CERTAIN EFFECTS OF SALTS ON THE PENETRATION OF BRILLIANT CRESYL BLUE INTO NITELLA

The effect of various substances on living cells may be advantageously studied by exposing them to such substances and observing their subsequent behavior in solutions of a basic dye, brilliant cresyl blue. The rate of penetration of the basic dye, brilliant cresyl blue, is decreased when cells are exposed to salts with monovalent cations before they are placed in the dye solution (made up with borate buffer mixture). This inhibiting effect is assumed to be due to the effect of the salts on the protoplasm. This effect is not readily reversible when cells are transferred to distilled water, but it is removed by salts with bivalent or trivalent cations. In some cases it disappears in dye made up with phosphate buffer mixture, or with borate buffer mixture at the pH value in which the borax predominates, and in the case of NaCl it disappears in dye containing NaCl. No inhibiting effect is seen when cells are exposed to NaCl solution containing MgCl₂ before they are placed in the dye solution. The rate of penetration of dye is not decreased when cells are previously exposed to salts with bivalent and trivalent cations. The rate is slightly increased when cells are placed in the dye solution containing a salt with monovalent cation and probably with bivalent or trivalent cations. In the case of the bivalent and trivalent salts the increase is so slight that it may be negligible.     

SPECTROPHOTOMETRIC STUDIES OF PENETRATION : IV. PENETRATION OF TRIMETHYL THIONIN INTO NITELLA AND VALONIA FROM METHYLENE BLUE.

Spectrophotometric measurements show that it is chiefly the trimethyl thionin that is present in the sap extracted from the vacuoles of uninjured cells of Nitella or Valonia which have been placed in methylene blue solution at a little above pH 9. Whether these measurements were made immediately or several hours later the same results were obtained. Methylene blue is detected in the sap (1) when the cells are injured or (2) when the contamination of the sap from the stained cell wall occurs at the time of extraction. The sap is found to be incapable of demethylating methylene blue dissolved in it even on standing for several hours. It is somewhat uncertain as to whether the trimethyl thionin penetrated as such from the external methylene blue solution which generally contains this dye as impurity (in too small concentration for detection by spectrophotometer but detectable by extraction with chloroform), or whether it has formed from methylene blue in the protoplasm. The evidences described in the text tend to favor the former explanation. Theory is discussed on basis of more rapid penetration of trimethyl thionin (in form of free base) than of methylene blue, or of trimethyl thionin in form of salt.     

THE EFFECT OF ACETATE BUFFER MIXTURES,ACETIC ACID,AND SODIUM ACETATE,ON THE PROTOPLASM,AS INFLUENCING THE RATE OF PENETRATION OF CRESYL BLUE INTO THE VACUOLE OF NITELLA

When living cells of Nitella are exposed to a solution of sodium acetate and are then placed in a solution of brilliant cresyl blue made up with a borate buffer mixture at pH 7.85, a decrease in the rate of penetration of dye is found, without any change in the pH value of the sap. It is assumed that this inhibiting effect is caused by the action of sodium on the protoplasm. This effect is not manifest if the dye solution is made up with phosphate buffer mixture at pH 7.85. It is assumed that this is due to the presence of a greater concentration of base cations in the phosphate buffer mixture. In the case of cells previously exposed to solutions of acetic acid the rate of penetration of dye decreases with the lowering of the pH value of the sap. This inhibiting effect is assumed to be due chiefly to the action of acetic acid on the protoplasm, provided the pH value of the external acetic acid is not so low as to involve an inhibiting effect on the protoplasm by hydrogen ions as well. It is assumed that the acetic acid either has a specific effect on the protoplasm or enters as undissociated molecules and by subsequent dissociation lowers the pH value of the protoplasm. With acetate buffer mixture the inhibiting effect is due to the action of sodium and acetic acid on the protoplasm. The inhibiting effect of acetic acid and acetate buffer mixture is manifested whether the dye solution is made up with borate or phosphate buffer mixture at pH 7.85. It is assumed that acetic acid in the vacuole serves as a reservoir so that during the experiment the inhibiting effect still persists.     

