THE PERMEABILITY OF CELLS FOR OXYGEN AND ITS SIGNIFICANCE FOR THE THEORY OF STIMULATION

It can be demonstrated by an indicator method that living cells are as freely permeable to oxygen as dead cells, and that sudden admission of oxygen to the cell cannot account for increased oxidation as a result of stimulation. Oxygen penetrates as readily as carbon dioxide among the acids and ammonia among the alkalies. Exposure of living plant cells to high oxygen pressures does not initiate certain oxidations (except after some hours), which proceed readily in dead plant cells in the air. In the light of the preceding statement, about the permeability of cells for oxygen, this is interpreted to mean that more oxygen enters the cell at high pressure, but that the reacting substances (chromogen and oxidase) are kept apart by some phase boundary as long as the cell is alive. Increased oxygen concentration eventually produces injury to the cell.     

THE EFFECTS OF SULFATHIAZOLE AND OF PROPYL CARBAMATE ON THE RATE OF OXYGEN CONSUMPTION AND GROWTH IN ESCHERICHIA COLI

1. The rates of growth and of oxygen consumption by cells of E. coli have been measured under identical conditions, and the effects of sulfathiazole (ST) and of n-propyl carbamate (PC) on these two processes have been compared. 2. The rate of growth was measured by (a) the increase in the viable cell count, (b) the increase in the optical density of the culture, (c) the increase in the rate of oxygen consumption, and (d) the decrease in the ammonia of the medium. The results as indicated by these several measures were identical under the conditions of these experiments. 3. Concentrations of ST or of PC which are just sufficient to stop growth completely, lower the rate of oxygen consumption per unit of bacterial protoplasm to a value approximately 50 per cent of that seen in the absence of the inhibitor. 4. It is shown that the rate of oxygen consumption in cells from old cultures is less affected by ST than is the rate of oxygen consumption by cells from young cultures. It is probable that the rate of oxygen consumption by "old" cells is lower than that of "young" cells. 5. The effects of ST and PC on both the rate of oxygen consumption and the rate of growth are very similar, indicating in a general way, that the mechanism of the actions of these two inhibitors is similar. Furthermore, since both of them produce appreciable inhibition of the rate of oxygen consumption while they are inhibiting growth, the possibility that the effect on oxygen consumption is the immediate cause of the effect on growth must be entertained.     

MEASUREMENTS OF THE METABOLISM OF TWO PROTOZOANS

1. The respiration of Amoeba proteus was measured. 10 c. mm. of cells were found to use about 1.6 mm.³ of oxygen per hour at 20°C. The respiratory quotient was found to be nearly unity. 2. No anaerobic metabolism was found for Amoeba. 3. The respiration of Blepharisma was found to be from 3 to 7 mm.³ oxygen per hour for 10 mm.³ cells. The respiratory quotient was about 1. 4. Blepharisma was shown to have a definite anaerobic metabolism. 80 mm.³ cells caused the evolution of 12.5 mm.³ carbon dioxide per hour at 20°C. in the presence of bicarbonate.     

THE OXYGEN CONSUMPTION ASSOCIATED WITH GROWTH IN ESCHERICHIA COLI AND THE EFFECT OF SULFATHIAZOLE AND OF n-PROPYL CARBAMATE ON IT

1. The rate of oxygen consumption by E. coli has been observed both in the presence and absence of ammonia which substance is used by this organism in these experiments as the sole source of nitrogen for growth. 2. After the ammonia has been completely taken up in a culture of E. coli, the rate of oxygen consumption by the culture is observed to fall rapidly. It becomes relatively constant again at a rate approximately 45 per cent of that existing immediately prior to the exhaustion of the nitrogen source. It appears that the fixation of ammonia, that is, growth, requires approximately 55 per cent of the oxygen consumed by the growing cell. 3. Inhibition of the oxygen consumption which is associated with ammonia fixation, by both sulfathiazole (ST) and n-propyl carbamate (PC) closely parallels the inhibition of growth by these compounds (as measured by viable cell counts, etc.). 4. The concentrations of ST and PC which inhibit growth exert little or no inhibitory effect on the rate of oxygen consumption by cells after the rate has fallen to the resting value. 5. It is pointed out that the above observations would be adequately accounted for if growth depended on a discrete fraction of the total oxygen consumption of the growing cell. 6. It is noted that PC, but not ST, has a significant accelerating effect on the oxygen consumption of the resting cell; and that for a given inhibition of growth, PC produces less inhibition of the total oxygen consumption of the cells, than does ST. The latter of these two observations would follow from the former if the resting oxygen consumption were a discrete entity.     

