Imitational modeling of process of evolution: From organic macromolecules to protocell and animal cell

A dynamic imitational model of initial stages of cell evolution has been developed based on role of environmental calcium concentration. The model is designed from our hypothesis about the medium of the appearance of protocells, which could be potassium water reservoirs rather than sea salt water with its predominance of sodium salts. The necessary elements of the appearance of the protocells served organic molecules, code of their synthesis, and formation of macromolecules under favorable ion concentration in environment: a high K⁺ and Mg²⁺ and a low Na⁺ concentration. The model is based on an assumption that one of the first stages in evolution of life was the appearance in the potassium-magnesium water reservoirs of organic molecules capable for self-replication on the basis of genetic code and formation of protocell with the potassium cytoplasm. The model has demonstrated necessity of formation of cell envelope for development of the protocell. Replacement of the dominant cation in water reservoirs—potassium by sodium—required the appearance of ion-transporting devices in plasma membrane and their participation in adaptation of cells to environment. This stage of evolution was accompanied by the most important morphofunctional event—formation of the plasma membrane instead of cell envelope. The membrane provided the ion asymmetry in the cell (preservation of K⁺ in it) relatively to the sodium external medium for maintaining optimal intracellular medium. In the model system, predecessors of animal cells elaborated mechanism of maintenance of the potassium cytoplasm with the sodium counterion dominating in the environment.     

Physico-chemical determinants of physiOlogical evolution: from protocell to the human

One of the key physiological problems of evolution is elucidation of the role of environmental inorganic factors in origin of life. A statement is substantiated that a highly significant event in evolution of life was the appearance of protocells with the K cytoplasm in K water reservoirs with their subsequent adaptation to the environment in which Na dominated. This step was accompanied by replacement of the cell envelope with the cell plasma membrane. Precursors of animals' elaborated mechanism of maintenance of the K-cytoplasm with Na-counterkation in the environment. In plants, Na remains a trace element, in animals the Na and K contents are approximately equal, but they are present in different fluid phases. In animals presence of Na+ in the external medium, K+ inside cells has become an initial pre-requisition for electrogenesis and appearance of asymmetrical cells. Electrogenesis of these cells has become a physiological pre-requisition for cell differentiation, the appearance of the nerve cell. Asymmetrical cell has provided development of absorption, digestion, excretion, respiration. Formation of the inner medium has become a prerequisite for establishment of homeostasis. Serum osmolality, sodium concentration, pH, Ca2+ are the most rigidly maintained constants in the blood serum of humans.     

The origin of membranes

The physicochemical conditions of the environment in which life arose are discussed, along with the appearance of protocells, their membranous envelope and the subsequent appearance of plasma membranes. The hypothesis that the first cells originated in reservoirs where potassium and magnesium salts (necessary for protein synthesis and thus for the formation of a cellular membrane) dominated, is substantiated. This was followed by adaptation of these cells to an external ocean-like environment, where sodium salts were prevalent. This stage of evolution required a plasma membrane capable of providing ion asymmetry between the cell’s cytoplasm and the external environment. At this stage of evolution in the predecessors of animals, the process of removal of sodium ions and accumulation of potassium ions began functioning in the plasma membrane. The problem of multicellular organisms was solved differently by animals and plants: animals developed a system of the extracellular fluids that provided stable physicochemical conditions on the external surface of the plasma membrane. Sodium ions were the stimulus for the formation of the polar cell, where sodium channels are situated on one side of the plasma membrane, and sodium pumps on the other, allowing the development of the absorption, excretion and breathing functions. The formation of fluids of the internal environment enabled the development of homeostasis and facilitated the biological progress of the animal kingdom.     

