Cholinesterase activity per unit surface area of conducting membranes

According to theory, the action of acetylcholine (ACh) and ACh-esterase is essential for the permeability changes of excitable membranes during activity. It is, therefore, pertinent to know the activity of ACh-esterase per unit axonal surface area instead of per gram nerve, as it has been measured in the past. Such information has now been obtained with the newly developed microgasometric technique using a magnetic diver. (1) The cholinesterase (Ch-esterase) activity per mm² surface of sensory axons of the walking leg of lobster is 1.2 x 10^-3 µM/hr. (σ = ± 0.3 x 10^-3; SE = 0.17 x 10^-3); the corresponding value for the motor axons isslightly higher: 1.93 x 10^-3 µM/hr. (σ = ± 0.41 x 10^-3; SE = ± 0.14 x 10^-3). Referred to gram nerve, the Ch-esterase activity of the sensory axons is much higher than that of the motor axons: 741 µM/hr. (σ = ± 73.5; SE = ± 32.6) versus 111.6 µM/hr. (σ = ± 28.3; SE = ± 10). (2) The enzyme activity in the small fibers of the stellar nerve of squid is 3.2 x 10^-4 µM/mm²/hr. (σ = ± 0.96 x 10^-4; SE = ± 0.4 x 10^-4). (3) The Ch-esterase activity per mm² surface of squid giant axon is 9.5 x 10^-5 µM/hr. (σ = ± 1.55 x 10^-5; SE = ± 0.38 x 10^-5). The value was obtained with small pieces of carefully cleaned axons after removal of the axoplasm and exposure to sonic disintegration. Without the latter treatment the figurewas 3.85 x 10^-5 µM/mm²/hr. (σ = ± 3.24 x 10^-5; SE = ± 0.93 x 10^-5). The experiments indicate the existence of permeability barriers in the cell wall surrounding part of the enzyme, since the substrate cannot reach all the enzyme even when small fragments of the cell wall are used without disintegration. (4) On the basis of the data obtained, some tentative approximations are made of the ratio of ACh released to Na ions entering the squid giant axon per cm² per impulse.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : III. OXYGEN CONSUMPTION AND CELL DIVISION OF FERTILIZED SEA URCHIN EGGS IN THE PRESENCE OF RESPIRATORY INHIBITORS

1. The effects of a number of respiratory inhibiting agents on the cell division of fertilized eggs of Arbacia punctulata have been determined. For eggs initially exposed to the reagents at 30 minutes after fertilization at 20°C., the levels of oxygen consumption prevailing in the minimum concentrations of reagents which produced complete cleavage block were (as percentages of the control): In 0.4 per cent O₂-99.6 per cent N₂, 32; in 0.7 per cent O₂-99.3 per cent CO, 32; in 1.6 x 10^–4 M potassium cyanide, 34; in 1 x 10^–3 M phenylurethane, 70; in 4 x 10^–3 M 5-isoamyl-5-ethyl barbituric acid, 20; in 3 x 10^–4 M iodoacetic acid, 53. 2. The carbon monoxide inhibition of oxygen consumption and cell division was reversed by light. The percentage inhibition of oxygen consumption by carbon monoxide in the dark is described by the usual mass action equation with K, the inhibition constant, equal to approximately 60, as compared to values of 5 to 10 for yeast and muscle. In 20 per cent O₂-80 per cent CO in the dark there was a slight stimulation of oxygen consumption, averaging 20 per cent. 3. Spectroscopic examination of fertilized and unfertilized Arbacia eggs reduced by hydrosulfite revealed no cytochrome bands. The thickness and density of the egg suspension was such as to indicate that, if cytochrome is present at all, the amount in Arbacia eggs is extremely small as compared to that in other tissues having a comparable rate of oxygen consumption. 4. Three reagents poisoning copper catalyses, potassium dithio-oxalate (10^–2 M), diphenylthiocarbazone (10^–4 M), and isonitrosoacetophenone (2 x 10^–3 M) produced no inhibition of division of fertilized Arbacia eggs. 5. These results indicate that the respiratory processes required to support division in the Arbacia egg may perhaps differ in certain essential steps from the principal respiratory processes in yeast and muscle.     

