 0.075 ml water and 0.5 ml NCS tissue solubilizer (Amersham/Searle Corp., Arlington Heights, Illinois) was added, followed by addition of 10 ml of a toluene solution containing 3g/1 PPO, 0.lg/ldimethyl-POPOP, and counted in a Packard Tricarb scintillation instrument. Nithin experimental accuracy the intracellular pH did not depend upon the length of incubation time which was varied from 2 minutes to 2 hours. (Figure 10). Principle of Using 0M0 Distribution to Measure Intracellularng The principle of employing the distribution of a weak organic acid as a monitor for intracellular pH is based on the assumption that the non-ionized form of the acid permeates passively across the cell membrane and attains equilibrium during incubation, while the ionized form of the weak acid remains practically impermeable to the cellular membrane (124, 50 14C-DMO inside the cell and in the Figure 10. The concentration of extracellular pellet space were calculated, and their ratio was related to the incubation time. (0M0). 51 (0M0)e 2 b . 5 30 60 120 Minutes Incubation Time Figure 10 52 125). Because 0M0 has a pK value of 6.1 at 56°C (125), almost all DMO molecules are in the non-ionized form (designated as DMOH) at pH 2 or 4. If the molecule permeates to the interior of the cell, and if the intra- cellular pH is higher than the pK value, some of the molecules will dissociate from the non-ionized form into the ionized form (designated as DMOT). The ratio of the two forms at equilibrium can be described by the Henderson-Hasselbach equation (124,125). 0M0- DMOH .......... (6) Intracellular pH = pK + log Moreover, if 0M0 passively enters the cell, the intracellular concentra- tion of the non-ionized form will be the same as that of the extracellu- lar non-ionized form. The net result will be an accumulation of DMO in the cell, provided the intracellular pH is higher than the pK value. But the intracellular concentration of DMO' and DMOH can not be measured directly. Only the counts of DMO in the supernatant and pellets can be directly measured. If we know the extracellular pellet water and intracellular pellet water we can calculate the intracellular DMO’ and DMOH concentration by the following relationships. These equations hold when extracellular pH< to memeeoo as opomicmu omu:_wo mp A—E\ms o. mproo mo commcoomom moooooo co cog: om>cmmoo cwopogo to omompmm x 11x .Iooz zoo.o mo cowuwooo omwzooco coo: APE.mE m. cowmcmomom ppoo mzoozoo co cw omcoso zaoillo .mvme ppmo so In to Hometm .m. ocomw. 64 PROTEIN RELEASE (ug ml) 3.0 - 1.0 - 2 (lw) C1300V HOPN :10 BWITIOA H Figure 13 p 65 .5: com po Iowpoo ovuoEcho one .uo mm no mcoewoo ucocoemwo Loom cw omxommo .opwzaooeoo oEmoFQoEcmce Eocw mmocomocoxcmo mooFoE owEmoFoopxo wo m_wmoco In one .4. 8:38.. E 93o: 66 In N d a . x x xi 3234 x 2232:. o 3.020 32.0 I Stem 8.2.6 4 I If) 9 ‘32 p.614: - l-Ulw-mv v) MMNOV aluoeds 67 Influence of Temperature and External pH on Intracellular pH Nithin experimental accuracy, the intracellular pH as measured by the DMO distribution method is not affected by temperature changes (56°, 24°), or by alteration of the extracellular pH from 2 to 6 (Figure 15). At pH 4 and 6, the cells were suspended in TC-buffer, which is TG-buffer, where citrate replaced the glycine. Moreover, to calculate the intracellular pH of cells in TC-buffer at pH 6, we assumed that in the extracellular medium, half of the number of DMO molecules are in ionized form, and the other half are in the non-ionized form. Thus, the cells are capable to maintain the intracellular pH when sub- jected to those environmental alterations. How Is the Hydrogen Ion Concentration Gradient Maintained? We explored whether Thermoplasma acidophila cells require active metabolic processes to maintain the hydrogen ion concentration gradient of 4.5 pH units, or whether passive mechanisms play an important role. When Thermoplasma acidophila cells were boiled for five hours at 100°, the intracellular pH measured at the end of the harsh treatment was still neutral (Table 2). These boiled cells were no longer viable as demonstrated by inoculating them into culture medium. By exposing viable cells to general metabolic inhibitors, such as 0.1 mM 2,4-dinitrophenol, l0 mM iodoacetate and l0 mM sodium azide, growth of cultured cells was prevented. None of these inhibitors altered the intracellular pH within experimental accuracy (Table 2). 