OXIDATION-REDUCTION POTENTIALS AND THE POSSIBLE RESPIRATORY SIGNIFICANCE OF THE PIGMENT OF THE NUDIBRANCH CHROMODORIS ZEBRA

1. Oxidation-reduction potential methods have been applied to a study of the blue-purple pigment present in solution in the blood and in the tissue cells of the nudibranch Chromodoris zebra. 2. The blue-purple pigment and its yellow reduction product form a reversible system whose E_o'' = x0.102 volts at pH 7.0 and whose valence change from oxidant to reductant appears to be one. 3. The system is unlike oxyhemoglobin-hemoglobin in the mode of oxygen transfer. Its rôle as a possible respiratory material is discussed.     

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.     

THE ACTIVITY OF YEAST INVERTASE AS A FUNCTION OF OXIDATION-REDUCTION POTENTIAL

The activity of yeast invertase as a function of oxidation-reduction potential has been investigated using a large number of oxidants and reductants. The activity is constant over the range of E_h from –270 to +600 mv., but above E_h = +600 mv. there is a sharp decrease in activity reaching 0 at E_h = +1,000 mv. The inhibiting action of strong oxidants is upon the enzyme rather than on the substrate and appears to be essentially irreversible Experiments indicate that the inhibiting action of strong oxidants on invertase is primarily related to their high oxidation-reduction potential rather than to a specific toxic action unrelated to E_h. The effects of oxidation-reduction potential upon invertase activity are independent of the purity of the enzyme, since they are the same for commercial invertases, fresh bakers'' yeast, powdered bakers'' yeast, brewers'' yeast, and highly purified invertase. Possible mechanisms involved in the inactivation of invertase by oxidants are discussed.     

Exposure of the Isolated Frog Skin to High Potassium Concentrations at the Internal Surface : II. Changes in epithelial cell volume,resistance and response to antidiuretic hormone

Isolated frog skin epithelia undergo marked, but reversible swelling when the external skin surface is bathed with conventional NaCl Ringer''s and the internal surface with KCl Ringer''s solutions. In 2 hours, epithelial thickness increased by over twofold. When NaCl Ringer''s was replaced on both sides of the skin, volume returned to control levels in less than 1 hour. When sulfate, rather than chloride, was the predominant anion, exposure of the internal surface to high potassium concentrations did not evoke changes in epithelial cell volume. With both KCl and K₂SO₄ Ringer''s, an immediate drop in DC resistance across the skin occurred. This was followed by partial recovery. Both the immediate drop and partial recovery were unrelated to changes in volume. A slow, sustained secondary drop in resistance was observed with KCl but not K₂SO₄ Ringer''s. This slower drop was associated temporally with swelling. When epithelial cell swelling occurred (i.e. with KCl Ringer''s), the characteristic response of the skin to vasopressin was abolished. However, with sulfate as anion, vasopressin elicited an increase in short-circuit current and/or in cell volume despite high internal potassium concentrations. It is concluded that epithelial swelling increased the permeability of the sodium-selective barrier at the external surface of the cells; and the possibility exists that stretching of cell membranes altered dimensions of pathways through which Na and water move, thereby mimicking the effects of vasopressin.     

CONTRIBUTION TO THE THEORY OF PERMEABILITY OF MEMBRANES FOR ELECTROLYTES

On page 39, Vol. viii, No. 2, September 18, 1925, multiply the right-hand side of formula (2) by the factor See PDF for Equation. On page 44, immediately after formula (1) the text should be continued as follows: Let us suppose a membrane to be separated by two solutions of KCl of different concentrations K₁ and K₂ and these concentrations and the corresponding concentrations of K⁺ within the membrane, which are in equilibrium with the outside solutions, to be so high that the H⁺ ions may be neglected. When a small electric current flows across the system, practically the K⁺ ions alone are transferred and that in a reversible manner. Therefore the total P.D. is practically See PDF for Equation This P.D. is composed of two P.D.''s at the boundaries and the diffusion potential within the membrane. Suppose the immobility of the anions is not absolute but only relative as compared with the mobility of the cations, KCl would gradually penetrate into the membrane to equal concentration with the outside solution on either side and no boundary potential would be established. In this case the diffusion P.D. within the membrane is the only P.D., amounting to See PDF for Equation but, V being practically = 0, it would result that See PDF for Equation So the definitive result is the same as in the former case. Now cancel the printed text as far as page 48, line 13 from the top of the page, but retain Fig. 1. On page 50, line 19 from the top of the page, cancel the sentence beginning with the word But and ending with the words of the chain.     