Sarcoplasmic reticulum. IX. The permeability of sarcoplasmic reticulum membranes

Fragmented sarcoplasmic reticulum (FSR) membranes isolated from rabbit skeletal muscle are impermeable to inulin-¹⁴C (mol wt 5,000), and dextran-¹⁴C (mol wt 15,000–90,000) at pH 7.0–9.0, yielding an excluded space of 4–5 µl/mg microsomal protein. In the same pH range urea and sucrose readily penetrate the FSR membrane. EDTA or EGTA (1 mM) increased the permeability of microsomes to inulin-¹⁴C or dextran-¹⁴C at pH 8–9, parallel with the lowering of the FSR-bound Ca⁺⁺ content from initial levels of 20 nmoles/mg protein to 1–3 nmoles/mg protein. EGTA was as effective as EDTA, although causing little change in the Mg⁺⁺ content of FSR. The permeability increase caused by chelating agents results from the combined effects of high pH and cation depletion. As inulin began to penetrate the membrane there was an abrupt fall in the rate of Ca⁺⁺ uptake and a simultaneous rise in ATPase activity. At 40°C inulin penetration occurred at pH 7.0 with 1 mM EDTA and at pH 9.0 without EDTA, suggesting increased permeability of FSR membranes. This accords with the higher rate of Ca⁺⁺ release from FSR at temperatures over 30°C. The penetration of microsomal membranes by anions is markedly influenced by charge effects. At low ionic strength and alkaline pH acetate and Cl are partially excluded from microsomes when applied in concentrations not exceeding 1 mM, presumably due to the Donnan effect. Penetration of microsomal water space by acetate and Cl occurs at ionic strengths sufficiently high to minimize charge repulsions.     

THE EFFECTS OF CAFFEINE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE FERTILIZED EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. By means of the Warburg-Barcroft microrespirometer apparatus and the Warburg direct method, the relative effect of caffeine upon the O₂ consumption of the fertilized egg of Arbacia punctulata was shown for the following concentrations in sea water: 0.002 per cent (M/10,000), 0.004 per cent (M/5,000), 0.02 per cent (M/1,000), 0.1 per cent (M/200), 0.2 per cent (M/100), 0.5 per cent (M/40), and 2 per cent (M/10). 2. In comparison with the normal eggs (uninhibited, non-caffeine-treated controls), caffeine in concentrations including and greater than 0.1 per cent (M/200) depressed the average uptake from approximately 25 to 61 per cent over the 3 hour period. In a number of instances, as typified by Experiment 10, the effective inhibitory concentration ranged from 0.02 per cent (M/1,000) upward and the degree of depression of the O₂ consumption ranged from 10.6 per cent to 60.6 per cent. 3. All caffeine concentrations including and above 0.02 per cent (M/1,000) in the series used, resulted in decreasing the normal rate of cleavage division in the fertilized Arbacia eggs. 4. The higher concentrations (0.5 and 2 per cent) produced a complete blockage of the cleavage process. 5. Complete cleavage inhibition was noted only when the O₂ uptake had been depressed to 50 per cent or more of the normal controls. 6. O₂ consumption-time relationship data indicate an average depression, in O₂ consumption over a 3 hour period, ranging from 25 per cent with a caffeine concentration of 0.1 per cent to a 61 per cent inhibition with a concentration of 2 per cent. 7. Concentrations of less than 0.1 per cent (certainly of less than 0.02 per cent) give variable results and indicate no significant effect. 8. It is inferred from the respiration data presented that it is probable that the inhibition of the O₂ consumption in fertilized Arbacia eggs is due to the influence of caffeine upon the main (activity or primary) pathway. It will be observed that there are certain similarities of the caffeine data to the degree of inhibition accomplished by sodium cyanide. Moreover, it has been demonstrated that the cyanide probably acts on the cytochrome oxidase step in the cytochrome oxidase-cytochrome chain of reactions constituting the O₂ uptake phase of respiratory metabolism. It is not improbable, therefore, that caffeine also may act upon the cytochrome oxidase enzyme. 9. From the viewpoint of environmental conditions influencing reproductive phenomena, it is of interest that caffeine can affect the normal metabolism of the zygote.     

