THE ISOELECTRIC POINTS OF THE PROTEINS IN CERTAIN VEGETABLE JUICES

The state in which a protein substance exists depends upon the nature of its combination with acids or bases and is changed by change in the protein compound. The nature of the compound of a protein that exists at any hydrogen ion concentration can be ascertained if the isoelectric point of the protein is known. Accordingly information regarding the isoelectric points of vegetable proteins is of importance for operations in which it may be desirable to change the state of protein substances, as in the dehydration of vegetables. The Protein in Potato Juice.—The hydrogen ion concentration of the filtered juice of the potato is in the neighborhood of 10^–7 N. Such juice contains the globulin tuberin to the extent of from 1 to 2 per cent. The character of the compound of tuberin that exists in nature was suggested by its anodic migration in an electric field. The addition of acid to potato juice dissociated this compound and liberated tuberin at its isoelectric point. The isoelectric point of tuberin coincided with a slightly lower hydrogen ion concentration than 10^–4 N. At that reaction it existed most nearly uncombined. The flow of current during cataphoresis was greatest in the neighborhood of the isoelectric point. This evidence supplements that of the direction of the migration of tuberin, since it also suggests the existence of the greatest number of uncombined ions near this point. At acidities greater than the isoelectric point tuberin combined with acid. The compound that was formed contained nearly three times as much acid as was needed to dissociate the tuberin compound that existed in nature. At such acidities tuberin migrated to the cathode. Though never completely precipitated tuberin was least soluble in the juice of the potato in the neighborhood of its isoelectric point. Both the compounds of tuberin with acids and with bases were more soluble in the juice than was uncombined tuberin. The nature of the slight precipitate that separated when potato juice was made slightly alkaline was not determined. The Protein in Carrot Juice.—The isoelectric point of the protein in carrot juice coincided with that of tuberin. Remarkably similar also were the properties of carrot juice and the juice of the potato. Existing in nature at nearly the same reaction they combined with acids and bases to nearly the same extent and showed minima in solubility at the same hydrogen ion concentrations. The greatest difference in behavior concerned the alkaline precipitate which, in the carrot, was nearly as great as the acid precipitate. The Protein in Tomato Juice.—The protein of the tomato existed in a precipitated form near its isoelectric point. Accordingly it was not present to any extent in filtered tomato juice. If, however, the considerable acidity at which the tomato exists was neutralized the protein dissolved and was filterable. It then migrated to the anode in an electric field. The addition of sufficient acid to make the hydrogen ion concentration slightly greater than 10^–5 N again precipitated the protein at its isoelectric point. At greater acidities migration was cathodic.     

STIMULATION BY MINERAL AND FATTY ACIDS IN THE BARNACLE BALANUS BALANOIDES

1. Stimulation in the rock barnacle Balanus balanoides by hydrochloric, sulfuric, and nitric acids, and by the first seven members of the normal aliphatic acid series has been studied. The hydrogen ion concentrations of the solutions tested varied from 3.2 x 10^–8 to 5.889 x 10^–6. The criterion of response was percentage closure in groups of individuals, recorded at 1 minute intervals until maximum closure occurred. 2. The intensity of stimulation by these acids is proportional to the effects of two forces, one related to the change in the (H⁺), and the other to the field of force around the anion of the acid added to the environment. 3. A preliminary interpretation of the results led to the development of the following expression which fits approximately the data obtained at the end of 4 minutes: Per cent closure = 100 – 100e ^{–0.1z+(0.003125)2–0.1z+(0.003125)2n(z–0.4)} where z is the (H⁺) x 10⁷ and n is the number of carbon atoms (if present) in the anion of the acid. This equation assumes that the anions of the mineral acids enter into the reaction stoichiometrically, and emphasizes the difference in the fields of force around the anion of the fatty acids, a difference which is correlated with the length of the carbon chain. 4. A further analysis of the data revealed the presence of three or more receptor groups which appeared to be differentially affected by forces originating from the anions of the acids. 5. The order of stimulating efficiency for the mineral acids was found to be: HCl>H₂SO₄>HNO₃. 6. The order of stimulating efficiency for the fatty acids was found to be: heptylic>caproic>valeric>butyric = acetic>propionic = formic.     