STUDIES ON PHOTOSYNTHESIS : SOME EFFECTS OF LIGHT OF HIGH INTENSITY ON CHLORELLA

1. The effect on oxygen evolution of Chlorella vulgaris produced by light intensities up to about 40,000 f.-c. has been studied by the use of the Warburg technique. 2. Above a certain critical intensity, which is determined by the previous history of the cells, the rate of oxygen evolution decreases from the maximum to another constant rate. This depression is at first a completely reversible effect. 3. With a sufficiently high intensity this constant rate represents an oxygen uptake greater than the rate of dark respiration. During such a constant rate of oxygen uptake a progressive injury to the photosynthetic mechanism takes place. After a given oxygen consumption the rate falls off, approaching zero, and the cells are irreversibly injured. 4. The constant rate of oxygen evolution (2 and 3) decreases in a continuous manner with increasing light intensities, approaching a value which is approximately constant for all lots of cells regardless of previous history. 5. Two alternative hypotheses have been presented to explain the observed phenomena. The more acceptable of these proposes quick inactivation of the photosynthetic mechanism, the extent of inhibition depending on the light intensity. 6. In Chlorella vulgaris solarization is influenced by the previous history of the cells.     

THE KINETICS OF THE DECOMPOSITION OF PEROXIDE BY CATALASE

It has been shown that the experimental results obtained by Morgulis in a study of the decomposition of hydrogen peroxide by liver catalase at 20°C. and in the presence of an excess of a relatively high concentration of peroxide are quantitatively accounted for by the following mechanisms. 1. The rate of formation of oxygen is independent of the peroxide concentration provided this is greater than about 0.10 M. 2. The rate of decomposition of the peroxide is proportional at any time to the concentration of catalase present. 3. The catalase undergoes spontaneous monomolecular decomposition during the reaction. This inactivation is independent of the concentration of catalase and inversely proportional to the original concentration of peroxide up to 0.4 M. In very high concentrations of peroxide the inactivation rate increases. 4. The following equation can be derived from the above assumptions and has been found to fit the experiments accurately. See PDF for Equation in which x is the amount of oxygen liberated at the time t, A is the total amount of oxygen liberated (not the total amount available), and K is the inactivation constant of the enzyme.     

THE INSENSITIVITY OF PARAMECIUM TO CYANIDE AND EFFECTS OF IRON ON RESPIRATION

1. The effects of KCN and iron salts on oxygen consumption has been studied in the cell of Paramecium caudatum by manometric methods. 2. KCN solutions of strengths from M/200 to M/10,000 have been shown to produce no decrease in oxygen consumption, but have in most cases produced a very slight increase in the respiration rate. 3. The pH values were found to have little or no effect on these results. 4. Iron salts produce either no effect or a great diminution of oxygen consumption, in no case causing stimulation of rates of respiration. 5. Iron salts in neutral solutions do not penetrate the Paramecium cell nor do they cause so marked an effect as in an acid state. 6. The iron-content of Paramecium was found to be extremely small and not demonstrable by delicate tests. It is believed that iron is not combined in the cell in the form of a respiration-catalyst sensitive to cyanide.     

TEMPERATURE CHARACTERISTICS FOR THE OXYGEN CONSUMPTION OF GERMINATING SEEDS OF LUPINUS ALBUS AND ZEA MAYS

The rate of oxygen consumption by germinating seeds of Lupinus albus and of Zea mays was studied as a function of temperature (7–26°C.). The Warburg manometer technique was used, with slight modifications. Above and below a critical temperature at 19.5°C. the temperature characteristic for oxygen consumption by Lupinus albus was found to be µ = 11,700± and 16,600 respectively. The same critical temperature was encountered in the case of Zea mays, with temperature characteristics µ = 13,100± above and µ = 21,050 below that temperature.     

THE OXIDATION-REDUCTION POTENTIAL OF THE LUCIFERIN-OXYLUCIFERIN SYSTEM

The oxidation-reduction potential of the Cypridina luciferin-oxyluciferin system determined by a method of "bracketing" lies somewhere between that of anthraquinone 2-6-di Na sulfonate (E_o ^'' at pH of 7.7 = –.22) which reduces luciferin, and quinhydrone (E_o ^'' at pH of 7.7 = +.24), which oxidizes luciferin. Systems having an E_o ^'' value between –.22 and +.24 volt neither reduce oxyluciferin nor oxidize luciferin. If the luciferin-oxyluciferin system were truly reversible considerable reduction and oxidation should occur between –.22 and +.24. The system appears to be an irreversible one, with both "apparent oxidation" and "apparent reduction potentials" in Conant''s sense. Hydrosulfites, sulfides, CrCl₂, TiCl₃, and nascent hydrogen reduce oxyluciferin readily in absence of oxygen but without luminescence. Luminescence only appears in water solution if luciferin is oxidized by dissolved oxygen in presence of luciferase. Rapid oxidation of luciferin by oxygen without luciferase or oxidation by K₃Fe(CN)₆ in presence of luciferase but without oxygen never gives luminescence.     