A study of the kinetic properties of the stable semiquinone of the reaction-center secondary acceptor in chromatophores of non-sulfur purple bacteria

Flash-induced absorption changes at 450 nm were investigated in isolated chromatophores of Rhodopseudomonas sphaeroides and Rhodospirillum rubrum non-sulfur purple bacteria to follow the redox changes of the semiquinone species of the secondary quinone acceptor of the photosynthetic reaction center. Excitation of a dark-adapted chromatophore suspension by a series of successive flashes in the presence of electron donors capable of rapidly reducing the photooxidized reaction-center pigment causes the formation of a stable semiquinone species (Q^⨪_B) with a lifetime which is shown to be proportional to the amount of the oxidized redox mediator in the incubation medium. It is shown that the disappearance of the flash-induced absorption changes at 450 nm on lowering the ambient redox potential (E_h) to 200–300 mV is the result of increasing the lifetime of Q^⨪_B, as the amount of the oxidized mediator diminishes; consequently, in these circumstances, the 2–5 min dark interval between the flash cycles appears insufficient for Q^⨪_B recovery. After the addition of redox mediators with a low midpoint potential, acting as an oxidant for Q^⨪_B, the flash-induced redox changes of Q^⨪_B were observed at low E_h values unless E_h reached a value at which Q_B underwent reduction at equilibrium to form Q_BH₂. The data provide evidence that reaction centers with a fully oxidized secondary acceptor can donate electrons to the cyclic electron-transport chain only after two turnovers, leading to the formation of the doubly reduced ubiquinone species (Q_BH₂) of the secondary acceptor.     

Electron transport across glycerol monooleate bilayer lipid membranes facilitated by magnesium etiochlorin.

The transport of electrons across biological membranes is believed to play an important role in many biophenomena. Although there have been many examples of systems which may be transporting electrons across Mueller-Rudin bilayer lipid membranes (blm), none has been well characterized. The system we describe here comprises a glycerol monooleate blm containing a magnesium etiochlorin (Mg-C) separating two aqueous phases each containing ferricyanide, ferrocyanide, KCl, and a platinum electrode. The E0s for the Mg-C+/Mg-C and ferri-/ferrocyanide couples are 0.22 and 0.24 V vs. SCE. Thus the MG-C+/Mb-C system is easily poised by the ferri-/ferrocyanide system. When the potentials of the ferri-/ferrocyanide couples are different on each side of the blm we show that the open-circuit membrane potential nearly equals the difference between the redox potentials. This is unequivocal evidence that electrons are being transferred across the blm from one aqueous phase to the other. On the basis of these experiments we deduce that electron transport is the major charge transport mechanism. When redox potentials are the same on each side of the blm, the conductance of the membrane can be greater than 10(-3) S/cm2. The conductance is proportional to the second power of the concentration of Mg-C in the membrane-forming mixture. A number of additional experiments are described which attempt to elucidate the mechanism of electron transfer. We believe that our data are consistent with the idea of an electron-hopping mechanism in which the transmembrane electron transport occurs by a series of second-order electron transfers between membrane-bound electron donors (Mg-C) and acceptors (Mg-C+). Alternative explanations are presented.     

Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress

Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS) is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite''s defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs) and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite''s cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica''s functions and indicate a new role for arginase in E. histolytica''s resistance to OS.     

THE PENETRATION OF STRONG ELECTROLYTES

The entrance of strong electrolytes into Valonia is very slow unless the cells are injured. This, together with the very high electrical resistance of the protoplasm, suggests that they may penetrate largely as undissociated molecules formed at the surface of the protoplasm by the collision of ions. Under favorable circumstances KCl may be absorbed to the extent of 3 x 10^–8 mols per hour per sq. cm. of surface together with about 0.17 as much NaCl. Other substances which seem to penetrate to some extent are Li, Rb, Br, BrO₃, I, IO₃, and selenite. Little or no penetration is shown by SCN, ferricyanide, ferrocyanide, formate, salicylate, tungstate, seleniate, NO₂, SO₃, Sb, glycerophosphate, and many heavy metals and the alkaline earths. In sea water whose specific gravity had been increased by CsCl cells of Valonia floated for over a year and there was little or no penetration of Cs except as the result of injury. The penetration of NH₄Cl decreases the specific gravity of the sap and causes the cells to float: under these circumstances they live indefinitely. It is probable that NH₃ or NH₄OH penetrates and is subsequently changed to NH₄Cl. It would seem that if the sea contained a little more ammonia this would be a floating organism.     