COMPARATIVE STUDIES ON RESPIRATION : IX. THE EFFECTS OF ANTAGONISTIC SALTS ON THE RESPIRATION OF ASPERGILLUS NIGER.

1. In the presence of 0.05 per cent dextrose the respiration of Aspergillus niger is increased by NaCl in concentrations of 0.25 to 0.5M, and by 0.5M CaCl₂. 2. Stronger concentrations, as 2M NaCl and 1.25M CaCl₂, decrease the respiration. The decrease in the higher concentrations is probably an osmotic effect of these salts. 3. A mixture of 19 cc. of NaCl and 1 cc. of CaCl₂ (both 0.5M) showed antagonism, in that the respiration was normal, although each salt alone caused an increase. 4. Spores of Aspergillus niger did not germinate on 0.5M NaCl (plus 0.05 per cent dextrose) while they did on 0.5M CaCl₂ (plus 0.05 per cent dextrose) and on various mixtures of the two. This shows that a substance may have different effects on respiration from those which it has upon growth.     

EFFECTS OF GUAIACOL AND HEXYLRESORCINOL IN THE PRESENCE OF BARIUM AND CALCIUM

Guaiacol was applied at two spots on the same cell of Nitella. At one spot it was dissolved in 0.01 M NaCl, at the other in 0.01 M CaCl₂ or BaCl₂. The effect was practically the same in all cases, i.e. a similar change of P.D. in a negative direction, involving a more or less complete loss of P.D. (depolarization). When hexylresorcinol was used in place of guaiacol the result was similar. That Ca⁺⁺ and Ba⁺⁺ do not inhibit the effect of these organic depolarizing substances may be due to a lack of penetration of Ca⁺⁺ and Ba⁺⁺. The organic substances penetrate more rapidly and their effect is chiefly on the inner protoplasmic surface which is the principal seat of the P.D.     

THE ACTIVATION OF STARFISH EGGS BY ACIDS : II. THE ACTION OF SUBSTITUTED BENZOIC ACIDS AND OF BENZOIC AND SALICYLIC ACIDS AS INFLUENCED BY THEIR SALTS.

1. Comparison of the rates of activation of unfertilized starfish eggs in pure solutions of a variety of parthenogenetically effective organic acids (fatty acids, carbonic acid, benzoic and salicylic acids, chloro- and nitrobenzoic acids) shows that solutions which activate the eggs at the same rate, although widely different in molecular concentration, tend to be closely similar in C_H. The dissociation constants of these acids range from 3.2 x 10^–7 to 1.32 x 10^–3. 2. In the case of each of the fourteen acids showing parthenogenetic action the rate of activation (within the favorable range of concentration) proved nearly proportional to the concentration of acid. The estimated C_H of solutions exhibiting an optimum action with exposures of 10 minutes (at 20°) lay typically between 1.1 x 10^–4 M and 2.1 x 10^–4 M (pH = 3.7–3.96), and in most cases between 1.6 x 10^–4 M and 2.1 x 10^–4 M (pH = 3.7–3.8). Formic acid (C_H = 4.2 x 10^–4 M) and o-chlorobenzoic acid (C_H = 3.5 x 10^–4 M) are exceptions; o-nitrobenzoic acid is ineffective, apparently because of slow penetration. 3. Activation is not dependent on the penetration of H ions into the egg from without, as is shown by the effects following the addition of its Na salt to the solution of the activating acid (acetic, benzoic, salicylic). The rate of activation is increased by such addition, to a degree indicating that the parthenogenetically effective component of the external solution is the undissociated free acid. Apparently the undissociated molecules alone penetrate the egg freely. It is assumed that, having penetrated, they dissociate in the interior of the egg, furnishing there the H ions which effect activation. 4. Attention is drawn to certain parallels between the physiological conditions controlling activation in the starfish egg and in the vertebrate respiratory center.     