Na,K-ATPase in the nuclear envelope regulates Na+: K+ gradients in hepatocyte nuclei

Evidence is emerging that the nuclear envelope itself is responsible for transport and signaling activities quite distinct from those associated with the nuclear pore. For example, the envelope has a Ca2+-signaling pathway that, among other things, regulates meiosis in oocytes. The nuclear envelope's outer membrane also contains K+ channels. Here we show that Na+/K+ gradients exist between the nuclear envelope lumen and both cytoplasm and nucleoplasm in hepatocyte nuclei. The gradients are formed by Na,K-ATPases in the envelope's inner membrane, oriented with the ATP hydrolysis site in the nucleoplasm. We further demonstrate nucleoplasm/cytoplasm Na+ and K+ gradients, of which only the Na+ gradient is dissipated directly by Na,K-ATPase inhibition with ouabain. Finally, our results demonstrate that nuclear pores are not freely permeable to sodium and potassium. Based on these results and numerous in vitro studies, nuclear monovalent cation transporters and channels are likely to play a role in modulation of chromatin structure and gene expression.     

The salting-in hypothesis of post-hypertonic lysis

The phenomenon of slow cooling cryoinjury has remained one of the primary areas of research in cryobiology since the early 1950s when it was first investigated thoroughly. Lovelock demonstrated that cell death from freezing and thawing was mainly due to exposure to hypertonic solutions and the subsequent dilution back to isotonic conditions. He suggested that the cell became permeable to sodium in hypertonic conditions leading to a loading of sodium during the hypertonic exposure, which caused the cell to swell past its elastic limit during resuspension in isotonic media (post-hypertonic lysis). This idea was pursued by Zade-Oppen, Farrant, and others who were able to show that the membrane became leaky to cations in hypertonic media but they could not provide any mechanism that would cause the cell to load up with sodium (other than an exchange of extracellular sodium for intracellular potassium, leaving the cell with the same cation concentration that it started out with). In the absence of such a mechanism, predicting post-hypertonic lysis from osmotic simulations cannot be done.A simplified model is proposed in which the intracellular milieu is composed of both KCl and a proteinaceous component that normally forms many salt bridges between amino acids with fixed charges. When the intracellular salt concentration increases, the proteins are “salted in” to solution (salt bridges are replaced with ionic interactions) thereby decreasing the intracellular cation concentration. Cation channels in the plasma membrane are opened by exposure to a high salt concentration (either inside or outside the membrane) allowing extracellular sodium to take the place of the intracellular potassium that is interacting with anionic groups on the proteins. Dilution of the external medium (which also occurs during melting) causes water to move into the cells, diluting the cytoplasm. The proteins are then “salted out” of solution and release the salt back to free ions in solution. The cell has an excess of intracellular ions and may swell past its elastic limit due to water influx. A simulation engine is developed based on the model and compared to results in the literature for freeze–thaw injury in human red blood cells.     

Computer simulation on the cooperation of functional molecules during the early stages of evolution

It is very likely that life began with some RNA (or RNA-like) molecules, self-replicating by base-pairing and exhibiting enzyme-like functions that favored the self-replication. Different functional molecules may have emerged by favoring their own self-replication at different aspects. Then, a direct route towards complexity/efficiency may have been through the coexistence/cooperation of these molecules. However, the likelihood of this route remains quite unclear, especially because the molecules would be competing for limited common resources. By computer simulation using a Monte-Carlo model (with "micro-resolution" at the level of nucleotides and membrane components), we show that the coexistence/cooperation of these molecules can occur naturally, both in a naked form and in a protocell form. The results of the computer simulation also lead to quite a few deductions concerning the environment and history in the scenario. First, a naked stage (with functional molecules catalyzing template-replication and metabolism) may have occurred early in evolution but required high concentration and limited dispersal of the system (e.g., on some mineral surface); the emergence of protocells enabled a "habitat-shift" into bulk water. Second, the protocell stage started with a substage of "pseudo-protocells", with functional molecules catalyzing template-replication and metabolism, but still missing the function involved in the synthesis of membrane components, the emergence of which would lead to a subsequent "true-protocell" substage. Third, the initial unstable membrane, composed of prebiotically available fatty acids, should have been superseded quite early by a more stable membrane (e.g., composed of phospholipids, like modern cells). Additionally, the membrane-takeover probably occurred at the transition of the two substages of the protocells. The scenario described in the present study should correspond to an episode in early evolution, after the emergence of single "genes", but before the appearance of a "chromosome" with linked genes.     