0n the basis of these heat and inhibitor treatments, we therefore conclude that the major portion of the pH gradient is not maintained by active Figure 15. 68 The effect of external pH on the intracellular pH of Thermoplasma acidophila. For pH 2, the suspension mediunlwas composed of 0.02N KCl, 0.04M sucrose, l.5mM .7H (NH4)ZSO4, 4.2mM MgSO 20, l.7mM CaCl2.2H 0 and 4 2 0.0lN glycine buffer. For pH 4 and 6, the glycine buffer was replaced by citrate buffer. Intracellular pH l- 69 b External pH Figure l5 4 7O processes, but rather by passive properties of the cell. A Donnan potential across the membrane generated by charged macromolecules impermeant to the cell membrane, can account for maintenance of the huge hydrogen ion concentration gradient without participation of active mechanisms. CHAPTER IV MEMBRANE POTENTIAL AND SURFACE CHARGE OF THERMOPLASMA ACIDOPHILA Introduction Thermpplasma acidophila is a mycoplasma-like organism which grows optimally at 59°C and pH 2, and stops growing above pH 4 (86). The intracellular pH lies between 6.4 and 6.9 as determined by the distribution of a radioactive weak organic acid, 5,5-dimethyl-2,4- oxazolidine-dione (DMO). The cell can maintain this pH gradient of about 4.5 pH units when subjected to metabolic inhibitors, such as iodoacetate, NaN3, and 2,4-dinitrophenol (134). Because the cell can passively maintain such a huge pH gradient across the membrane, we propose that a Donnan potential exists, possibly generated by charged macromolecules impermeable to the cell membranes. To test this hypothesis, the membrane potential was measured by the distribution of radioactive KS14 CN, which is known to permeate biological membrane (l35,l36). Furthermore, a radioactive lipophilic cation tetraethyl- ammonium (TEA+) was used to determine the polarity of the cell. The surface charge density and thez;potential, i.e., the potential difference from the cell surface of shear relative to the bulk medium can be estimated from the electrophoretic mobility of the cell. The 71 72 movement of the cell in an applied electric field can be observed directly under a microscope (137-139). The Helmholtz-Smoluchowski equation 0 C 4fln and a = l 2855 x \/2ci[exp(-zie/kT) - 1] (13) can be used for the calculation of the zeta-potential and the surface (12) charge density (138-142). Here, u = electrophoretic mobility (velo- city/unit field strength),c = potential difference between the surface of shear and the bulk of the liquid, D = dielectric constant, n = viscosity of the suspension medium, a = surface charge density. Ci are molar concentrations of ions with charge Zi’ N = Avogadro number, k = Boltzmann constant, e = electron charge. These equations are valid for large smooth particles at an ionic strength such that no > 100. Here, a is the radius of curvature of the cell surface. K is the Debye-Hbckel constant defined as _ 8 N e K‘ [10002 ma fzzci' '12 (14) For Thermoplasma acidophila with a diameter of 0.5 to 1 pm, this condition generally holds for the ionic strengths used in this inves- tigation. From the potential difference between the bulk phase of the external medium and that of the cytoplasm, and the zeta-potential, the potential profile between the two interfaces of the plasma membrane can be constructed. 