ACID PENETRATION INTO LIVING TISSUES

The threshold concentrations for sourness of nine acids have been determined with an accuracy of about 8 per cent, and the H⁺ ion concentration of these acids measured. Calculations have been made of the relative concentration gradients of the undissociated acid across the cell membrane for a series of acids having equal sourness and also for a series of acids having equal penetration velocity as determined from experiments by Crozier on Chromodoris and on Allolobophora. For solutions of equal pH a high degree of sourness has been found to be associated with a high penetration velocity of the undissociated acid or of the anion. A comparison of these gradients with the results of adsorption experiments on charcoal indicates that the acids are taken into the tissues by an adsorption process. Polar groups such as OH and Cl and Br are found to have a very marked effect in reducing the ability of organic acids to penetrate living tissues. The important rôle of optical activity of the acids in determining their physiological action has been noted.     

Regulation of Lipid Droplet Size in Mammary Epithelial Cells by Remodeling of Membrane Lipid Composition—A Potential Mechanism

Milk fat globule size is determined by the size of its precursors—intracellular lipid droplets—and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control) or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P < 0.0001). When cultured with oleic acid, 22% of the cells contained large lipid droplets (>3 μm) and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001). In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001). In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 μm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content.     

STIMULATION OF FUNDULUS BY OXALIC AND MALONIC ACIDS AND BREATHING RHYTHM AS FUNCTIONS OF TEMPERATURE

1. Chemical stimulation as a function of temperature was studied by using oxalic acid in fresh and salt water and malonic acid in salt water as stimulating agents on Fundulus. According to the Arrhenius equation the following µ values were obtained for the various acid solutions between 0 and 29°C.: for 0.002N oxalic in fresh water—15,800; 33,000; for 0.0008N oxalic in fresh water—15,800; 33,000; 48,000; for 0.002N oxalic in salt water—19,400; 24,100; 56,500; for 0.004N and 0.002N malonic in salt water—20,600; 65,000. At a critical temperature there is a sharp transition from one thermal increment to another. 2. The chemical processes controlling stimulation do not change with concentration, for different normalities of a single acid yield the same µ values. Distinctly different temperature characteristics were obtained for stimulation by oxalic in salt and fresh water. Likewise stimulation by oxalic and malonic in salt water yielded very different increments. This temperature study indicates that the controlling chemical reactions determining rate of response are different for the same acid in two different environments, or for two dibasic acids in the same environment. Other work indicates, however, that the fundamental stimulation system is the same for all the adds in both environments. Chemical rather than physical processes limit the rate of response since all the values are above 15,000. Stimulation depends upon a series of interrelated chemical reactions, each with its own temperature characteristic. Under varying conditions (e.g. change of temperature, environment, or acid) different chemical reactions may become the slowest or controlling process which determines the rate of response. 3. The variation of response, as measured by the probable error of the mean response time of the fish, is the same function of temperature as reaction time itself. Hence variability is not independent of reaction time and is controlled by the same catenary series of events which determine rate of response to stimulation. 4. Breathing rhythm of Fundulus as related to temperature was studied in both salt and fresh water. In salt water the temperature characteristic is 8,400 while in fresh water it is 16,400 below 9.5°C., and 11,300 above this critical temperature. These µ values are typical of those which have been reported by other workers for respiratory and oxidative biological phenomena. A change in thermal increment with an alteration in environment indicates that different chemical reactions with characteristic velocity constants are controlling the breathing rhythm in salt and fresh water.     

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.     