Monensin-induced cation movements in bovine erythrocytes

Monensin is a carboxylic ionophore that has been observed to increase cation permeability across the membrane of several cell types. Additionally, it is used commercially as an anticoccidial agent and has been found to increase feed efficiency in cattle. The objectives of these experiments were to determine the ability of monensin to stimulate cation (Na and K) transport across the bovine erythrocyte membrane and determine the effects of anion substitution on the action of the compound. Erythrocyte cation analyses revealed that all of the animals used in this study were low potassium (LK). Red cells were incubated in an artificial medium in the presence or absence of monensin, and cell sodium, potassium and water were determined at several time periods. It was observed that monensin stimulated the movement of sodium and potassium down their respective concentration gradients. Cell water content ("D") was observed to increase in response to an elevation in cell cation content. In synthetic media containing acetate, sulfate, citrate, thiocynate and gluconate substituted for chloride as the anion specie in the presence of monensin, there were measureable differences in intracellular sodium and water during the incubation period. The addition of DIDS to the control media containing chloride was observed to inhibit from 60 to 80 percent of the monensin-stimulated sodium movements. The results of this study show that monensin stimulates cation movements in bovine erythrocytes and anion substitutes may alter the action of this ionophore. Additionally, it was demonstrated that the action of monensin can be modified by inhibition of Band 3.     

Effects of nonsteroidal anti-inflammatory drugs on the uptake of various cations by lymphoid cells.

Several acidic nonsteroidal anti-inflammatory drugs (NSAID) as well as their corresponding alcohol molecules which are known to induce swelling of isolated lymphocytes by changing cell membrane permeability to water, are demonstrated also to induce changes of membrane permeability of lymphoid cells to one divalent cation, calcium, and to three monovalent cations, rubidium, cesium and sodium. According to the cells ionic environment, they increase or decrease the cellular uptake of cation which is itself also closely dependent on the ionic composition of the incubation medium. This drug-effect is very rapid, directly related to the medium NSAID concentration and almost totally reversible except to the most potent drugs such as flufenamic acid. Changes in intracellular ionic balance could have important catalytic effects on the metabolism of normal as well as of pathological cells. This fact could explain side-effects of these drugs as well as some of their therapeutic effects.     

THE SIGNIFICANCE OF IONIC CONCENTRATIONS IN THE INTERNAL MEDIA OF ANIMALS

1. The electrical and ionic gradients across a cell membrane depend on its permeability properties, on the concentration and net valency of the organic constituents of the cytoplasm and on the critical energy barrier to the extrusion of sodium. Such considerations do not, however, explain the small extent to which the concentration of potassium varies in myoplasm which may, instead, be related to the effects of potassium on particular enzymes. 2. The fact that the apparent optimum level of potassium cannot usually be maintained in animals in which the extracellular level of sodium is below about 140 mM may explain why so many non-marine animals have internal media of about that concentration, for more concentrated body fluids would require more work for their regulation. 3. In axoplasm, the concentration of potassium is more nearly proportional to the concentration of sodium in the internal medium and this may partly explain the general correlation between the extracellular levels of sodium and potassium. 4. The relation between pH and temperature in poikilothermic vertebrates is such as to suggest that the prime function of acid-base regulation is to control the ionization of imidazole groups. 5. High tensions of carbon dioxide cannot be maintained in water-breathing animals because of the high solubility of this gas in water as compared with oxygen. Bicarbonate levels are correspondingly low to give a suitable pH. Higher tensions are possible in air-breathing animals, and also necessary if water and heat are to be conserved, but an uncertain upper limit is set by the need for oxygen. The associated higher levels of bicarbonate confer the advantage of better buffering. 6. Calcium and bicarbonate levels are not obviously limited by the solubility of calcium carbonate and a more general limitation on the composition of body fluids seems to arise from the low solubilities of calcium phosphates. 7. The pattern of ionic balance in vertebrate plasma, reflected in a nearly constant value to the molar ratio ([Ca] + 5 × 10^--⁴)/([K] +0.034 [Na]), may be explained in terms of the maintenance of a constant electrical gradient across certain areas of cell membrane, between the inner and outer double layers. 8. The patterns of cation balance in the haemolymphs of molluscs, crustacea and insects are also reviewed, with emphasis on the correlations existing between the concentrations of different cations. An attempt is made to relate the correlations in the mollusca to the concentrations of cations at the surfaces of cells.     