A new cytochrome b-like pigment with a peak at 567 nm and a low redox potential in denitrifying bacteria

Dithionite reduced difference spectra of extracts of denitrifying pseudomonads revealed small absorption maxima at 567 and 539 nm, suggestive of α and β bands of a new b type cytochrome. The new pigment was present in cells grown both aerobically and anaerobically and was located in the particulate fraction of extracts. These extracts also contained, in much higher concentrations, additional pigments resembling cytochromes c₅₅₃ and b₅₅₉, which were readily reduced by NADH or endogenous substrates, although a small proportion of the b₅₅₉ required dithionite for complete reduction. In contrast, most of the new 567 pigment was not readily reduced by NADH, succinate, or endogenous substrates, and it was most easily visualized with dithionite in the sample cuvette, and either endogenous substrates or NADH in the reference cuvette. Dyes of low redox potential such as benzyl viologen (E_m,7 = ?359 mV), phenosafranine (E_m,7 = ?250 mV) and reduced janus green (E_m,7 = ?225 mV) could substitute for dithionite as reductant for the new 567 pigment. Cresyl violet (E_m,7 = ?160 mV) caused partial reduction. However, redox compounds of higher potential such as reduced indigo carmine, (E_m,7 = ?125 mV) reduced methylene blue (E_m,7 = ?11 mV), ferrooxalate and ascorbate could not replace dithionite as reductant. Most of the cytochrome b₅₅₉ and the c₅₅₃ were reduced by ascorbate. Thus the new 567 pigment appears to have a mid-point potential between ?225 and ?125 mV, well below most of the cytochrome b₅₅₉. The new 567-nm pigment was rapidly oxidized by brief but vigorous aeration and was also slowly and partially re-reduced when concentrated extracts were allowed to stand without aeration. A more complete reduction of the 567 pigment was readily obtained by the addition of a mixture of NADH and FAD. The 567 pigment was observed in several denitrifying pseudomonads, P. fluorescens, P. stutzeri and also in Micrococcus denitrificans, but was not detectable in the non-denitrifiers Escherichia coli or Aerobacter aerogenes.     

An investigation of hemopexin redox properties by spectroelectrochemistry: biological relevance for heme uptake

Hemopexin (HPX) has two principal roles: it sequesters free heme in vivo for the purpose of preventing the toxic effects of this moiety, which is largely due to heme’s ability to catalyze free radical formation, and it transports heme intracellularly thus limiting its availability as an iron source for pathogens. Spectroelectrochemistry was used to determine the redox potential for heme and meso-heme (mH) when bound by HPX. At pH 7.2, the heme-HPX assembly exhibits E _1/2 values in the range 45–90 mV and the mH-HPX assembly in the range 5–55 mV, depending on environmental electrolyte identity. The E _1/2 value exhibits a 100 mV positive shift with a change in pH from 7.2 to 5.5 for mH-HPX, suggesting a single proton dependent equilibrium. The E _1/2 values for heme-HPX are more positive in the presence of NaCl than KCl indicating that Na⁺, as well as low pH (5.5) stabilizes ferro-heme-HPX. Furthermore, comparing KCl with K₂HPO₄, the chloride salt containing system has a lower potential, indicating that heme-HPX is easier to oxidize. These physical properties related to ferri-/ferro-heme reduction are both structurally and biologically relevant for heme release from HPX for transport and regulation of heme oxygenase expression. Consistent with this, when the acidification of endosomes is prevented by bafilomycin then heme oxygenase-1 induction by heme-HPX no longer occurs.     

The cytochromes of a marine Beggiatoa

Naturally grown Beggiatoa filaments, occurring in massive near-mono-cultures at a “black smoker” wall of the Guaymas Basin hydrothermal vent site, were harvested and used for the analysis of their cytochromes. The cytochromes have been characterized by gel permeation chromatography, optical spectroscopy and redox potentiometry. Only c-type cytochromes were detected; a small, high potential cytochrome c that seems typical of its class, and a large complex (M_r 210,000) containing at least four thermodynamically distinct c-type hemes, which was partially dissociated by chromatography on DEAE-Sepharose. The hemes of the large complex have appropriate oxidation-reduction midpoint potentials (E_m7 +240 mV, +15 mV,-160 mV,-340 mV) to be involved in the metabolism of sulfide, which is presumed to be the source of reductant for this organism.     