ON THE QUANTA OF LIGHT PRODUCED AND THE MOLECULES OF OXYGEN UTILIZED DURING CYPRIDINA LUMINESCENCE

A study of the oxygen consumed per lumen of luminescence during oxidation of Cypridina luciferin in presence of luciferase, gives 11.4 x 10^–5 gm. oxygen per lumen or 88 molecules per quantum of λ = 0.48µ, the maximum in the Cypridina luminescence spectrum. For reasons given in the text, the actual value is probably somewhat less than this, perhaps of the order of 6.48 x 10^–5 gm. per lumen or 50 molecules of oxygen and 100 molecules of luciferin per quantum. It is quite certain that more than 1 molecule per quantum must react. On the basis of a reaction of the type: luciferin + 1/2 O₂ = oxyluciferin + H₂O + 54 Cal., it is calculated that the total efficiency of the luminescent process, energy in luminescence/heat of reaction, is about 1 per cent; and that a luciferin solution containing 4 per cent of dried Cypridina material should rise in temperature about 0.001°C. during luminescence, and contain luciferin in approximately 0.00002 molecular concentration.     

THE RESPIRATION OF LUMINOUS BACTERIA AND THE EFFECT OF OXYGEN TENSION UPON OXYGEN CONSUMPTION

1. The respiration of luminous bacteria has been studied by colorimetric and manometric methods. 2. Limulus oxyhaemocyanin has been used as a colorimetric indicator of oxygen consumption and indicator dyes were used for colorimetric determination of carbon dioxide production. 3. The Thunberg-Winterstein microrespirometer has been used for the measurement of the rate of oxygen consumption by luminous bacteria at different partial pressures of oxygen. 4. The effect of oxygen concentration upon oxygen consumption has been followed from equilibrium with air to low pressures of oxygen. 5. Luminous bacteria consume oxygen and produce carbon dioxide independent of oxygen pressures from equilibrium with air (152 mm.) to approximately 22.80 mm. oxygen or 0.03 atmosphere. 6. Dimming of a suspension of luminous bacteria occurs when oxygen tension is lowered to approximately 2 mm. Hg (0.0026 atmosphere) and when the rate of respiration becomes diminished one-half. 7. Pure nitrogen stops respiratory activity and pure oxygen irreversibly inhibits oxygen consumption. 8. The curve for rate of oxygen consumption with oxygen concentration is similar to curves for adsorption of gasses at catalytic surfaces, and agrees with the Langmuir equation for the expression of the amount of gas adsorbed in unimolecular layer at catalytic surfaces with gas pressure. 9. A constant and maximum rate of oxygen consumption occurs in small cells when oxygen concentration becomes sufficient to entirely saturate the surface of the oxidative catalyst of the cell.     

THE EFFECT OF CARBON DIOXIDE TENSION ON THE METABOLISM OF CEREBRAL CORTEX AND MEDULLA OBLONGATA

Manometric measurements were made of oxygen uptake (Q _OO2) and aerobic lactic acid output (Q_G) by slices of cerebral cortex and medulla oblongata of the cat in the presence of mixtures of 1, 5, and 20 volumes per cent of carbon dioxide in oxygen. The concentrations of NaHCO₃ and NaCl in the medium were varied to maintain constant pH and sodium ion concentrations. The calcium ion concentration was 0.0002 M. At pH 7.5 under these conditions, an increase in carbon dioxide from 1 per cent to 5 per cent doubled the Q_G of both tissues but did not alter Q _OO2; an increase from 5 per cent to 20 per cent carbon dioxide had no further effect on Q_G in either tissue or Q _OO2 of cortex, but did depress the Q _OO2 of medulla. At pH 8.1, an increase in carbon dioxide from 1 per cent to 5 per cent raised the Q _OO2 and Q_G of cortex by about 60 per cent. Measurements at low oxygen tension carried out previously in phosphate medium were repeated in bicarbonate medium to obtain data for the combined output of lactic acid and carbon dioxide (Q_A). When the oxygen in the gas phase was decreased from 95 to 3 volumes per cent, the lactic acid output as measured colorimetrically increased by 114 mg./gm. in cortex and by 8 mg./gm. in medulla; Q_A increased from 12.3 to 13.5 in cortex and decreased from 5.1 to 3.8 in medulla.     