The salting-in hypothesis of post-hypertonic lysis

The phenomenon of slow cooling cryoinjury has remained one of the primary areas of research in cryobiology since the early 1950s when it was first investigated thoroughly. Lovelock demonstrated that cell death from freezing and thawing was mainly due to exposure to hypertonic solutions and the subsequent dilution back to isotonic conditions. He suggested that the cell became permeable to sodium in hypertonic conditions leading to a loading of sodium during the hypertonic exposure, which caused the cell to swell past its elastic limit during resuspension in isotonic media (post-hypertonic lysis). This idea was pursued by Zade-Oppen, Farrant, and others who were able to show that the membrane became leaky to cations in hypertonic media but they could not provide any mechanism that would cause the cell to load up with sodium (other than an exchange of extracellular sodium for intracellular potassium, leaving the cell with the same cation concentration that it started out with). In the absence of such a mechanism, predicting post-hypertonic lysis from osmotic simulations cannot be done.A simplified model is proposed in which the intracellular milieu is composed of both KCl and a proteinaceous component that normally forms many salt bridges between amino acids with fixed charges. When the intracellular salt concentration increases, the proteins are “salted in” to solution (salt bridges are replaced with ionic interactions) thereby decreasing the intracellular cation concentration. Cation channels in the plasma membrane are opened by exposure to a high salt concentration (either inside or outside the membrane) allowing extracellular sodium to take the place of the intracellular potassium that is interacting with anionic groups on the proteins. Dilution of the external medium (which also occurs during melting) causes water to move into the cells, diluting the cytoplasm. The proteins are then “salted out” of solution and release the salt back to free ions in solution. The cell has an excess of intracellular ions and may swell past its elastic limit due to water influx. A simulation engine is developed based on the model and compared to results in the literature for freeze–thaw injury in human red blood cells.     

Effect of the sodium/potassium ratio on glyceraldehyde 3-phosphate dehydrogenase interaction with red cell vesicles.

Binding of glyceraldehyde 3-phosphate to glyceraldehyde-3-phosphate dehydrogenase, the membrane protein known as Band 6, causes shifts in the 31P nuclear magnetic resonance spectrum of the substrate (Fossel, E.T. and Solomon, A.K (1977) Biochim. Biophys. Acta 464, 82--92). We have studied the resonance shifts produced by varying the sodium/potassium ratio, at constant ionic strength, in order to examine the relationship between the cation transport system and glyceraldehyde-3-phosphate dehydrogenase. Alteration of the potassium concentration at the extracellular face of the vesicle affects the conformation of glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face, thus showing that a conformation changed induced by a change in extracellular potassium can be transmitted across the membrane. Alterations of the sodium concentration at the cytoplasmic face also affect the enzyme conformation, whereas sodium changes at the extracellular face are without effect. In contrast, there is no sidedness difference in the effect of potassium concentrations. The half-values for these effects are like those for activation of the red cell (Na4 + K+)-ATPase. We have also produced ionic concentration gradients across the vesicle similar to those Glynn and Lew (1970) J. Physiol. London 207, 393--402) found to be effective in running the cation pump backwards to produce adenosine triphosphate in the human red cell. The sodium/potassium concentration dependence of this process in red cells is mimicked by 31P resonance shifts in the (glyceraldehyde 3-phosphate/glyceraldehyde-3-phosphate dehydrogenase/inside out vesicle) system. These experiments provide strong support for the existence of a functional linkage between the membrane (Na+ + K+)-ATPase and the glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face.     