73 Material and Methods Growth of Cells and Harvest Thermoplasma acidophila was grown as described previously (134). Aerated 18 l cultures were harvested at late log phase, after 22 hours of growth, by centrifugation at 9,000 x g, at 15°C for 5 minutes. Then the cells were washed twice and resuspended in l.5 mM (NH4)ZSO4, 4.2 mM M9304.7H20, l.7 mM CaClZ. 2 H20, 0.04 M sucrose and 0.0l M glycine buffer, pH 2. This medium has the same ionic strength, osmolarity, and divalent cation concentration as the growth medium. An l8 l culture usually yielded 35 to 40 ml of a cellular su5pension containing 20 to 30 mg/ml protein. In aliquots of 1 ml, that cellular suspension was distributed into a set of centrifugation tubes (size 4 ml), followed by addition of 1 ml of the suspension buffer mentioned above to each tube. Procedure of Measuring Membrane Potential by the Distribution of SCN‘ In a typical experiment (Figure l6), 8 tubes were used for gravimetrically determining the total pellet water, Vt, 8 tubes were 14 incubated with C-dextran (molecular weight 60,000 - 90,000), 0.1 pCi, specific activity l.3l mCi/mM (New England Nuclear, Boston, Massachusetts), to measure the extracellular pellet water, Ve’ In addition, 8 tubes were l4 incubated with 0.l uCi KS CN (60 mCi/mM specific activity, Amersham/ Searle, Arlington Heights. Illinois), and 8 tubes with 0.1 uCl ‘4 C- labelled tetraethylammonium bromide (TEAB) (2.8 mCi/mM, New England Nuclear, Boston, Massachusetts). After incubation for one hour at 56 °C (the amount of KSCN accumulated in the cells did not depend upon the length of incubation which was varied from 10 minutes to 2 hours), all 74 .uzum mo cowuzowcpmwu mg» »n meucmpoa mcmgasme mcp mcwgzmmms Lo» mgznmumcg vcm mFQwucwga mgu we msmcum Popcmswgqum c< .m_ mz=m_a 75 o_ weaned S: a? mohoTsHéomw :5 no a 507289 mumsk monEmHZMU D 91.8 ETD; swzomH— m.mlm.w n In. map 28.x Eh“5...“. .I. u>fio esp pcwmmgamc mpocucou mgp _Pm new m o“ H .paxm .mmzupma ucogmwmwu sage mew; m op H yo asocm m seem nmuumpmm wasp mecwm m we «you Pmuwaxp pcmmmgamg a .35 £33 :8 mag 93 so: F828 93 3 3:23 umgsmmms mew: $.55qu 6:29.65 93 :8: mLOpwowgcw use mcowpmcucmucou zumx sue; we mucmzpycH .N In use comm um meow «Lo; mucmewgmaxm F_< .<. .AHV .cm soc; umpmpzopmu mm; cwmizuma :owpmgucmucou cowco mg» mews: psofi-zum. psofi-zuma a me u c_ u s CWH-zomH Hm c._-zomH soc» nmumpzupwo we; meucwuoa mcmanmE age . m 6 F womam Lopez Lepsppmumgucw mg» m? > i > u .> .mumam cmgmz umFFma Lm_:F~mumprm mza m? m> .mumam Lopez meFma ~mpop ecu we p> .mcanEmE mammFa mzu mmogum 20 .4. l VH cowusgwcpmwv mcu m:_gammws an m_w;qovwom mEmMEmOELmLH cw mepcmuoa meanEmE we» mo cowgmcwEgmpmo m m>wuomowumg mo m mppwh mnNHH manH mnNHH mnefifi Nnmofi QHNHH enem mama Nnmofi Naomfi NHVHH mnmma >5 .s Nanak who“ mama Nude mace momm fine NHNN mums coon enem ofinom paofl-zum_\=_fi-zuma N.m o.oH ¢.m N.m N.HH m.oH 4.“ m.HH “.mfi o.NN m.wm m.mH moflxaau.flzuqflmxvau o.m¢ ¢.mo o.Hm N.NN o.mm m.mm .eofi ¢.mo m.¢m m.2m H.Nm N.mm monEeo.AzueHm¥vmu Nae Nam mam macs en sum mnmfi mnofl onefl enofl anew ens .1..> finom Hnmm enom enfim mnme mneq mnom mnmm mace mnmm cums mnem Fa.m> finqm finem sham Hafio HnNm Hnmm Name 3445 fine“ Home Nnoofi some P:.p> mH.H mH.H NH.N mo.~ mm.H mm.H HN.N NH.N mN.N mm.~ mH.H NN.N mofixeau.fluefi-emcoxmnvau H.4m N.eN o.mw H.mm N.Nm m.mm H.mm m.e~ m.me o.mm N.