THE INFLUENCE OF HYDROGEN ION CONCENTRATION ON THE INACTIVATION OF PEPSIN SOLUTIONS

1. Pepsin in solution at 38°C. is most stable at a hydrogen ion concentration of about 10^–5 (pH 5.0). 2. Increasing the hydrogen ion concentration above pH 5.0 causes a slow increase in the rate of destruction of pepsin. 3. Decreasing the hydrogen ion concentration below pH 5.0 causes a very rapid increase in the rate of destruction of the enzyme. 4. Neither the purity of the enzyme solution nor the anion of the acid used has any marked effect on the rate of destruction or on the zone of hydrogen ion concentration in which the enzyme is most stable. 5. The existence of an optimum range of hydrogen ion concentration for the digestion of proteins by pepsin cannot be explained by the destruction of the enzyme by either too weak or too strong acid.     

AMPHOTERIC COLLOIDS : I. CHEMICAL INFLUENCE OF THE HYDROGEN ION CONCENTRATION.

1. It has been shown in this paper that while non-ionized gelatin may exist in gelatin solutions on both sides of the isoelectric point (which lies for gelatin at a hydrogen ion concentration of C_H = 2.10^–5 or pH = 4.7), gelatin, when it ionizes, can only exist as an anion on the less acid side of its isoelectric point (pH > 4.7), as a cation only on the more acid side of its isoelectric point (pH < 4.7). At the isoelectric point gelatin can dissociate practically neither as anion nor as cation. 2. When gelatin has been transformed into sodium gelatinate by treating it for some time with M/32 NaOH, and when it is subsequently treated with HCl, the gelatin shows on the more acid side of the isoelectric point effects of the acid treatment only; while the effects of the alkali treatment disappear completely, showing that the negative gelatin ions formed by the previous treatment with alkali can no longer exist in a solution with a pH < 4.7. When gelatin is first treated with acid and afterwards with alkali on the alkaline side of the isoelectric point only the effects of the alkali treatment are noticeable. 3. On the acid side of the isoelectric point amphoteric electrolytes can only combine with the anions of neutral salts, on the less acid side of their isoelectric point only with cations; and at the isoelectric point neither with the anion nor cation of a neutral salt. This harmonizes with the statement made in the first paragraph, and the experimental results on the effect of neutral salts on gelatin published in the writer''s previous papers. 4. The reason for this influence of the hydrogen ion concentration on the stability of the two forms of ionization possible for an amphoteric electrolyte is at present unknown. We might think of the possibility of changes in the configuration or constitution of the gelatin molecule whereby ionized gelatin can exist only as an anion on the alkaline side and as a cation on the acid side of its isoelectric point. 5. The literature of colloid chemistry contains numerous statements which if true would mean that the anions of neutral salts act on gelatin on the alkaline side of the isoelectric point, e.g. the alleged effect of the Hofmeister series of anions on the swelling and osmotic pressure of common gelatin in neutral solutions, and the statement that both ions of a neutral salt influence a protein simultaneously. The writer has shown in previous publications that these statements are contrary to fact and based on erroneous methods of work. Our present paper shows that these claims of colloid chemists are also theoretically impossible. 6. In addition to other physical properties the conductivity of gelatin previously treated with acids has been investigated and plotted, and it was found that this conductivity is a minimum in the region of the isoelectric point, thus confirming the conclusion that gelatin can apparently not exist in ionized condition at that point. The conductivity rises on either side of the isoelectric point, but not symmetrically for reasons given in the paper. It is shown that the curves for osmotic pressure, viscosity, swelling, and alcohol number run parallel to the curve of the conductivity of gelatin when the gelatin has been treated with acid, supporting the view that these physical properties are in this case mainly or exclusively a function of the degree of ionization of the gelatin or gelatin salt formed. It is pointed out, however, that certain constitutional factors, e.g. the valency of the ion in combination with the gelatin, may alter the physical properties of the gelatin (osmotic pressure, etc.) without apparently altering its conductivity. This point is still under investigation and will be further discussed in a following publication. 7. It is shown that the isoelectric point of an amphoteric electrolyte is not only a point where the physical properties of an ampholyte experience a sharp drop and become a minimum, but that it is also a turning point for the mode of chemical reactions of the ampholyte. It may turn out that this chemical influence of the isoelectric point upon life phenomena overshadows its physical influence. 8. These experiments suggest that the theory of amphoteric colloids is in its general features identical with the theory of inorganic hydroxides (e.g. aluminum hydroxide), whose behavior is adequately understood on the basis of the laws of general chemistry.     