Studies on streaming. I. Inhibition of protoplasmic streaming and cytokinesis of Chaos chaos by adenosine triphosphate and reversal by magnesium and calcium ions

In Chaos chaos streaming, motility, and cytokinesis were inhibited nearly 100% for several hours by 2.5–5 mM sodium adenosine triphosphate (ATP)¹ added to culture fluid. All three effects were completely prevented by the addition of equimolar Mg⁺⁺ or Ca⁺⁺ ions but not Na⁺ to the ATP/culture fluid solution. The effects of ATP were not reproduced by EDTA, EGTA, colchicine, or AMP. Sodium pyrophosphate produced about 50% inhibition at 5 mM. Studies with ¹⁴C-ATP showed that 5 x 10^-5 to 5 x 10^-4 mmole of ATP was firmly associated with each milliliter of packed cells after an hour''s incubation at 24°C. Labeling studies also showed that prevention of the ATP effects by Mg⁺⁺ ions was not due to a decrease in the amount of ATP associated with the cells.     

INHIBITION OF PHOTOSYNTHESIS IN CHLORELLA PYRENOIDOSA BY THE IODO-ACETYL RADICAL

Photosynthesis in Chlorella pyrenoidosa is inhibited by iodo-acetic acid and iodo-acetamide, both of which attack the Blackman reaction. Since acetamide is without effect, the iodo-acetyl radical must be responsible. The study of the action of the acid is complicated by the fact that its ions penetrate slowly, if at all, so that negative results with this agent are without significance unless penetration can be established. The absorption spectrum of the cells is not affected by concentrations of iodo-acetamide which completely inhibit photosynthesis. This establishes that the chromophore groups of chlorophyll are not involved, and renders it unlikely that any other part of the molecule is. Inasmuch as cyanide likewise inhibits by way of the Blackman reaction, it would seem necessary to postulate that this complex can be attacked at two different loci, which may or may not be on the same molecule. The presence of the iodo-acetyl radical also gives rise to three other effects. (1) Concentrations (10^–5 M or less) too small to inhibit photosynthesis may increase the rate by interacting with the photochemical complex. (2) Concentrations (ca. 10^–4 M) which inhibit photosynthesis increase the rate of respiration. (3) Concentrations (10^–3 M or more) higher than those required to inhibit photosynthesis inhibit respiration.     

MOLECULAR WEIGHT AND ELECTROPHORESIS OF CRYSTALLINE RIBONUCLEASE

Electrophoretic studies on purified crystalline ribonuclease showed the absence of any impurities differing in mobility from the bulk of material. The isoelectric point of ribonuclease was found by electrophoresis to be at about pH 7.8. Ultracentrifuge studies indicated fair homogeneity of ribonuclease in solution. Only one moving component has been observed. The molecular weight of ribonuclease was found to be 12,700 from rate of sedimentation (S ²⁵ = 1.85 x 10^–13 in 0.5 M (NH₄)₂SO₄) and diffusion measurement (D = 1.36 x 10_–6 in 0.5 M (NH₄)₂SO₄), in good agreement with the average value of 13,000 found from equilibrium measurements. This low value for the molecular weight of a protein would seem to discredit the value 17,600 as representing a universal unit weight for proteins in general.     

Cation regulation in the smooth muscle of frog stomach

Cation composition of frog smooth muscle cells was investigated. Fresh stomach muscle rings resembled skeletal muscle, but marked Na gain and K loss followed immersion. Mean Na (49.8–79.7 mM/kg tissue) and K (61.8–80.1 mM/kg tissue) varied between batches, but were stable for long periods in vitro. Exchange of 6–30 mM Na/kg tissue with ²²Na was extremely slow and distinct. Extracellular water was estimated from sucrose-¹⁴C uptake. Calculated exchangeable intracellular Na was 9 mM/kg cell water, and varied little. Thus steady-state transmembrane cation gradients appeared to be steep. K-free solution had only slight effects. Ouabain (10^-4 M) caused marked Na gain and reciprocal K loss; at 30°C, Na and K varied linearly with time over a wide range of contents, indicating constant net fluxes. Net fluxes decreased with temperature decrease. ²²Na exchange in ouabain-treated tissue at 20–30°C was rapid and difficult to analyze. The best minimum estimates of unidirectional Na fluxes at 30°C were 10–12 times the constant net flux; constant pump efflux may explain these findings. The rapidity of Na exchange may not reflect very high permeability, but it does require a high rate of transport work.     

