The combination between hemolysins and red cells or ghosts,as studied with a radioactive lysin and with new color reactions

The quantity of a radioactive hemolysin, sodium dodecyl sulfonate-S³⁵, taken up by red cells from concentrations too small to produce hemolysis varies with the lysin concentration, and does so in a way which can be described by an adsorption isotherm. Attempts to use color reactions or surface tension measurements to determine the quantity of digitonin, saponin, and the bile salts taken up by red cells from hypolytic concentrations have failed, principally because chromogenic, and also surface-active, substances are liberated from the cells when the lysin is added. Color reactions with the anthrone reagent show that digitonin and saponin are both taken up by or fixed to red cell ghosts; the extent of the uptake, however, is uncertain, again because of the liberation of chromogenic substances. Comparison of the results of the various methods which measure the apparent amount of lysin fixed, or utilized in reactions between lysins and red cells or ghosts show discrepancies between results given by direct methods (measurement of radioactivity or of color) and indirect methods (addition of a second population after lysis of a first, and dependence of the position of the asymptote of the time-dilution curve on the number of red cells). The discrepancies are traceable to the inhibitory effects of substances liberated from the red cells or ghosts. The ease with which a lysin, once taken up by red cells, can be detached by diluting the system determines the extent to which the hemolytic reaction is "progressive," but has no observed connection with the quantity taken up in the first place. There is now ample evidence that lysis in systems containing simple hemolysins is a process involving two stages in time and two phases, and that it is usually complicated by reactions between the hemolysin and liberated inhibitory material.     

The kinetics of progressive reactions in systems containing saponin,digitonin, and sodium taurocholate

The relations between lysin concentration, percentage hemolysis at the moment at which the lysin concentration is reduced by dilution, and the amount of hemolysis which follows the dilution as a result of the reaction being "progressive" point to there being an "internal" phase at the red cell surfaces, in which the lysin is less affected by the dilution than in the system as a whole. A second possibility, i.e. that the combination of lysin molecules with certain components of the cell surface has an ultimate effect on neighboring components which depend on the former for their stability cannot, however, be ruled out. In systems containing digitonin or sodium taurocholate, this internal phase, once formed, seems to be almost unaffected by the dilution of the system; i.e., these lysins are very firmly held at the cell surfaces. In systems containing saponin the lysin is less firmly attached, so that dilution of the system affects its concentration appreciably.     

THE PROLYTIC LOSS OF K FROM HUMAN RED CELLS

The prolytic loss of K., i.e. the loss of K which takes place from red cells exposed to hypolytic concentrations of lysins, has been measured in systems containing distearyl lecithin, sodium taurocholate, sodium tetradecyl sulfate, saponin, and digitonin, by means of the flame photometer. The lysins are added in various concentrations to washed red cells from heparinized human blood, and the K in the supernatant fluids is determined after various intervals of time and at various temperatures. The prolytic loss K_p is compared in every experiment with the loss K_s into standard systems containing isotonic NaCl alone, with no lysin. The losses K_s and K_p increase with time, so that new steady states are approached logarithmically. The values of K_p which correspond to the new steady states depend on the lysin used, being greatest with taurocholate and smallest with digitonin. The temperature coefficient of the loss is positive, and the extent and course of the losses have no apparent relation to the prolytic shape changes. In systems in which the loss of K is appreciable, it can be inhibited by the addition of plasma or of either cholesterol or serum albumin. Of these two substances, even when used in quantities which have an approximately equal effect in inhibiting hemolysis, serum albumin is much the more effective. Just as the prolytic loss of K occurs without the loss of any Hb, so in concentrations of lysin sufficient to produce hemolysis the loss of K, expressed as a percentage of the total red cell K, increases much more rapidly with lysin concentration than does the loss of Hb expressed as a percentage of the total Hb. The explanation of these relations depends on whether the loss of K is treated as being all-or-none in the case of the individual cell or as being the result of the loss of part of the K from all of the cells. This point has still to be decided.     