Elucidation of toxicity of organic acids inhibiting growth of Escherichia coli W

The toxic effects of 3-hydroxypropionic acid (3-HP) at high concentrations on cell growth and cellular metabolism are a great challenge to its commercial production. This study has examined and compared the toxic effects of 3-HP on cell growth with other similar weak acids, especially lactic acid, under various concentrations, temperatures and pH using Escherichia coli W as the test strain. 3-HP was approximately 4.4-times more toxic than lactic acid due to the 4.4-fold weaker acidity or 0.64 higher pKa value. The two acids presented no appreciable difference when the growth inhibition was correlated with the undissociated or protonated free acid concentration calculated by the Henderson-Hasselbalch equation. The growth inhibition by other small organic acids, such as acetic acid, pyruvic acid, propionic acid, 2-hydroxybutyric acid (2-HB) and 3-hydroxybutyric acid (3-HB), was also well correlated with their pKa values or protonated free acid concentrations. This study suggests that the growth inhibition by small weak acids is mainly caused by the socalled proton effect (rather than the anion effect), i.e., an increase in the intracellular proton concentration. An appropriate increase in the medium pH was suggested to alleviate the acid toxicity by reducing the free acid concentration in the culture medium.     

THE COUPLED REDOX POTENTIAL OF THE LACTATE-ENZYME-PYRUVATE SYSTEM

1. The term "coupled redox potential" is defined. 2. The system lactic ion See PDF for Equation pyruvic ion + 2H⁺ + 2e is shown to be reversible (when the enzyme is lactic acid dehydrogenase) and its coupled redox potential between pH 5.2 and 7.2 at 32°C. is: See PDF for Equation 3. The free energy of the reaction: lactic ion (1m) → pyruvic ion (1m) = -ΔF = –14,572. 4. The standard free energy of formation (ΔF ₂₉₈) of pyruvic acid (l) is estimated at –108,127. This is merely an approximation as some necessary data are lacking. 5. The importance of coupled redox potentials as a factor in the regulation of the equilibrium of metabolites is indicated.     

Changes in Serum Free Amino Acids and Muscle Fatigue Experienced during a Half-Ironman Triathlon

The aim of this study was to investigate the relationship between changes in serum free amino acids, muscle fatigue and exercise-induced muscle damage during a half-ironman triathlon. Twenty-six experienced triathletes (age = 37.0 ± 6.8 yr; experience = 7.4 ± 3.0 yr) competed in a real half-ironman triathlon in which sector times and total race time were measured by means of chip timing. Before and after the race, a countermovement jump and a maximal isometric force test were performed, and blood samples were withdrawn to measure serum free amino acids concentrations, and serum creatine kinase levels as a blood marker of muscle damage. Total race time was 320 ± 37 min and jump height (-16.3 ± 15.2%, P < 0.001) and isometric force (-14.9 ± 9.8%; P = 0.007) were significantly reduced after the race in all participants. After the race, the serum concentration of creatine kinase increased by 368 ± 187% (P < 0.001). In contrast, the serum concentrations of essential (-27.1 ± 13.0%; P < 0.001) and non-essential amino acids (-24.4 ± 13.1%; P < 0.001) were significantly reduced after the race. The tryptophan/BCAA ratio increased by 42.7 ± 12.7% after the race. Pre-to-post changes in serum free amino acids did not correlate with muscle performance variables or post-race creatine kinase concentration. In summary, during a half-ironman triathlon, serum amino acids concentrations were reduced by > 20%. However, neither the changes in serum free amino acids nor the tryptophan/BCAA ratio were related muscle fatigue or muscle damage during the race.     