Cations in molluscan tissues at sharply different hemolymph osmolality

Experiments on different bivalve and gastropod species living in fresh, brackish, and sea water of different salinity demonstrated a direct correlation between the osmolality of the hemolymph and interstitial fluid and the concentration of sodium ions in it. A direct correlation between the osmolality of the interstitial fluid and the content of potassium and magnesium ions in the tissues (adductor and foot) was revealed. The significance of physicochemical indices of the environment and formation of eukaryotic cells at the initial stages of animal evolution are discussed.     

X-ray microanalysis of cockroach fat body in relation to ion and water regulation

X-Ray microanalysis of fat body from the cockroach (Periplaneta americana) showed that the crystals in the urate cells sequestered potassium and sodium during water deprivation. Postassium sequestration appeared to occur by the formation of additional crystals whereas sodium appeared to be taken up by pre-existing crystals of the “amorphous granule” type. Urate cells increased in cytoplasmic density i.e. mass per unit volume as a result of water deprivation and lost potassium from the cytoplasm but not from the nucleus. Trophocyte cells also showed an increase in density and a loss of potassium from the cytoplasm. The nucleus in trophocyte cells however, did not increase in density and showed an increase in chlorine concentration. A previously undescribed type of crystal in the trophocyte cell cytoplasm also increased markedly in chlorine concentration. The mycetocyte cells did not appear to increase in density but chlorine concentration was elevated in symbionts, cytoplasm and nucleus. An increase in density is considered to be a consequence of a reduction in volume due to water loss. It is confirmed that the fat body plays a major role in the maintenance of ion balance during water deprivation.     

Solvent drag across gramicidin channels demonstrated by microelectrodes

The competition of ion and water fluxes across gramicidin channels was assessed from the concentration distributions of both pore-impermeable and -permeable cations that were simultaneously measured by double-barreled microelectrodes in the immediate vicinity of a planar bilayer. Because water movement across the membrane led to accumulation of solutes on one side of the membrane and depletion on the other, the permeable cation was not only pushed by water across the channel (true solvent drag); it also flowed along its concentration gradient (pseudo-solvent drag). For the demonstration of true solvent drag, a difference between the bulk concentrations on the hypertonic and the hypotonic sides of the membrane was established. It was adjusted to get equal cation concentrations at both membrane/water interfaces. From the sodium and potassium fluxes measured along with membrane conductivity under these conditions, approximately five water molecules were found to be transported simultaneously with one ion through the channel. In diphytanoyl phosphatidylcholine membranes, a single-channel hydraulic permeability coefficient of 1.6 x 10(-14) cm(3) s(-1) was obtained.     

Lymphocyte monovalent cation metabolism: cell volume, cation content and cation transport