The inhibition of hemolysis,as studied by the technique used for investigating progressive reactions,and by a technique using radioactive hemolysins

Inhibition of hemolysis by plasma has been studied in systems containing saponin, digitonin, and sodium lauryl sulfate, using the methods developed for the study of the kinetics of progressive reactions. The results are that the progressive nature of the hemolytic reaction in saponin systems becomes less when the inhibitor is added, that the addition of inhibitor to digitonin systems has no effect on the final result although the velocity of the progressive reaction is reduced, and that the effect of plasma in lauryl sulfate systems is intermediate between the effects in saponin systems and digitonin systems. A simple explanation is that the lysin is very strongly fixed, to form an internal phase, to the cell surfaces in digitonin systems, less strongly in laurate systems, and still less strongly in saponin systems. To answer the question as to whether, in a system in which some of the lysin forms as internal phase, the addition of an inhibitor results in a redistribution of the lysin between the internal phase and the bulk phase, sodium lauryl sulfate-S³⁵ and sodium cetyl sulfate-S³⁵ were prepared, and their distribution between the internal phase and the bulk phase was measured before and after the addition of plasma, the lysins being added to the cells either before or after the addition of the inhibitor. The results show that there is a large uptake of these lysins at the red cell surfaces when they are added first, and that the subsequent addition of plasma greatly reduces the quantity of lysin held in the internal phase. Further, if the inhibitor is added first and the lysin subsequently, the internal lysin phase is very incompletely formed. Serum albumin, used in place of plasma, gives essentially similar results.     

HEMOLYTIC SYSTEMS CONTAINING ANIONIC DETERGENTS

1. The members of the homologous series of anionic detergents, the sodium salts of the sulfated straight chain alcohols with the general formula C_nH_2n+1·SO₃·Na, are hemolytic, the lytic activity being at a maximum when the compound contains 14 carbon atoms in the chain. In systems in which lysis is comparatively rapid, the hemolytic effect increases with increasing pH, but in systems containing quantities of lysin near the asymptotic concentrations the pH dependence of the activity is reversed. The effect of temperature is principally one on the velocity constant of the lytic reaction, with smaller effects on the position of the asymptotes of the time-dilution curves and on their shape. 2. The quantities of the detergents which produce disk-sphere transformations are approximately one-tenth of those required to produce complete hemolysis. In most cases, the shape change occurs when there are too few detergent molecules present to cover the red cell surfaces with a monolayer. 3. Plasma inhibits the hemolytic action of these detergents, and, in the quantities in which they occur in plasma, lecithin, serum globulin, cholesterol, and serum albumin, produce inhibitory effects which increase in that order in systems containing the C-14 sulfate. It can be inferred from these inhibitory effects that the anionic detergents can form compounds or complexes with lipid, lipoprotein, and protein components of the red cell ultrastructure.     

A progressive reaction occurring with a radioactive hemolysin,sodium oleate-I 131

Sodium oleate reacts progressively with human red cells at pH 7. By progressive is meant a reaction which is not adequately described as reversible or irreversible; such reactions cannot be stopped once they are under way, and are probably associated with a more or less stable "internal" lysin phase at the cell surfaces. The uptake of the lysin and the effect of dilution on the uptake can be studied by converting sodium oleate into the radioactive form, sodium oleate-I¹³¹. The uptake is a parabolic function of the lysin initially present in the system, and the effect of a tenfold dilution of systems in which red cells have remained in contact with the lysin for 2 minutes is to reduce the lysin taken up at the cell surfaces twofold. The lysin rapidly forms a relatively stable layer at the cell interfaces, and this layer is little affected by the dilution of the system as a whole.     

HEMOLYSIS BY SAPONIN AND SODIUM TAUROCHOLATE,WITH SPECIAL REFERENCE TO THE SERIES OF RYVOSH

1. The series of Ryvosh is obtained when hemolysis of the red cells of the animals concerned occurs with saponin as the lytic agent. 2. The series of Ryvosh is not obtained when R _∞ is taken as the resistance constant and sodium taurocholate is used to hemolyse the cells of the same animals. 3. The hemolysin sodium taurocholate has been found to differ from saponin in that the time-dilution curves are found to approach their respective asymptotes with different values of κ.     