THE ACTIVATION OF STARFISH EGGS BY ACIDS

1. Exposure of unfertilized starfish eggs to dilute solutions of weak acids (fatty acids, benzoic and carbonic acids) in isotonic balanced salt solution causes complete activation with the proper durations of exposure. For each acid the rate of activation (reciprocal of optimum duration) varies with concentration and temperature; at a given temperature and within a considerable range of concentrations (e.g. 0.00075 to 0.004 M for butyric acid), this rate is approximately proportional to concentration. We may thus speak of a molecular rate of action characteristic of each acid. 2. In general the molecular rate of action increases with the dissociation constant and surface activity of the acids. In the fatty acid series (up to caproic), formic acid has the most rapid effect, acting about four times as rapidly as acetic; for the other acids the order is: acetic = propionic ≦ butyric < valeric < caproic. Carbonic acid acts qualitatively like the fatty acids, but its molecular rate of action is only about one-fourteenth that of acetic acid. 3. Hydrochloric and lactic acids are relatively ineffective as activating agents, apparently because of difficulty of penetration. Lactic acid is decidedly the more effective. The action of both acids is only slightly modified by dissolving in pure (isotonic NaCl and CaCl₂) instead of in balanced salt solution. 4. The rate of action of acetic acid, in concentrations of 0.002 M to 0.004 M is increased (by 10 to 20 per cent) by adding Na-acetate (0.002 to 0.016) to the solution. The degree of acceleration is closely proportional to the estimated increase in undissociated acetic acid molecules. Activation thus appears to be an effect of the undissociated acid molecules in the external solution and not of the ions. Acetate anions and H ions acting by themselves, in concentrations much higher than those of the solutions used, have no activating effect. The indications are that the undissociated molecules penetrate rapidly, the ions slowly. Having penetrated, the molecules dissociate inside the egg, yielding the ions of the acid. 5. When the rate of activation is slow, as in 0.001 M acetic acid, the addition of Na-acetate (0,008 M to 0.016 M) has a retarding effect, referable apparently to the gradual penetration of acetate ions to the site of the activation reaction with consequent depression of dissociation. 6. An estimate of the C_H of those solutions (of the different activating acids) which activate the egg at the same rate indicates that their H ion concentrations are approximately equal. On the assumptions that only the undissociated molecules penetrate readily, and that the conditions of dissociation are similar inside and outside the egg, this result indicates (especially when the differences in adsorption of the acids are considered) that the rate of activation is determined by the C_H at the site of the activation reaction within the egg.     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : I. MEMBRANE POTENTIALS.

1. It is shown that a neutral salt depresses the potential difference which exists at the point of equilibrium between a gelatin chloride solution contained in a collodion bag and an outside aqueous solution (without gelatin). The depressing effect of a neutral salt on the P.D. is similar to the depression of the osmotic pressure of the gelatin chloride solution by the same salt. 2. It is shown that this depression of the P.D. by the salt can be calculated with a fair degree of accuracy on the basis of Nernst''s logarithmic formula on the assumption that the P.D. which exists at the point of equilibrium is due to the difference of the hydrogen ion concentration on the opposite sides of the membrane. 3. Since this difference of hydrogen ion concentration on both sides of the membrane is due to Donnan''s membrane equilibrium this latter equilibrium must be the cause of the P.D. 4. A definite P.D. exists also between a solid block of gelatin chloride and the surrounding aqueous solution at the point of equilibrium and this P.D. is depressed in a similar way as the swelling of the gelatin chloride by the addition of neutral salts. It is shown that the P.D. can be calculated from the difference in the hydrogen ion concentration inside and outside the block of gelatin at equilibrium. 5. The influence of the hydrogen ion concentration on the P.D. of a gelatin chloride solution is similar to that of the hydrogen ion concentration on the osmotic pressure, swelling, and viscosity of gelatin solutions, and the same is true for the influence of the valency of the anion with which the gelatin is in combination. It is shown that in all these cases the P.D. which exists at equilibrium can be calculated with a fair degree of accuracy from the difference of the pH inside and outside the gelatin solution on the basis of Nernst''s logarithmic formula by assuming that the difference in the concentration of hydrogen ions on both sides of the membrane determines the P.D. 6. The P.D. which exists at the boundary of a gelatin chloride solution and water at the point of equilibrium can also be calculated with a fair degree of accuracy by Nernst''s logarithmic formula from the value pCl outside minus pCl inside. This proves that the equation x² = y ( y + z) is the correct expression for the Donnan membrane equilibrium when solutions of protein-acid salts with monovalent anion are separated by a collodion membrane from water. In this equation x is the concentration of the H ion (and the monovalent anion) in the water, y the concentration of the H ion and the monovalent anion of the free acid in the gelatin solution, and z the concentration of the anion in combination with the protein. 7. The similarity between the variation of P.D. and the variation of the osmotic pressure, swelling, and viscosity of gelatin, and the fact that the Donnan equilibrium determines the variation in P.D. raise the question whether or not the variations of the osmotic pressure, swelling, and viscosity are also determined by the Donnan equilibrium.     