THE KINETICS OF PENETRATION : XIV. THE PENETRATION OF IODIDE INTO VALONIA

When 0.1 M NaI is added to the sea water surrounding Valonia iodide appears in the sap, presumably entering as NaI, KI, and HI. As the rate of entrance is not affected by changes in the external pH we conclude that the rate of entrance of HI is negligible in comparison with that of NaI, whose concentration is about 10⁷ times that of HI (the entrance of KI may be neglected for reasons stated). This is in marked contrast with the behavior of sulfide which enters chiefly as H₂S. It would seem that permeability to H₂S is enormously greater than to Na₂S. Similar considerations apply to CO₂. In this respect the situation differs greatly from that found with iodide. NaI enters because its activity is greater outside than inside so that no energy need be supplied by the cell. The rate of entrance (i.e. the amount of iodide entering the sap in a given time) is proportional to the external concentration of iodide, or to the external product [N⁺]_o [I^-l_o, after a certain external concentration of iodide has been reached. At lower concentrations the rate is relatively rapid. The reasons for this are discussed. The rate of passage of NaI through protoplasm is about a million times slower than through water. As the protoplasm is mostly water we may suppose that the delay is due chiefly to the non-aqueous protoplasmic surface layers. It would seem that these must be more than one molecule thick to bring this about. There is no great difference between the rate of entrance in the dark and in the light.     

SEPARATION OF POTASSIUM ISOTOPES IN VALONIA AND NITELLA

The ratio of K³⁹ ÷ K⁴¹ appears to be lower in the sap of Valonia and Nitella than in the environment, indicating that the living cell can separate these isotopes to some extent. Experiments with a mixture of guaiacol and p-cresol suggest that a similar separation may occur here but further experiments are needed.     

ON THE ACCUMULATION OF DYE IN NITELLA

Living cells of Nitella were placed in different concentrations of brilliant cresyl blue solutions at pH 6.9. It was found that the greater the concentration of the external dye solution, the greater was the speed of accumulation of the dye in the cell sap and higher was the concentration of dye found in the sap at equilibrium. Analysis of the time curves showed that the process may be regarded as a reversible pseudounimolecular reaction. When the concentration in the sap is plotted as ordinates and the concentration in the outside solution as abscissae the curve is convex toward the abscissae. There is reason to believe that secondary changes involving injury take place as the dye accumulates and that if these changes did not occur the curve would be concave toward the abscissae. The process may be explained as a chemical combination of the dye with a constituent of the cell. This harmonizes with the fact that the temperature coefficient is high (about 4.9). Various other possible explanations are discussed.     

STIMULATION OF FUNDULUS BY HYDROCHLORIC AND FATTY ACIDS IN FRESH WATER,AND BY FATTY ACIDS,MINERAL ACIDS,AND THE SODIUM SALTS OF MINERAL ACIDS IN SEA WATER

1. Fundulus heteroclitus was found to be a reliable experimental animal for studies on chemical stimulation in either fresh or sea water. 2. The response of Fundulus to hydrochloric, acetic, propionic, butyric, valeric, and caproic acids was determined in fresh water, while the same acids plus sulfuric and nitric, as well as the sodium salts of the mineral acids, were tested in sea water. 3. Stimulation of Fundulus by hydrochloric acid in fresh water is correlated with the effective hydrogen ion concentration. Stimulation by the n-aliphatic acids in the same environment is correlated with two factors, the effective hydrogen ion concentration and the potential of the non-polar group in the molecule. However, as the number of CH₂ groups increases the stimulating effect increases by smaller and smaller amounts, approaching a maximum value. 4. Stimulation of Fundulus by hydrochloric, sulfuric, and nitric acids in sea water is correlated with the forces of primary valence which in turn are correlated with the change in hydrogen ion concentration of the sea water. The n-aliphatic acids increase in stimulating efficiency in sea water as the length of the carbon chain increases, but a limiting value is not reached as soon as in fresh water. 5. Only a slight difference in stimulation by hydrochloric acid is found in sea water and in fresh water. However, there is a significant difference in stimulation by the fatty acids in fresh and in sea water, which is partly explained by the different buffering capacities of the two media. It is to be noted that in the same environment two different fish, Fundulus and Eupomotis, give different results, while the same fish (Fundulus) in two different environments responds similarly to mineral acids but differently to fatty acids. These results illustrate that stimulation is a function of the interaction between environment and receptors, and that each is important in determining the response. 6. Stimulation by sodium chloride, nitrate, and sulfate is correlated with equivalent concentrations of the salts added to sea water, or with the forces of primary valence. Although the threshold for stimulation by the salts is considerably higher than for the acids, the efficiency of stimulation by the salts is greater.     