EFFECTS OF pH AND OF VARIOUS CONCENTRATIONS OF SODIUM,POTASSIUM, AND CALCIUM CHLORIDE ON MUSCULAR ACTIVITY OF THE ISOLATED CROP OF PERIPLANETA AMERICANA (ORTHOPTERA)

1. Twenty-five solutions which contained KCl (0.0, 0.2, 0.4, 0.6, and 0.8 gm. per liter), in combination with CaCl₂ (0.0, 0.2, 0.4, 0.6, and 0.8 gm. per liter), 10.0 gm. of NaCl, and 0.2 gm. of NaHCO₃ per liter of solution were tested in order to determine satisfactory KCl/CaCl₂ ratios in an insect physiological salt mixture for the maintenance of muscular activity by the isolated crop of the American roach. Satisfactory activity products (0.390 to 0.549) were obtained in seven mixtures with KCl/CaCl₂ ratios of 0.2/0.2, 0.4/0.4, 0.6/0.6, 0.8/0.8, 0.2/0.4, 0.4/0.6, and 0.6/0.8, expressed as gram per liter. These ratios lie between 0.50 and 1.00. In solutions which contained calcium, but no potassium, approximately 50 per cent of the crops exhibited an initial tone increase and were arrested in rigor. See Fig. 2. In solutions which contained potassium, but no calcium, all crops showed an initial loss of tone and arrest in relaxation. See Fig. 2. 2. Seven KCl/CaCl₂ ratios (see paragraph 1 above) were tested with eight NaCl concentrations (1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, and 1.8 per cent) at a pH of 8.0. In these mixtures, the ones with KCl/CaCl₂ ratios of less than 1.0 produced higher activity products than those with ratios equal to 1.00. The highest average activity product (0.849) was obtained in the solutions with 0.2 gm. of KCl and 0.4 gm. of CaCl₂ per liter. 3. Four KCl/CaCl₂ ratios (0.2/0.2, 0.4/0.4, 0.2/0.4, and 0.4/0.6 gm. per liter) were tested with 1.4, 1.5, and 1.6 per cent NaCl at a pH of 7.5. When analyzed with data from comparable solutions at a pH of 8.0, it was found that 1.4 per cent NaCl afforded an optimum environment for isolated crop activity. 4. Effects of hydrogen and hydroxyl ion concentrations were studied at pH values of 6.8, 7.5, 8.0, and 8.9. The highest average activity product, 1.011, was produced at a pH of about 8.0. 5. A satisfactory physiological salt solution for the isolated foregut of the American roach, Periplaneta americana, would contain 14.0 gm. of NaCl, 0.4 gm. of CaCl₂, 0.2 gm. of KCl, and 0.2 gm. of NaHCO₃ per liter of solution. This mixture should have a pH value between 7.8 and 8.2. 6. Durations of crop activity extending over periods as long as 25 hours were quite common, and several crops maintained contractions for more than 30 hours. The greatest longevity was for crop 814, from a female, which continued activity for slightly more than 47 hours. 7. A significant difference between the activity products of the crops from males and the crops from females was recorded. Although there was not a significant difference in the amount of food ingested by males and females, 12 hours after feeding there was more food in the females'' crops, and the food progressed more rapidly through the males'' crops than through the females''. In addition, crops from the two sexes reacted differently to the effects of day old solutions. This sex difference is apparently related to an inherently increased activity of the crop from the male roach.     

A possible relationship between the effect of factors on photoactivation of photosystem II depleted of functional Mn and cytochrome b559

The photoassembly of the Mn₄CaO₅ cluster in Mn-depleted photosystem II preparations (photoactivation) was studied under the influence of oxidants, reductants and pH. New data on the effect of these factors on the photoactivation yield are presented. The presence of the oxidant, ferricyanide, negatively affected the photoactivation yield over the entire concentration range studied (0–1 mM). In contrast to ferricyanide, the addition of the reductant, ferrocyanide, up to 1 mM resulted in an increase in the photoactivation yield. Other reductants either did not significantly affect (diphenylcarbazide) or suppressed (ascorbate) the photoactivation yield. The effect of ferrocyanide on photoactivation were found to be similar dichlorophenolindophenol. Investigation of the photoactivation yield as a function of pH revealed that the maximum yield was observed at pH 6.5 in the presence of ferrocyanide and DCPIP, and at pH 5.5 without additives. In addition, the photoactivation yield at pH 5.5 was the same without and with the addition of ferrocyanide or dichlorophenolindophenol. Although ferricyanide suppressed the photoactivation, the photoactivation yield increased in the presence of ferricyanide by shifting the pH to the acidic region. The samples contained approximately 25 % of the HP cyt b₅₅₉, which was in the reduced state, as the absorbance at 559 nm was decreased upon addition of ferricyanide and subsequent addition of ferrocyanide returned the spectrum to the baseline. A possible relationship between the effect of factors on the photoactivation and the involvement of cyt b₅₅₉ in the protection of PSII from oxidative damage on the donor side is discussed.     