Sodium fluxes in internally dialyzed squid axons

The effects which alterations in the concentrations of internal sodium and high energy phosphate compounds had on the sodium influx and efflux of internally dialyzed squid axons were examined. Nine naturally occurring high energy phosphate compounds were ineffective in supporting significant sodium extrusion. These compounds were: AcP, PEP, G-3-P, ADP, AMP, GTP, CTP, PA, and UTP.¹ the compound d-ATP supported 25–50% of the normal sodium extrusion, while ATP supported 80–100%. The relation between internal ATP and sodium efflux was nonlinear, rising most steeply in the range 1 to 10 µM and more gradually in the range 10 to 10,000 µM. There was no evidence of saturation of efflux even at internal ATP concentrations of 10,000 µM. The relation between internal sodium and sodium efflux was linear in the range 2 to 240 mM. The presence of external strophanthidin (10 µM) changed the sodium efflux to about 8–12 pmoles/cm² sec regardless of the initial level of efflux; this changed level was not altered by subsequent dialysis with large concentrations of ATP. Sodium influx was reduced about 50 % by removal of either ATP or Na and about 70 % by removing both ATP and Na from inside the axon.     

THE COMBINATION OF GELATIN WITH HYDROCHLORIC ACID : II. NEW DETERMINATIONS OF THE ISOELECTRIC POINT AND COMBINING CAPACITY OF A PURIFIED GELATIN.

1. Cooper''s gelatin purified according to Northrop and Kunitz exhibited a minimum of osmotic pressure and a maximum of opacity at pH 5.05 ±0.05. The pH of solutions of this gelatin in water was also close to this value. It is inferred that such gelatin is isoelectric at this pH and not at pH 4.70. 2. Hydrogen electrode measurements with KCl-agar junctions were made with concentrated solutions of this gelatin in HCl up to 0.1 M. The combination curve calculated from these data is quite exactly horizontal between pH 2 and 1, indicating that 1 gm. of this gelatin can combine with a maximum of 9.35 x 10^–4 equivalents of H⁺. 3. Conductivity titrations of this gelatin with HCl gave an endpoint at 9.41 (±0.05) x 10^–4 equivalents of HCl per gram gelatin. 4. E.M.F. measurements of the cell without liquid junction, Ag, AgCl, HCl + gelatin, H₂, lead to the conclusion that this gelatin in 0.1 M HCl combines with a maximum of 9.4 x 10^–4 equivalents of H⁺ and 1.7 x 10^–4 equivalents of Cl^- per gram gelatin.     

Some properties of bound and soluble dynein from sea urchin sperm flagella

Axonemes were isolated from sperm of Colobocentrotus by a procedure involving two extractions with 1% Triton X-100 and washing The isolated axonemes contained 7 x 10¹⁵ g protein per µm of their length. Treatment of the axonemes with 0 5 M KCl for 30 min extracted 50–70% of the flagellar ATPase protein, dynein, and removed preferentially the outer arms from the doublet tubules. Almost all of the dynein (85–95%) could be extracted from the axonemes by dialysis at low ionic strength. In both cases the extracted dynein sedimented through sucrose gradients at 12–14S, and no 30S form was observed The enzymic properties of dynein changed when it was extracted from the axonemes into solution. Solubilization had a particularly marked effect on the KCl- and pH-dependence of the ATPase activity. The pH-dependence of soluble dynein was fairly simple with a single peak extending from about pH 6 to pH 10. The pH-dependence of bound dynein was more complex. In 0.1 M KCl, the bound activity appeared to peak at about pH 9, and dropped off rapidly with decreasing pH, reaching almost zero at pH 7; an additional peak at pH 10 0 resulted from the breakdown of the axonemal structure and solubilization of dynein that occurred at about this pH. A similar curve was obtained in the absence of KCl, except for the presence of a further large peak at pH 8 Measurement of the kinetic parameters of soluble dynein showed that both K_m and V_max increased with increasing concentrations of KCl up to 0.5 M When bound dynein was assayed under conditions that would induce motility in reactivated sperm (0 15 M KCl with Mg⁺⁺ activation), it did not obey Michaelis-Menten kinetics, although it did when assayed under other conditions. The complex enzyme-kinetic behavior of bound dynein, and the differences between its enzymic properties and those of soluble dynein, may result from its interactions with tubulin and other axonemal proteins     