METHODS OF STUDYING THE RESPIRATORY EXCHANGE IN SMALL AQUATIC ORGANISMS,WITH PARTICULAR REFERENCE TO THE USE OF FLAGELLATES AS AN INDICATOR FOR OXYGEN CONSUMPTION

1. Flagellates are positively chemotactic to a certain concentration of dissolved oxygen which is lower than that in water saturated with oxygen under atmospheric partial pressure. Consequently, when a small aquatic animal is held motionless between cover-slip and slide in a suspension of flagellates in water saturated with oxygen, the flagellates are attracted to those parts of the animal which are absorbing oxygen. The relative sizes of the flagellate aggregations then show the relative activities of the different surfaces of the animal in absorbing oxygen. 2. Applying this method to the red Chironomus larva it was found that the animal respires by the whole body surface except by the head and the "ventral gills" and that the relative intensity of oxygen intake by the different parts of the body varies in different individuals and in the same individual at different times. 3. The absence of oxygen intake by the "ventral gills" was confirmed with the microspectroscope. In oxygen-free water all the hemoglobin of the blood becomes reduced. When an air bubble is now introduced so that it touches the "ventral gills" oxyhemoglobin first appears in the nearest body segment to the bubble, not in the "gills." 4. When a small aquatic animal is held motionless between cover-slip and slide in a solution of an indicator which changes color about the neutral point of water the relative extent of color change at different surfaces of the animal''s body indicates the relative amounts of carbon dioxide given off by these surfaces. 5. Using this method with the red Chironomus larva similar conclusions were reached for carbon dioxide output as for oxygen intake.     

DEVELOPMENT OF EYE COLORS IN DROSOPHILA: SOME PROPERTIES OF THE HORMONES CONCERNED

The substance inducing the production of pigment in the eyes of vermilion brown mutants of Drosophila melanogaster has been shown to be a relatively stable chemical entity possessing true hormone-like activity. A simple method for obtaining hormone solutions has been developed involving extraction of dried wild type Drosophila pupae with ethyl alcohol and water. A logarithmic proportionality has been found to exist between the amount of hormone and the induced eye color. This relationship provides a simple method for the quantitative determination of hormone concentration in given extracts. Larvae and pupae of D. melanogaster contain an intracellular enzyme which inactivates the hormone in the presence of molecular oxygen. The hormone is not oxidized under ordinary conditions with either molecular oxygen or hydrogen peroxide. The hormone has been found to be an amphoteric compound with both acidic and basic groups and with a molecular weight between 400 and 600. The solubility and precipitation reactions of the hormone suggest its amino acid-like nature. However, the instability to heat, acid, and alkali, and its rather restricted occurrence indicate a rather complex specific structure.     

TEMPERATURE CHARACTERISTICS FOR THE PRODUCTION OF CO2 BY GERMINATING SEEDS OF LUPINUS ALBUS AND ZEA MAYS

The rates of production of CO₂ by germinating seeds of Lupinus albus and Zea mays were studied between temperatures 12.5° and 25°C. with the HCl-Ba(OH)₂ titration method. The temperature characteristics found are different from those previously obtained for the oxygen consumption of the same seeds germinated in the same manner. For Lupinus, the temperature characteristics above and below the critical temperature of 20° are 16,100 ± and 24,000 ± calories respectively. For Zea, no evidence of a critical temperature was found in this region, and the temperature characteristic is 20,750 ± calories throughout the range of temperature tested. The possible interpretations of the difference in the values of temperature characteristics for oxygen consumption and for production of CO₂ are noted.     

TEMPERATURE CHARACTERISTICS FOR THE METABOLISM OF CHLORELLA : II. THE RATE OF RESPIRATION OF CULTURES OF CHLORELLA PYRENOIDOSA AS A FUNCTION OF TIME AND OF TEMPERATURE

The respiration of the green alga Chlorella pyrenoidosa, suspended in Knop''s solution, has been studied in the dark as a function of time and of temperature. The rates of oxygen consumption and of carbon dioxide production (at constant temperature) decline for about 25 hours to a low, constant level. From an analysis of the curves it is suggested that two substances, A and B, are utilized, whose respiratory quotients are 1 and 0.65 respectively. The values of the temperature characteristics were found to be: for oxidation of A, 19,500 (0.6 to 11.5°C.) and 3,500 (11.5 to 28°C.); for oxidation of B, 5,600 (23.4 to 0.6°C.).     