¢m 0.0m mofix5au.Au¢H-=mguxaevmu smug FEESQZDAC _ocpeou mzaz _oepeou zume zumx Pozpcou m N H :5 m :5 H :5 OH 2H.o 2H0.o .paxm .paxm .paxm A.u.pcouv m m_neh 81 concentration should depress the observed potential, because it is lowered by any passively permeant anion present at high concentrations. Because HSCN is a rather strong acid with a pK a -1.85 (144). At pH 2, more than 99% of SCN_ should be in the ionized form. To test whether the SCN_ anion passively permeates the cell membrane, two kinds of experiment were performed. First, the SCN- accumulated inside the cell can be readily washed out by the same buffer applying three washes (Figure 17). For comparison, acetate cannot be washed out (Figure 17). Secondly, increasing the external nonradioactive KSCN concentration to 1 mM, the amount accumulated depends linearly on the KSCN concentration (Figure 18). However, upon increase of the external SCN— concentration to 0.01 N, the membrane potential was reduced to 87 mV; upon further increase to 0.1 N, the membrane potential was lowered to 54 mV (Table 3). 'This behavior confirms the notion that at lower concentrations the SCN' distribution does indeed monitor the membrane potential as we proposed. Influence of Metabolic Inhibitors on Membrane Potential In the presence of 10 mM NaN3, an electron transport inhibitor, or 1 mM 2,4-dinitrophenol, or 5 mM carbonyl cyanide, m-chlorophenyl hydrazone (CCCP), which are proton conducting uncouplers, the measured membrane potential remained unchanged (Table 3). As reported previously (134), similar inhibitors exerted no measurable influence upon the intracellular pH. Therefore, both the membrane potential and the pH gradient are maintained passively. The 120 mV membrane potential, (positive inside), compensates only partially the pH gradient of 4.5 pH Figure 17. Removal of radioactive material by washing Thermoplasma acidophila cells with suspension buffer. x . _"‘ ..... x KSMCN. o . '— ..... o MC-labelled Acetate. Acetate included for comparison. 83 \ ‘NK Io5 ‘ \ 0 Acetate ‘\. ‘\ )( P CD 00 C) Ifii r 939’.“ (DON TI [\3 05 r I . . . . r- r- r- 7‘ 7' 9° “9 9° NAmmOmbmaooivb r (3 9l Figure 20 92 Figure 21. Dependence of electrophoretic mobility on the cation concentrations. For pH 2, 0.01N glycine buffer, for pH 6, 0.01N glycyl-glycine buffer. 93 4.0~ 3.8- 3.6 3.4 3.2- 3.0- 2.8;; 2.6- 2.4- 2.2- 2.01- l.8-— l.6- l.4- l2- 1.0- 1 er PH=2, KCI PH=2,COC|2 PH=6, KCI PH=6, COCIZ I xi). I Mobility (um/secl/(volt/cm) .6 .4 - 2 10'5 lO'4 10:3 lo-2 10'| [Concentration] Figure 21 94 increased to 0.01 N or 0.1 N, which is presumably an ionic strength effect. At pH 6, the effect of Ca++ concentration of the electro— phoretic mobility of Thermoplasma acidophila cells was dramatic. The mobility had a value of -2.7 (pm/sec)/(volt/cm) when the concentration of Ca++ was lower than 5 x 10-5N, and changed to -0.5 (pm/sec)/(volt/ cm), i.e., a 5-fold decrease, when the Ca++ concentration increased to 0.01N and higher values. The Ca++ ion concentration which reduced 4N. At pH 6, K+ ions of the mobility to half its value was 5 x 10' concentrations similar to Ca++ concentration had little effect on the electrophoretic mobility of Thermoplasma acidophila cells. Discussion Based on the membrane potential measured by the method of SCN- distribution, the surface charge density, and the zeta-potential estimated from electrophoretic mobility, a model for a Therm0plasma acidophila was drawn (Figure 22). The SCN' distribution measured the potential difference between the bulk phase of the cytoplasm relative to that of the external medium. The electrophoretic mobility measurement gave the surface potential difference between the outer surface of the cell and the bulk phase of the external medium. The surface charge of Thermoplasma acidophila was inferred to be negative. This conclusion is based on several facts: (1) The cells move from the negative electrode to the positive electrode. (2) When the external pH was raised above pH 6, the mobility increased. This was presumably due to the fact that more negative charges were exposed by raising the pH, since the pK of some carboxyl groups lies around 95 Figure 22. A model for Thermoplasma acidophila; the surface is negatively charged, and the potential difference between two bulk phases is 120 mV (positive inside), a zeta-potential of about 8 mV exists between the external surface of shear to the bulk phase of the external medium, when the external medium is at the same pH, ionic strength and divalent ion concentra- tions of the growth medium. 