THE COMBINATION OF SALTS AND PROTEINS. I

1. It has been found that the ratios of the total concentrations of Ca, Mg, K, Zn, inside and outside of gelatin particles do not agree with the ratios calculated according to Donnan''s theory from the hydrogen ion activity ratios. 2. E.M.F. measurements of Zn and Cl electrode potentials in such a system show, however, that the ion activity ratios are correct, so that the discrepancy must be due to a decrease in the ion concentration by the formation of complex ions with the protein. 3. This has been confirmed in the case of Zn by Zn potential measurements in ZnCl₂ solutions containing gelatin. It has been found that in 10 per cent gelatin containing 0.01 M ZnCl₂ about 60 per cent of the Zn⁺⁺ is combined with the gelatin. 4. If the activity ratios are correctly expressed by Donnan''s equation, then the amount of any ion combined with a protein can be determined without E.M.F. measurements by determining its distribution in a proper system. If the activity ratio of the hydrogen ion and the activity of the other ion in the aqueous solution are known, then the activity and hence the concentration of the ion in the protein solution can be calculated. The difference between this and the total molar concentration of the ion in the protein represents the amount combined with the protein. 5. It has been shown that in the case of Zn the values obtained in this way agree quite closely with those determined by direct E.M.F. measurements. 6. The combination with Zn is rapidly and completely reversible and hence is probably not a surface effect. 7. Since the protein combines more with Zn than with Cl, the addition of ZnCl₂ to isoelectric gelatin should give rise to an unequal ion distribution and hence to an increase in swelling, osmotic pressure, and viscosity. This has been found to be the case.     

Decreased K+ conductance produced by Ba++ in frog sartorius fibers

The action of Ba⁺⁺ on membrane potential (E_m) and resistance (R_m) of frog (R. pipiens) sartorius fibers was studied. In normal Cl^- Ringer''s, Ba⁺⁺ (<9 mM) did not depolarize or induce contractions, but increased R_m slightly above the control value of 3.8 ± 0.6 KΩ-cm². In Cl^--free Ringer''s (methane sulfonate) R_m was 28.8 ± 2.8 KΩ-cm², and low concentrations of Ba⁺⁺ (0.05–5.0 mM) depolarized and induced spontaneous contractions (fibrillation), even in tetrodotoxin. To stop disturbance of the microelectrodes, contractions were prevented by using two Cl^--free solutions: (a) twice hypertonic with sucrose (230 mM), or (b) high K⁺ (83 mM) partially replacing Na⁺. In the hypertonic solution, the fiber diameters decreased, E_m increased slightly, and R_m decreased to 9.0 ± 0.6 KΩ-cm² (perhaps due to swelling of sarcotubules). Ba⁺⁺ (0.5 mM) rapidly increased R_m to 31.3 ± 3.8, decreased E_m (e.g., to -30 mv), and induced spontaneous "action potentials;" Sr⁺⁺ had no effect. In the high K⁺ solution, the fibers were nearly completely depolarized, and R_m was decreased markedly to 1.5 ± 0.2 KΩ-cm²; Ba⁺⁺ increased R_m to 6.7 ± 0.5 KΩ-cm². The Ba⁺⁺ actions usually began within 0.5 min and reached a maximum within 5 min. Addition of SO₄ ⁼, to precipitate the Ba⁺⁺, rapidly reversed the increase in R_m. Ba⁺⁺ must act by decreasing K⁺ conductance (g_K). In Cl^- Ringer''s, the high g_Cl/g_K ratio masked the effect of Ba⁺⁺ on g_K. Thus, small concentrations of Ba⁺⁺ specifically and rapidly decrease g_K.     

CHEMICAL ANTAGONISM OF IONS : IV. EFFECT OF SALT MIXTURES ON GLYCINE ACTIVITY.