STUDIES ON CELL METABOLISM AND CELL DIVISION : II. STIMULATION OF CELLULAR OXIDATION AND REVERSIBLE INHIBITION OF CELL DIVISION BY DIHALO AND TRIHALOPHENOLS

The dihalo and trihalophenols, and phenols containing both halo and nitro substituents in the same molecule, produce, in fertilized eggs of Arbacia punctulata, a rise in rate of oxygen consumption and a reversible block to cell division. To define the conditions which affect the degree of this activity, the following factors have been varied: the arrangement of substituents in the molecule, the concentration of reagent, and the time after fertilization at which the reagent is added. The stimulation of oxygen consumption and reversible block to cell division produced by the dihalophenols are qualitatively the same as those previously produced in fertilized Arbacia eggs by certain dinitrophenols. To yield optimum respiratory effect and maximum division block, it usually requires a higher concentration of dihalo than of the corresponding dinitrophenol. For example, with fertilized Arbacia eggs at 20°C. 2,4-dinitrophenol, in optimum concentration of 3 x 10^–5 molar, raises oxygen consumption to 292 per cent of normal (4). The corresponding values for two dihalo analogues are: 2,4-dichlorophenol, 10^–4 molar and 236 per cent; 2,4-dibromophenol, 6 x 10^–5 molar and 282 per cent. The halophenols differ from the nitrophenols in two interesting respects: (a) The monohalophenols produce little or no oxidative stimulation or division block in fertilized Arbacia eggs; p-nitrophenol is very active in both respects. (b) The symmetrical trihalophenols have an appreciable ability to stimulate oxygen consumption and block division; symmetrical trinitrophenol is inactive in both respects (4). The increases in oxygen consumption produced in fertilized Arbacia eggs by 2,4-dichloro and 2,4-dinitrophenol are larger than the percentage increases given by methylene blue and o-cresol indophenol under the same experimental conditions. The dihalo and dinitrophenols produce a reversible block to the cell division of fertilized marine eggs. The oxidation-reduction indicators, in contrast to the dihalo and dinitrophenols, block cell division irreversibly and fertilized eggs of Arbacia do not recover from optimum respiratory stimulating concentrations of these oxidation-reduction dyes. The present experiments with halophenols are in harmony with and lend considerable support to the hypothesis (4) that nitro and similarly substituted phenols derive their biological activity from the presence and properties of the phenolic OH group, as modified by proper substitution in the phenolic benzene ring.     

OBSERVATIONS ON THE RESPIRATION OF TRYPANOSOMA CRUZI IN CULTURE

1. The oxygen consumption of cultural forms of Trypanosoma cruzi decreases in intensity with increasing age of the cultures; no correlation with any other factor studied could be established. 2. The respiratory quotient was high for the first 10 days, i.e. as long as the population increased; with the onset of a decline in numbers, the R.Q. began to drop. It is believed that the flagellates consume in the beginning predominantly sugar and later predominantly protein. Observations on the pH of the cultures bear out this view. 3. The oxygen consumption was independent of the oxygen tension over a wide range of tensions. 4. The oxygen consumption increased in the temperature range 13° to 40°C., while a temperature of 44°C. proved to be lethal. Upon application of Arrhenius'' equation, two straight lines, intersecting at about 28°C., resulted. The µ values were 23,980 and 5275 for the lower and higher temperature range respectively. 5. Of the oxidase inhibitors tested, strong inhibition of the oxygen consumption was achieved with azide, cyanide, and hydrogen sulfide. Pyrophosphate had no influence at all. There is some probability that cytochrome oxidase is the chief oxidase present. 6. The strongest inhibitory influence due to dehydrogenase inhibitors was observed with propyl carbamate and high concentrations of ethyl carbamate. 7. A small fraction of the oxygen consumption, about 10 per cent, may be due to substances with sulfhydryl groups, as indicated by a slight but distinct inhibition due to dilute iodoacetate and to arsenite.     

ARSENATE AS A CATALYST OF OXIDATION

Arsenate exerts a catalytic effect on the oxidation of pyrogallol by atmospheric oxygen, on the catalytic oxidation of pyrogallol by metallic iron, and on the presumably enzymatic production of CO₂ by Elodea canadensis.     

COMPARATIVE STUDIES ON RESPIRATION : VI. INCREASED PRODUCTION OF CARBON DIOXIDE ACCOMPANIED BY DECREASE OF ACIDITY.

In petals of Salvia high concentrations of ether cause an increase in oxygen consumption and in the production of CO₂, while at the same time a decrease occurs in the acidity of the cell contents.