96 " (+) A - ? 64 PH= . - (+) pH-2 — ? '- lll=iZOmV :CPgtsrflLgl ----------- Outside _ cytoplasm Medium Membrane Figure 22 97 4.5. (3) Ca‘l’+ ions, in the concentration range of 1 mM and up, reduced the electrophoretic mobility at pH 6. This effect was explained by assuming that Ca++ ions are specifically bound to the negatively charged cell surface, probably to the phosphate head groups of the phospholipids, since Ca++ has the similar binding behavior to the monolayers and bilayers of phosphatidylserine (145)., We interpreted the data in terms of specific binding rather than unspecific ionic strength effects. This conclusion is based on two facts: First, KCl at concentrations of comparable ionic strength showed little effect in reducing the mobility. Secondly, the Ca++ ion concentration needed in reducing the mobility was rather low, 115,, 5 x 10-4 N Ca++ ion could reduce the electrophoretic mobility to half the value measured in the absence of Ca++ (Table 4, and Figure 21). At pH 2, most of the binding sites are protonated, and therefore few Ca++ ions can be bound. It is interesting to note that at pH 6, a high Ca++ ion concentration (higher than 0.01 N) could reduce the mobility of Thermoplasma acidophila cells to a level found at pH 2 (Table 4, Figure 21). Thus H+ and Ca++ ions have similar and competitive effects in reducing the mobility of the cells. This interpretation of Ca++ binding to the cell surface is consistent with the Ca++ effect on the phase transition temperature of Thermoplasma acidophila membranes measured by EPR spin technique (146). The total charge of the cell is negative, the bulk potential across the membrane is positive inside, and the surface is highly negatively charged. Therefore, inside the cell some positive ions or macromolecules may exist which cannot penetrate the cell membrane. These particles are responsible for the Donnan potential which is 98 positive inside the cell. We do not have any direct evidence for such an existence, but we do have some indicative evidence. From pH 7 to 10.5, the mobilities of the cells are abnormally high (Figure 20). This may be explained by cytoplasmic or membrane proteins leaking out of the cell (the proteins do leak out at pH higher than 7, see reference 135, also Figure 13, Chapter III of this dissertation). Because the mobility increased dramatically, the materials which leaked out must be positively charged. Will the negatively charged membrane pose some problems for the anion SCN' to penetrate into the cell? The answer is no. The question of how SCN' permeates the negatively charged biological membrane in general and the Thermoplasma acidophila membrane in particular can be explained by the fact that the estimated charge density of various biological membranes ranges from 100 to 40 000 A2 per elementary charge (147-150). For Thermoplasma acidophila cells, the estimated charge density ranges from 1000 to 6000 A2 per elementary charge (Table 4). With such a low density, the charge is probably distributed discretely (150-152). Thus, there is plenty of space for SCN' ion to diffuse through the membrane. We do not have any information about the surface on the cyt0" plasmic side of the membrane. It may be positive or negative. In Figure 22 it is tentatively drawn as positive. (This is purely an assumption, made to isslutrate a point). Thus, a zeta-potential may also exist on the inner surface of the membrane. Consequently, the "real potential across the bilayer" may be 15 mV higher than the bulk— to-bulk phase potential difference measured by SCN' distribution. The surface zeta-potential will also influence the