THE PENETRATION OF LUMINOUS BACTERIA BY THE AMMONIUM SALTS OF THE LOWER FATTY ACIDS : PART I. GENERAL OUTLINE OF THE PROBLEM,AND THE EFFECTS OF STRONG ACIDS AND ALKALIES.

It is shown that disappearance of the light of luminous bacteria may be used as a criterion of cell penetration; that luminous bacteria are cytolyzed by water, hypotonic solutions, and by freely penetrating solutions; that luminous bacteria are not injured by hydrogen or hydroxyl ions in the external solutions within the range of pH values employed with the ammonium salts and that therefore disappearance of the light in isotonic solutions of these salts must be due to penetration of the solute; and that there is a characteristic difference between the effects of strong and of weak acids and alkalies on luminous bacteria.     

THE REACTIONS OF HALICYSTIS AND OF VALONIA TO INJECTIONS OF CERTAIN PROTEINS

It is shown (1) that Valonia and Halicystis cells exhibit varying degrees of tolerance to injections of animal peptone, animal proteose, crystallized egg albumen, and diphtheria toxin; (2) that Valonia cells display decreased tolerance to egg albumen in increasing dosages, although Halicystis is completely tolerant of the highest dosage used; (3) that the mortality curves of Valonia injected with egg albumen and of both Valonia and Halicystis injected with diphtheria toxin show the delayed effect characteristic of laboratory mammals when treated similarly; (4) that Valonia cells injected twice with egg albumen exhibit no change in susceptibility to its effects; and (5) that neither species of algae gives evidence of having formed antibodies against the antigens used.     

THE SOLUBILITIES OF THE l-DIHALOGENATED TYROSINES IN ETHANOL-WATER MIXTURES AND CERTAIN RELATED DATA

1. The solubilities of l-tyrosine, dichloro-l-tyrosine (hydrated), dibromo-l-tyrosine (anhydrous), and diiodo-l-tyrosine in ethanol-water mixtures at 25° have been determined. 2. It was found that the solubilities of the dihalogenated substitution products of l-tyrosine are increased by addition of ethanol up to a certain concentration. Further addition of ethanol leads to a decrease in solubility. The solubility of l-tyrosine is decreased by addition of ethanol. 3. Dichloro-l-tyrosine (hydrated) was found to change to the anhydrous form when allowed to stand in the presence of ethanol. 4. The apparent heat of solution of diiodo-l-tyrosine in an ethanol-water mixture has been determined. 5. The solubility of dl-thyroxine at 30° has been determined in urea solution, ethanol, dioxan, ethylene glycol, and propylene glycol.     