A zone phenomenon and a progressive reaction occurring with a radioactive hemolysin,sodium erucate,I 131

Sodium erucate reacts progressively (i.e., once the reaction is started in a time which is so short that the lysin is in contact with the red cells for 30 seconds, it cannot be stopped even by being diluted 10-fold) with human red cells at pH 7. At the same time, systems containing the lysin and human red cells show a zone phenomenon, lysis occurring most readily in a certain concentration of lysin but more slowly in larger or smaller concentrations. Sodium erucate-I¹³¹ can be used to investigate both the zone phenomenon and the progressive character of the reaction. As regards the former, large concentrations of the lysin react relatively poorly with the red cell surfaces and the resistance of the red cells is relatively high. This may be due to the presence of an admixed inhibitor or to the development of an inhibitory state. The lysin is taken up and fixed by material in the red cell surface, so that the "internal phase" of lysin attached to the cell surfaces is so firmly fixed that a 10-fold dilution has no effect on it. It follows that lysis in these systems is progressive, as it is found to be.     

K-Na EXCHANGE ACCOMPANYING THE PROLYTIC LOSS OF K FROM HUMAN RED CELLS

In systems containing human red cells and sodium taurocholate as a lysin, or distearyl lecithin as a sphering agent, the prolytic loss of K at 25°C. is accompanied by a gain of Na by the cell, the gain being somewhat greater than the K loss. A small volume increase accompanies the exchange. The kinetics of the K loss and the Na gain are similar to those already described; i.e., the changes are rapid at first, and slow down so that after 12 to 20 hours it appears that a new steady state is being approached. Similar, but smaller, losses of K and gains of Na occur when the cells stand in isotonic NaCl at 25°C. without the addition of a lysin or sphering agent. On these and other experimental grounds, it is impossible to retain the idea that the mammalian red cell in general is impermeable to cations. The cells nevertheless seem to be in a steady state with respect to their environment, their ionic composition changing as the composition of the environment is changed. The possible processes by means of which one steady state can be exchanged for another—changes in the permeability of a surface membrane, changes in the velocity of an active ion transfer process dependent on red cell metabolism, and changes in the activity of the ions in the red cell interior as a result of changes in an orderly internal structure—are discussed.     

Sodium Transport in Turtle Erythrocytes : Apparent stimulation of exchange diffusion by anaerobiosis

Studies were performed on Na and K transport by red blood cells of the freshwater turtle under anaerobic and aerobic conditions. Although it had previously been assumed that cation transport in turtle red blood cells was dependent on respiration, the present data show greater Na efflux rates in N₂ than in O₂. However, ouabain inhibited Na transport by the same amount quantitatively in O₂ and N₂ gas phases. Thus there was no difference in ouabain-sensitive or "pump" Na transport rates. Na influx rates were higher in nitrogen than in air and potassium influx rates were not significantly different under aerobic and anaerobic conditions. Moreover in the absence of sodium in the bathing medium no difference between air and nitrogen could be discovered. Finally with ethacrynic acid plus ouabain there was an additional decrease in Na efflux but there was a persisting difference between air and nitrogen. These studies do not rule out the existence of a ouabain-insensitive ethacrynic acid-inhibitable flux; however, they suggest that at least part of the activation of Na efflux observed in N₂ was due to increased exchange diffusion.     

The permeability of human cells to cations after treatment with resorcinol,n-butyl alcohol,and similar lysins

The form of families of curves relating K loss to time in systems containing hypolytic concentrations of resorcinol and of n-butyl alcohol points to the human red cell's being slightly permeable to K and Na even when it is in isotonic NaCl (or plasma), and to the effect of the hypolytic concentrations of lysin being such as to increase this permeability. The rate of reentry of K into red cells which have lost it is more rapid than the rate of the previous loss. This may be due to the reimmersion of the lysin-treated cells in isotonic KCl producing further modifications of the ion-restricting mechanisms associated with the red cell structure. The volume changes observed in systems which show the large K-Na exchanges produced by resorcinol and by n-butyl alcohol are not the same as those which would be expected on the basis of the dual mechanism of hemolysis hypothesis or of the colloid-osmotic hemolysis hypothesis. Extensive swelling of the red cells occurs only when the concentrations of lysin are large enough to produce considerable hemolysis.     