THE DISTRIBUTION OF THE WATER-SOLUBLE INORGANIC ORTHOPHOSPHATE IONS WITHIN THE CELL: ACCUMULATION IN THE NUCLEUS : Electron Probe Microanalysis

Lead acetate treatment of unfixed cells immobilizes the intracellular water-soluble, inorganic orthophosphate ions as microcrystalline lead hydroxyapatite precipitates (see reference 1). These precipitates have been analyzed with the electron microprobe. A much higher concentration of phosphorus has been found in the nucleoli of maize root tip cells fixed in lead acetate-glutaraldehyde (organic phosphorus plus inorganic orthophosphate), as compared to the nucleoli of roots fixed in glutaraldehyde alone (organic phosphorus). The concentration of the inorganic orthophosphate pool in these nucleoli is three to five times as high as the concentration of the macromolecular organic phosphate. Since nearly all of the latter is in RNA, the concentration of inorganic phosphate in the nucleolus is calculated to be roughly 0.5–0.8 M. About 30%—and up to 50%—of the total cellular inorganic phosphate is accumulated in the nucleolus since the mean concentration per cell is about 10^-2 M. In the extranucleolar part of the nucleus the mean concentration was estimated by densitometry to be roughly six times less than in the nucleolus (⩽ 0.1 M), and appears more concentrated in the nucleoplasm than in the condensed chromatin. While there is no direct evidence for the concentration in the cytoplasm, it certainly must be much lower than the mean cellular level (i.e., < 10^-2 M) since the nucleus is about 10% of the total cell volume. The implications of this compartmentation in the intact cell are discussed in connection with (A) the availability of orthophosphate ions for the cytoplasm in those processes in which these ions affect the rate of enzymatic reactions, and (B) protein nucleic acid interactions within the nucleus and nucleolus.     

THE CONCENTRATION EFFECT IN NITELLA

A method distinguishing between the concentration effect due to the cell wall and that due to the protoplasm is described: the importance of this lies in the fact that if the protoplasm shows a concentration effect one or both ions of the salt must tend to enter its outer surface. Studies on the concentration effect of KCl with living protoplasm of Nitella show that when P.D. is plotted as ordinates and the logarithm of concentration as abscissæ the graph is not the straight line demanded in the ideal case by theory but has less slope and is somewhat concave to the axis of the abscissæ. With a variety of salts the dilute solution is positive, which indicates that the cation has a greater mobility in the protoplasm than the anion or that the partition coefficient of the cation (A_c) increases faster than that of the anion (A_a) as the concentration increases. If the result depended on the partition coefficients we should say that when A_c ÷ A_a increases with concentration the dilute solution is positive. When A_c ÷ A_a decreases as the concentration increases the dilute solution is negative. In either case the increase in concentration may be accompanied by an increase or by a decrease in the relative amount of salt taken up. Theoretically therefore there need be no relation between the sign of the dilute solution and the relative amount of salt taken up with increasing concentration. Hypothetical diagrams of the electrical conditions in the cell are given. If we define the chemical effect as the P.D. observed in leading off at two points with equivalent concentrations of different salts we may say that the chemical effect of the protoplasm is very much greater than that of the cell wall.     