The pH of a 0.01 molar solution of glycine, half neutralized with NaOH, is 9.685. Addition of only one of the salts NaCl, KCl, MgCl₂, or CaCl₂ will lower the pH of the solution (at least up to 1 µ). If a given amount of KCl is added to a glycine solution, the subsequent addition of increasing amounts of NaCl will first raise the pH (up to 0.007 M NaCl). Further addition of NaCl (up to 0.035 M NaCl) will lower the pH, and further additions slightly raise the pH. The same type of curve is obtained by adding NaCl to glycine solution containing MgCl₂ or CaCl₂ except that the first and second breaks occur at 0.015 M and 0.085 M NaCl, respectively. Addition of CaCl₂ to a glycine solution containing MgCl₂ gives the same phenomena with breaks at 0.005 M and 0.025 M CaCl; or at ionic strengths of 0.015 µCaCl₂ and 0.075 µCaCl₂. This indicates that the effect is a function of the ionic strength of the added salt. These effects are sharp and unmistakable. They are almost identical with the effects produced by the same salt mixtures on the pH of gelatin solutions. They are very suggestive of physiological antagonisms, and at the same time cannot be attributed to colloidal phenomena.     

DIFFERING RATES OF DEATH AT INNER AND OUTER SURFACES OF THE PROTOPLASM : I. EFFECTS OF FORMALDEHYDE ON NITELLA

When protoplasm dies it becomes completely and irreversibly permeable and this may be used as a criterion of death. On this basis we may say that when 0.2 M formaldehyde plus 0.001 M NaCl is applied to Nitella death arrives sooner at the inner protoplasmic surface than at the outer. If, however, we apply 0.17 M formaldehyde plus 0.01 M KCl death arrives sooner at the outer protoplasmic surface. The difference appears to be due largely to the conditions at the two surfaces. With 0.2 M formaldehyde plus 0.001 M NaCl the inner surface is subject to a greater electrical pressure than the outer and is in contact with a higher concentration of KCl. In the other case these conditions are more nearly equal so that the layer first reached by the reagent is the first to become permeable. The outer protoplasmic surface has the ability to distinguish electrically between K⁺ and Na⁺ (potassium effect). Under the influence of formaldehyde this ability is lost. This is chiefly due to a falling off in the partition coefficient of KCl in the outer protoplasmic surface. At about the same time the inner protoplasmic surface becomes completely permeable. But the outer protoplasmic surface retains its ability to distinguish electrically between different concentrations of the same salt, showing that it has not become completely permeable. After the potential has disappeared the turgidity (hydrostatic pressure inside the cell) persists for some time, probably because the outer protoplasmic surface has not become completely permeable.     

THE COMBINATION OF CERTAIN PROTEINS WITH HYDROCHLORIC ACID

Electromotive force measurements of cells without liquid junction, of the type Ag, AgCl, HCl + protein, H₂, have been made at 30°C. with the proteins gelatin, edestin, and casein in 0.1 M hydrochloric acid. The data are consistent with the assumptions of a constant combining capacity of each protein for hydrogen ion, no combination with chloride ion, and Failey''s principle of a linear variation of the logarithm of the mean activity coefficient of the acid with increasing protein concentration. The combining capacities for hydrogen ion so obtained are 13.4 x 10^–4 for edestin, 9.6 x 10^–4 for gelatin, and 8.0 x 10^–4 for casein, in equivalents of combined H⁺ per gm. of protein.     

THE PHYSIOLOGICAL EFFECTS OF l-TYROSINE AND l-PHENYL-ALANINE ON THE RATE AND QUALITY OF PULSATION IN THE SEVENTY-TWO HOUR EXTIRPATED EMBRYONIC CHICK HEART

1. 72 hour isolated chick hearts show an increase in pulsation rate when placed in M/1000, M/10,000, and M/50,000 l-tyrosine solutions. The optimal effect is seen in M/10,000 and M/50,000 l-tyrosine. 2. All hearts show disturbance of rhythm either in the form of irregular rhythm or heart block. 3. 62 hour isolated chick hearts are not susceptible to l-tyrosine while 96 hour hearts are markedly sensitive. 4. 72 hour isolated chick hearts placed in 1 part in 10,000 and 1 part in 50,000 l-epinephrine show approximately the same effects as were seen with l-tyrosine. 5. 72 hour isolated chick hearts placed in M/1000 and M/10,000 l-phenylalanine show an initial depression followed by an l-tyrosine effect.