Mechanisms which determine sodium and potassium content and volume of rat thymic and human chronic lymphocytic leukemia (CLL) lymphocytes have been studied. The deleterious effect of cell isolation on monovalent cation content was proven by comparing thymus sodium and potassium concentration to that of thymocytes prepared from autologous hemithymus. In vivo distribution ratios of sodium-24 and potassium-42 between thymus water and plasma water were very similar to the distribution ratios of non-radioactive isotopes (sodium-23 and potassium-39). The similar lymphocyte: thymocyte ratio of (a) cell volume (1.48), (b) cell sodium plus potassium (1.47) and (c) cell water (1.50) demonstrated the close correlation of lymphocyte volume with monovalent cation content and water content. Steady-state CLL lymphocyte sodium (32 ± 1.9 mM) and potassium (131 ± 5.1 mM) and thymocyte sodium (31 ± 1.2 mM) and potassium (136 ± 3.9 mM) were similar; however, these steady-state levels were maintained by quantitatively different membrane functions. Radiopotassium and radiosodium uptake by thymocytes was more rapid than by CLL lymphocytes. Ouabain-sensitive potassium influx was 2.4 times greater in thymic (8.70 ± 2.28 mmoles/cm²/min × 10^?8) than in CLL (3.24 ± 0.45 mmoles/cm²/min × 10^?8) lymphocytes. Potassium exodus was also slower in CLL lymphocytes as compared to thymocytes. Ouabain-sensitive sodium accumulation and ouabain-insensitive sodium accumulation were also slower in CLL lymphocytes than in rat thymocytes. Half-maximal ouabain inhibition of sodium entry was 7.5 × 10^?3 M in thymic and CLL lymphocytes. The inhibitory effect of ouabain on sodium and potassium transport was easily reversible. Oligomycin inhibited ouabain-sensitive potassium accumulation in both lymphocyte types. Four lines of evidence indicate the presence in the lymphocyte of a system of leaks and pumps, the latter subserved by a ouabain and oligomycin-sensitive (sodium-potassium) ATPase: (a) steady-state monovalent cation gradient (K ～ 20:1, Na ～ 5:1), (b) the inability to maintain normal sodium and potassium gradients at cold temperature and in the presence of ouabain, (c) the effect of ouabain and oligomycin on active potassium influx and (d) the restitution of steady-state sodium and potassium concentration after cell isolation, ouabain treatment and cold exposure. CLL lymphocytes as compared to rat thymocytes have a decreased rate of ouabain-insensitive sodium uptake and potassium exodus requiring a reduced rate of active sodium extrusion and potassium accumulation to maintain steady-state cation content. Ouabain-sensitive ATPase is difficult to locate in lymphocytes in vitro possibly because it comprises a very small proportion of membrane ATPase since magnesium activated ecto-ATPase in intact lymphocytes is 1500 to 2500 times that of the intact erythrocyte. The inhibition by ouabain of blast transformation, mitosis, amino acid accumulation and nucleic acid synthesis in vitro, may reflect the importance of ouabain-sensitive ATPase and monovalent cation transport in the function of lymphoid cells.     

Motile activities of Dictyostelium discoideum differ from those in Protista or vertebrate animal cells

Cell movement in the amoebae Dictyostelium discoideum has been examined in media differing in monovalent cation concentration (i.e. Na+ and K+). Under isotonic or even slightly hypertonic conditions, the cells move equally well in solutions in which either potassium or sodium ions dominate. However, in strongly hypertonic solutions the amoebae showed motility in a 2% potassium chloride solution, but remained motionless in a hypertonic 2% sodium chloride solution. This inhibition of D. discoideum amoebae movement in a hypertonic sodium chloride solution was fully reversible. Such behaviour corresponds to that of plant, fungi, and some invertebrate animal cells rather than protozoan or vertebrate cells. These observations suggest that studies using D. discoideum as a model for cell motility in vertebrate animal tissue cells should be considered with caution, and would seem to confirm the classification of cellular slime moulds as related rather to Fungi than to Protista. This also shows that the cell membrane models should consider the asymmetry in sodium/potassium ion concentrations found in vertebrate animal cells as one of various possibilities.     

An unexpected effect of an ouabain-sensitive ATPase activity on the amount of antigen-antibody complexes formed in situ.