THE VISUAL DISCRIMINATION OF INTENSITY AND THE WEBER-FECHNER LAW

1. A study of the historical development of the Weber-Fechner law shows that it fails to describe intensity perception; first, because it is based on observations which do not record intensity discrimination accurately, and second, because it omits the essentially discontinuous nature of the recognition of intensity differences. 2. There is presented a series of data, assembled from various sources, which proves that in the visual discrimination of intensity the threshold difference ΔI bears no constant relation to the intensity I. The evidence shows unequivocally that as the intensity rises, the ratio See PDF for Equation first decreases and then increases. 3. The data are then subjected to analysis in terms of a photochemical system already proposed for the visual activity of the rods and cones. It is found that for the retinal elements to discriminate between one intensity and the next perceptible one, the transition from one to the other must involve the decomposition of a constant amount of photosensitive material. 4. The magnitude of this unitary increment in the quantity of photochemical action is greater for the rods than for the cones. Therefore, below a certain critical illumination—the cone threshold—intensity discrimination is controlled by the rods alone, but above this point it is determined by the cones alone. 5. The unitary increments in retinal photochemical action may be interpreted as being recorded by each rod and cone; or as conditioning the variability of the retinal cells so that each increment involves a constant increase in the number of active elements; or as a combination of the two interpretations. 6. Comparison with critical data of such diverse nature as dark adaptation, absolute thresholds, and visual acuity shows that the analysis is consistent with well established facts of vision.     

Carrier-Mediated Uptake of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Vacuoles Isolated from Catharanthus roseus Cells

The uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, into vacuoles isolated from Catharanthus roseus cells has been studied by silicone layer floatation filtering. The transport across the tonoplast of MACC is stimulated fourfold by 5 millimolar MgATP, has a K_m of about 2 millimolar, an optimum pH around 7, and an optimum temperature at 30°C. Several effectors known to inhibit ATPase (N,N′-dicyclohexylcarbodiimide) and to collapse the transtonoplastic H⁺ electrochemical gradient (carbonylcyanide m-chlorophenylhydrazone, gramicidin, and benzylamine) all reduced MACC uptake. Abolishing the membrane potential with SCN⁻ and valinomycin also greatly inhibited MACC transport. Our data demonstrate that MACC accumulates in the vacuole against a concentration gradient by means of a proton motive force generated by a tonoplastic ATPase. The involvement of a protein carrier is suggested by the strong inhibition of uptake by compounds known to block SH—, OH—, and NH₂— groups. MACC uptake is antagonized competitively by malonyl-d-tryptophan, indicating that the carrier also accepts malonyl-d-amino acids. Neither the moities of these compounds taken separately [1-aminocyclopropane-1-carboxylic acid, malonate, d-tryptophan or d-phenylalanine] nor malate act as inhibitors of MACC transport. The absence of inhibition of malate uptake by MACC suggests that MACC and malate are taken up by two different carriers. We propose that the carrier identified here plays an important physiological role in withdrawing from the cytosol MACC and malonyl-d-amino acids generated under stress conditions.     

(—) S-adenosyl-L-methionine-magnesium Protoporphyrin Methyltransferase, an Enzyme in the Biosynthetic Pathway of Chlorophyll in Zea mays

The enzyme (—) S-adenosyl-L-methionine-magnesium protoporphyrin methyltransferase, which catalyzes the transfer of the methyl group from (—) S-adenosyl-L-methionine to magnesium protoporphyrin to form magnesium protoporphyrin monomethyl ester, has been detected in chloroplasts isolated from Zea mays.

Zinc protoporphyrin and free protoporphyrin also act as substrates in the system, although neither one is as active as magnesium protoporphyrin.

The following scheme of chlorophyll synthesis in higher plants is proposed: δ-aminolevulinic acid → → → protoporphyrin → magnesium protoporphyrin → magnesium protoporphyrin monomethyl ester → → → chlorophyll a.