MICROHETEROGENEITY AND PHOSPHOAMINO ACIDS IN THE CARBOXY-TERMINAL HALF OF MYELIN BASIC PROTEIN

—Three chromatographically distinct peptic peptides (F80-1, F80-2 and F80-3) derived from the C-terminal half of the bovine and guinea-pig myelin basic proteins were characterized. The three peptides of each animal species had the same N-terminal residue (phenylalanine) and essentially the same amino acid composition, but they differed in electrophoretic mobility at alkaline pH. The least basic peptide (F80-3) differed from the others in showing a deficit of C-terminal arginine residues and in containing phosphorus, 0·37 and 0·46 g-atom/mol of bovine and guinea-pig peptides, respectively. Other peptic peptides. derived from the N-terminal half of the basic protein, were essentially phosphorus-frcc. Analyses of partial acid hydrolyzates of peptide F80-3 by high voltage electrophoresis showed the presence of both phosphoserine and phosphothreonine. After incubation with E. coli alkaline phosphatase (EC 3.1.3.1). 34 and 40% of the bovine and guinea-pig F80-3 peptides. respectively, were converted to peptide F80-1. This reaction involved the loss of 2 net negative charges, and its extent corresponded to loss of essentially all of the phosphate originally present in the peptide. This result indicated that the phosphorylated species of peptide F80-3 contained one phosphate group per molecule.     

THE PRESENCE OF CYSTEINE IN FROG MYELIN BASIC PROTEIN

—Frog myelin basic protein, when subjected to ion-exchange chromatography at alkaline pH, underwent conversion to a higher molecular weight form. Treatment of the latter with 2-mercaptoethanol regenerated the monomeric basic protein. Amino acid analysis of the monomer and the higher molecular weight species after performic acid oxidation demonstrated the presence of approximately 1 mol of cysteic acid per mol of protein of molecular weight 19,700. Treatment of the monomer with the mild oxidizing agent azodicarboxylic acid bis dimethyl amide resulted in its partial conversion to the higher molecular weight form. These studies demonstrate that the frog myelin basic protein, unlike those of all other species hitherto examined, contains a single cysteinyl residue in its polypeptide chain.     

DETERMINATION OF CERTAIN AMINO ACIDS IN CHYMOTRYPSINOGEN,AND ITS MOLECULAR WEIGHT

1. A preparation of chymotrypsinogen, obtained from Dr. M. Kunitz, was analyzed for sulfur, the sulfur amino acids, tyrosine, and tryptophane. 2. The protein sulfur of chymotrypsinogen was accounted for as methionine, cysteine, and cystine. 3. A method is presented for calculating the minimum molecular weight of a protein from the distribution of the sulfur amino acids. In the case of chymotrypsinogen, the calculated minimum molecular weight was found to be the actual molecular weight. 4. The molecular weight of chymotrypsinogen is 36,700 by amino acid analysis as compared to 36,000 by osmotic pressure measurements of Kunitz and Northrop. Chymotrypsinogen contains per mol 17 atoms of sulfur, 3 residues of methionine, 4 of cysteine, 10 of half-cystine (i.e. 5 S—S linkages), 6 of tyrosine, and 10 of tryptophane. 5. The tryptophane content of chymotrypsinogen (5.51 per cent) is the highest of any protein so far on record. 6. Chymotrypsinogen contains no reactive SH groups, although it yields cysteine on hydrolysis. This may be due either to preformed but unreactive SH groups or to S—X groups. The term S—X group is used to denote the substitution of the sulfhydryl hydrogen by a constituent X; hydrolysis yields SH groups: S—X + HOH = SH + X—OH.     

THE STRUCTURE OF THE ULTRAVIOLET ABSORPTION SPECTRA OF CERTAIN PROTEINS AND AMINO ACIDS

1. The absorption spectra of a number of proteins in the region 2500 to 3000 A. have been found to comprise from six to nine narrow bands. In consequence of variation in the relative intensity of these bands from protein to protein, the absorption curve has a characteristic configuration for each protein. 2. These bands correspond closely in position with the narrow bands which appear in the absorption spectra of tryptophan, tyrosin, and phenylalanine. Tryptophan and tyrosin each present three bands, phenylalanine shows nine. 3. The bands in the proteins are accordingly attributed to these amino acids. In the proteins the bands are displaced from the positions which they occupy in the uncombined amino acids, in most instances, by 10 to 35 A. toward longer wavelengths. 4. The absorption spectrum of Pneumococcus Type I antibody resembles that of normal pseudoglobulin but shows characteristic differences.