A classical method of indirect immunofluorescence was applied on various kinds of lightly fixed and permeabilized cells to analyze the formation of the complexes between a nuclear antigen and its antibody (AAC). The amount of AAC decreased dramatically when the incubation with the first antibody was realized in the presence of ATP in a sodium-rich medium with 0.5 mM KCl. Addition of sodium vanadate, a general inhibitor of ATPases, ouabain or tetrabutylammonium ion, specific inhibitors of the Na+,K+-ATPase, prevented this effect. The established role of this enzyme is to increase free-K+ concentration and decrease free Na+ concentration in the cell. It is not surprising to find an ATPase still active since light fixation and permeabilization do not destroy phosphatases. But it is rather surprising to find something looking like Na+,K+-ATPase activity in permeabilized cells. The importance of potassium in this puzzling result is suggested by the fact that appearance of ACC was equally suppressed if the incubation was made in the absence of ATP in a potassium-rich medium without sodium. Results are discussed, taking into account the properties of cell-associated water and recently found interrelation between Na+,K+-ATPase and tubulin. In any case, the results seem interesting in the field of immunocytochemistry.     

Effect of the sodium/potassium ratio on glyceraldehyde-3-phosphate dehydrogenase interaction with red cell vesicles

Binding of glyceraldehyde 3-phosphate to glyceraldehyde-3-phosphate dehydrogenase, the membrane protein known as Band 6, causes shifts in the ³¹P nuclear magnetic resonance spectrum of the substrate (Fossel, E.T. and Solomon, A.K. (1977) Biochim. Biophys. Acta 464, 82–92). We have studied the resonance shifts produced by varying the sodium/potassium ratio, at constant ionic strength, in order to examine the relationship between the cation transport system and glyceraldehyde-3-phosphate dehydrogenase. Alteration of the potassium concentration at the extracellular face of the vesicle affects the conformation of glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face, thus showing that a conformation change induced by a change in extracellular potassium can be transmitted across the membrane. Alterations of the sodium concentration at the cytoplasmic face also affect the enzyme conformation, whereas sodium changes at the extracellular face are without effect. In contrast, there is no sidedness difference in the effect of potassium concentrations. The half-values for these effects are like those for activation of the red cell (Na⁺ + K⁺)-ATPase. We have also produced ionic concentration gradients across the vesicle similar to those Glynn and Lew ((1970) J. Physiol. London 207, 393–402) found to be effective in running the cation pump backwards to produce adenosine triphosphate in the human red cell. The sodium/potassium concentration dependence of this process in red cells is mimicked by ³¹P resonance shifts in the (glyceraldehyde 3-phosphate/glyceraldehyde-3-phosphate dehydrogenase/inside out vesicle) system. These experiments provide strong support for the existence of a functional linkage between the membrane (Na⁺ + K⁺)-ATPase and the glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face.     

Electron-microscopic study of the nuclear membrane of the PKEV cells during mitosis. 1. The breakdown of the nuclear membrane (prophase, prometaphase, metaphase)]

An ultrastructural study on dividing PKEV cells provided a possibility to distinguish between certain stages of their desintegration. The changes preceding fragmentation of the nuclear envelope commence with desorganization of its structural components: vanishing of granular peripherial chromatin layer; appearance of the pores without central granules; formation of deep invaginations of the nuclear membranes. The desintegration of the nuclear envelope starts from the disapearance of many pores and the appearance of perforations almost of the same size. Simultaneously, the number of polysomes is reduced on the outer membrane of the nuclear envelope and in the cytoplasm. Specific features of the nuclear envelope being lost it becomes undistinguishable from the reticulum elements. On serial sections, no contacts were observed between chromosomes and membranous elements.     

The specific requirement for sodium chloride for the active uptake of l-glutamate by Halobacterium salinarium

1. Uptake of l-glutamate by Halobacterium salinarium is dependent on high concentrations of sodium chloride in the environment. When the sodium chloride is replaced by isomolar concentrations of potassium chloride, sodium acetate or potassium acetate, only negligible uptake occurs. 2. Most of the glutamate taken up can be shown to be in the cells in the free state and at a concentration of at least 50 times that in the medium. Sodium chloride is therefore required for an active transport of the glutamate into the cells. 3. The question whether sodium chloride is essential for the actual migration of glutamate across the cell envelope or for the mechanism supplying energy for this migration is discussed on the basis of experiments on endogenous respiration and with inhibitors.