local pH near the surface of the membrane (153). If the cytoplasmic side of the 99 membrane really has a surface potential more positive than that of the bulk of the cytoplasm, the local pH near the inner surface is more alkaline than that of the bulk phase, and the local pH at the external surface is lower than that of the bulk of the medium. The pH and 9 profile of Thermoplasma acidophila might be important with respect to Mitchell's chemiosmotic hypothesis (this particular point, see reference 36). From kinetic experiments in chloroplasts, it was found that the H+ current generated by light would immediately return to trigger ATP synthesis, before the H+ to be equilibrated between the membrane solution interface and the bulk phase (154). This finding makes the zeta- potential and pH near the membrane surface an important and interesting parameter to investigate. Unfoutunately, the zeta-potential and surface charge density on the cytoplasmic side of the membrane cannot be measured by a simple direct technique. Lastly, we would like to raise the question of whether the chemi- osmotic theory of energy coupling, proposed by Mitchell (37,40,47) may be applied to this organism, i.e., can the respiratory transport of H+ and its return to intuce ATP synthesis be the mechanism for energy transduc- tion in Thermoplasma acidophila? At first glance, it seems that the data observed are incompatible with the Mitchell's hypothesis, since the total proton motive force needed for ATP synthesis is 270 mV (negative inside) (54,75,79). However, even at pH 2, the net electrochemical potential gradient in the resting state of the cell is -l70 mV (negative inside), (—290 mV from pH gradient, +120 mV from Donnan potential). Ne do not consider these data as incompatible with the Mitchell hypothesis, because the -270 mV required should be the proton electro- chemical potential gradient of a respiring energized cell. Our «170 mV was obtained from resting cells. 100 This observation of course does not provide any direct evidence which supports the chemiosmotic theory either. However, we do have some indirect hints that the chemiosmotic theory may still be applied to this organism. In Thermoplasma acidophila, the cell polarity is reversed in comparison to Escherichia cali cells or mitochondria, either of them is negative inside. Thermoplasma acidOphila constantly faces a very acidic environment, and consequently a huge hydrogen ion concentration gradient, normally not faced by other organisms. In the absence of a passive Donnan potential, Thermoplasma acidophila would then need a special kind of respiratory chain which would eject H+ against an abnormally high electrochemical potential barrier. The reversible H+ ion transporting ATPase would also face an abnormal high electrochemical potential barrier. Therefore, Thermgplasma acidophila, Using a passive Donnan potential (positive inside) to reverse its cell polarity, could partially balance the huge pH gradient. In this way, the remaining electrochemical potential barrier for both respiratory proton transport and the reversible proton transporting ATPase is of the same order of magnitude usually found in Escherichia coli or mitochondria. This aspect suggests that the Mitchell hypothesis may still be applicable for Thermoplasma acidophila. Nevertheless, Mitchell's hypothesis offers an explanation for the fact that Thermgplasma acidophila cells do not grow above pH 4.5, since at this pH value the membrane potential w drops to +40 mV. Consequently,lw - (RT/F) 1n (Apnfl (negative inside) becomes less than 80 mV. This makes the coupling mechanism of oxidative phosphorylation non-functional. Thus. the cell is flikely to be not just shielded passively from the acidic environment. BIBLIOGRAPHY 10. ll. l2. l3. 14. 15. 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