HEMOLYSIS OF CHICKEN BLOOD

1. The time-dilution curves are given for the hemolytic action of saponin, sodium taurocholate, and sodium oleate on nucleated chicken erythrocytes. 2. Saponin and sodium taurocholate cause hemolysis but leave the nuclei and ghosts in suspension, thereby making the end-point of hemolysis more arbitrary than the clear end-point for non-nucleated cell hemolysis. 3. The curves of hemolysis by saponin and taurocholate are shown to be of the same nature as are found in the hemolysis of non-nucleated cells. 4. Sodium oleate causes first hemolysis and then, in the stronger solutions, causes karyolysis. Two pairs of values for κ and c = ∞ are thus obtainable for the same reaction, one pair for the destruction of corpuscular membrane, the other pair for the destruction of the nucleus. 5. Viscosity changes are found in the lysin-cell system with strong concentrations of sodium taurocholate and sodium oleate. Time-viscosity curves are given for these changes. 6. Microscopically, the action of these lysins on the nucleated chicken red cell appears to be similar to their action on the non-nucleated erythrocytes.     

THE POTENTIAL AND RESPIRATION OF FROG SKIN : I. THE EFFECT OF THE HOMOLOGOUS CARBAMATES. II. THE EFFECT OF CERTAIN LYSINS

Measurements of the O₂ consumption and of the potential of frog skin, made under comparable conditions, show that the homologous carbamates (ethyl, propyl, butyl, and amyl) reduce both the O₂ consumption and the potential, but not in a similar manner. In this respect, the effect of the carbamates is like the effect of reduction in O₂ tension. The simple lysins (saponin and the bile salts), on the other hand, abolish the potential without reducing the O₂ consumption at all. Irrespective of whether one considers the concentration of carbamate in the entire system or the amount of carbamate adsorbed by the frog skin, Traube''s rule relating the effect of a carbamate to its position in the homologous series does not seem to apply.     

Hemolysis considered as a progressive reaction in a heterogeneous system

It is demonstrated, without the use of special assumptions, that red cells are heterogeneous with respect to their resistance to at least certain lysins, that the reaction between the cell components and the lysin is virtually irreversible in some cases but reversible, although to different extents, in others, and that the lysin initiates a process in the cell which is not adequately described by the terms reversible and irreversible, but rather by the term progressive. Progressive reactions, i.e. reactions which cannot be stopped once they are well under way, may be looked for in systems which have structure, and in which local reactions occurring at strategic points lead to disproportionate results.     

Volume changes in hemolytic systems containing resorcinol,taurocholate, and saponin

Simultaneous measurement of hemolysis, the volume of the intact cells, and the K lost from the intact cells of systems containing resorcinol, sodium taurocholate, and saponin shows that the volume increases may be conspicuously small while the K losses are large, and that the volume increases are un-equal for equal K losses produced by different lysins. In higher concentrations of the same lysins, the critical volume for hemolysis is a function of the nature of the lysin and of its concentration. It is impossible to say whether these observations are compatible with the current "dual mechanism" and "colloid osmotic" hypotheses of hemolysis, in which the swelling of the cell is supposed to result from the lysin having made it cation-permeable. The difficulty to be overcome is that the theory cannot be developed to describe volume changes in finite time unless we know what assumptions to make about the mobilities of K and Na, the forces driving them into and out of the cell, etc. The experimental results do not suggest, however, that any simple set of assumptions would be satisfactory. The conditions which regulate the upper limit of the swelling, i.e. the point at which a swelling phenomenon becomes a hemolytic phenomenon, are functions of the nature of the lysin and sometimes of its concentration. They require to be specified by an independent statement, over and above any statement which may be made about the rate at which swelling occurs in the system. The simplest view of the situation is that the conditions which regulate the critical volume and those which regulate the rate of swelling are both functions, as yet undefined, of the reaction which takes place between the lysin and the structural components of the red cell.     

Staphylococcal virolysin,a phage-induced lysin; its differentiation from the autolysis of normal cells

Virolysin is a lysin which appears in Staphylococcus aureus K₁ cells infected with phage P₁₄; together with phage, virolysin is released from phage-infected cells at the time of lysis. Autolysin is a lysin formed by uninfected cells of the K₁ strain; autolysin is released from uninfected cells by autolysis. They show the following similarities: Both agents act within the genus Micrococcus. They lyse cells only after the cell has been subjected to a damaging or "sensitizing" treatment, such as heat, bacteriophage, acetone, or ultraviolet irradiation. The course of lysis of heated cells by both lysins has been found to proceed in a similar manner. A constant percentage of cells is lysed, independent of the concentration of lysin; the residual cells remain resistant to either lysin. Lysis proceeds logarithmically with time, and the velocity constants K are proportional to the lysin concentration. K increases with increasing temperature. Both lysins are unaffected by antiserum to the phage. They are inhibited alike by a number of chemicals, including known enzyme inhibitors. Both agents are destroyed by proteolytic enzymes and are precipitated by 40 per cent saturation with (NH₄)₂SO₄. Both lysins are very thermolabile. The two lysins differ with respect to their pH optimum, antigenic relationship and specificity for Micrococcus lysodeikticus. These results suggest that (1) both lysins have many properties associated with enzymes, (2) the lysis of heated cells, which they produce, has some of the characteristics of a chemical reaction, (3) the lysin from the phage-infected cell is clearly different from the lysin of the uninfected cell.     

THE OXIDATION OF SODIUM LACTATE BY HYDROGEN PEROXIDE

By means of a modification of the technique of the Osterhout apparatus it is possible to follow the production of CO₂ from sodium lactate when acted upon by H₂O₂. The results of this process indicate that the reaction is not a simple one but is of an autocatalytic type. This conclusion is borne out by the fact that the determinations of H₂O₂ during the reaction show an increased amount of peroxide during the earlier stages of the reaction. This is considered to be due to the formation of a peroxide by the oxidation of the acetaldehyde (formed by the interaction of H₂O₂ and sodium lactate) with the oxygen of the air. When the reaction is carried out in an atmosphere of nitrogen no increase is observed. Further experiments in nitrogen tend to show that acetaldehyde is the end-product of the action of H₂O₂ alone. The effect of FeCl₃ upon the reaction depends upon the previous treatment of the iron salt. If the iron solution is added to the H₂O₂ before mixing with the lactate there is an increased amount of CO₂. If, however, the iron is added to the lactate before the addition of the peroxide, the action tends to inhibit the production of CO₂. The reaction of H₂O₂ with sodium lactate is comparable to the action of killed yeast and methylene blue as determined by Palladin and his coworkers.     

Respiration and sodium transport in rabbit urinary bladder

Respiration of rabbit urinary bladder was measured in free-floating pieces and in short-circuited pieces mounted in an Ussing chamber. Ouabain, amiloride, and potassium-free saline inhibited respiration approx. 20%; sodium-free saline depressed respiration approx. 40–50%. The coupling ratio between respiration and transport in short-circuited tissues was about two sodium ions per molecule O₂. Chloride-free saline depressed mean oxygen consumption 21% in free-floating tissue pieces; 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and furosemide had no effect. The effect of chloride-free saline in short-circuited tissues was variable; in tissues with low transport rates, respiration was stimulated about 21% while in tissue with high transport rates respiration was reduced about 24%. Nystatin and monensin, both of which markedly increase the conductance of cell membranes with a concomitant increase in sodium entry, stimulated respiration. These data indicate that 50–60% of the total oxygen consumption is not influenced by sodium, 20–25% is linked to (Na⁺ + K⁺)-ATPase transport, while the remaining 25–30% is sodium-dependent but not ouabain-inhibitable.     

Respiration rates, ATP content and ionic regulation in hepatocytes of starving lamprey during the pre-spawning period of their life cycle

During the winter pre-spawning migration, lampreys Lampetra fluviatilis stop feeding, and their liver metabolism is reduced substantially. Aerobic ATP production in hepatocytes decreased to one third and ATP content decreased by 50% as compared with the values in autumn. In spite of the decrease of endogenous and phosphorylating (oligomycin-sensitive) respiration in winter, the oxygen consumption used to drive sodium and potassium pumping through Na,K-ATPase activity (ouabain-sensitive respiration) remained virtually constant. Consequently its share in phosphorylating respiration increased from 16·3% in November to 54·2% in February. Potassium influx was similar within the range of ATP content between 2·5 and 1 nmol 10⁻⁶ cells and decreases only in hepatocytes which contained <0·8 nmol ATP 10⁻⁶ cells.