Localised corrosion induced by a marine vibrio

The corrision of mild steel in media with and without bacterial cultures was assessed using potentiostatic and potentiodynamic techniques and the production of biofilm on the metal surface was studied by scanning electron microscopy.Metal in a solution consisting of the inorganic components of Postgate's medium C was not passivated, but a passive surface was indiced by the addition of lactate, citrate, or phosphate. The breakdown potential (E_b of the passivated metal was most anodic for phosphate. No significant change in the electrochemical behaviour of the steel was seen when the formulation of Postgate's medium C was completed by the addition of yeast extract, but chloride, added to allow the growth of Vibrio alginolyticus, caused a reduction in the E_b value.Vibrio alginolyticus reduced the E_b value in Postgate's medium C from −0·37 to −0·43V, indicating its corrosive capacity. This value was reduced still further, to −0·60V, when sulphate-reducing bacteria were also present.Scanning electron microscopy showed the presence of colonies of V. alginolyticus on the metal surface. When cleaned, it was apparent that intense pitting had occurred beneath these colonies.It is suggested that V. alginolyticus may promote chemical or SRB-induced corrosion by removing a passive film from the metal, allowing aggressive species such as sulphides to affect the surface.     

Membrane potential difference of isolated plant vacuoles: positive or negative? I. Evidence for membrane binding of cationic probes

The binding of membrane potential cationic probes was studied on phospholipidic liposomes by equilibrium dialysis and microelectrophoresis. Surface binding of lipophilic cations (benzyltributylammonium or tetraphenylphosphonium) appears to be the major accumulation mechanism in liposomes and simulates the existence of a negative transmembrane potential (E_m), in absence of any transmembrane ionic gradient. Furthermore, this apparent negative potential has a classical response with regard to common E_m effectors, namely a depolarization induced by KCl or FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone). The relevance of these results to the study of transtonoplast potential difference on isolated vacuoles was investigated. Tetraphenylphosphonium was shown to bind to the tonoplast, the essential features of binding and interaction with E_m effectors being similar in vacuoles and liposomes. Therefore the assumption of negligible binding of cationic probe to vacuoles, classically admitted in determinations of vacuolar E_m using lipophilic cations, is untenable.     

THE GROWTH OF DUCKWEEDS IN MINERAL NUTRIENT SOLUTIONS WITH AND WITHOUT ORGANIC EXTRACTS

1. Detmer''s solution and a modified Knop''s solution are unfavorable culture media for the growth of Spirodela polyrhiza. 2. When the modified Knop''s solution was diluted to 10 times its volume, Spirodela polyrhiza and Lemna valdiviana grew and reproduced for periods of 26 months and 21 months, respectively. 3. Growth in the dilute Knop''s solution, which alone can support the growth of Spirodela indefinitely, was considerably stimulated over a period of 23 days by adding to every liter the water-soluble material of 0.4 gm. autolyzed yeast, or the material of 2.5 gm. peat soluble in a 1 per cent solution of NaHCO₃. 4. The nature of the stimulus or of the protection afforded by the organic material is as yet unknown. 5. The necessity of organic accessory foods (auximones) in the nutrition of green plants cannot be accepted as an established fact.     

Generation of a proton gradient in Desulfovibrio vulgaris

Washed cells of Desulfovibrio vulgaris strain Marburg oxidized H₂, formate, lactate or pyruvate with sulfate, sulfite, trithionate, thiosulfate or oxygen as electron acceptor. CuCl₂ as an inhibitor of periplasmic hydrogenase inhibited H₂ and formate oxidation with sulfur compounds, and lactate oxidation in H₂-grown, but not in lactate-grown cells. H₂ oxidation was sensitive to O₂ concentrations above 2% saturation. Carbon monoxide inhibited the oxidation of all substrates tested. Additions of micromolar H₂ pulses to cells incubated in KCl in the presence of various sulfur compounds (reductant pulse method) resulted in a reversible acidification. This proton release was stimulated by thiocyanate, methyl triphenylphosphonium (MTPP⁺) or valinomycin plus EDTA, and completely inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), CuCl₂ or carbon monoxide. The extrapolated H⁺/H₂ ratios obtained with sulfate, sulfite, trithionate or thiosulfate varied from 1.0 to 1.7. Micromolar additions of O₂ to cells incubated in the presence of excess of electron donor (oxidant pulse method) caused proton translocation with extrapolated H⁺/H₂ ratios of 3.9 with H₂, 1.6 with lactate and 2.4 with pyruvate. Since a periplasmic hydrogenase can release at maximum 2 H⁺/H₂, it is concluded that D. vulgaris is able to generate a proton gradient by vectorial proton translocation across the cytoplasmic membrane and by extracellular proton release by a periplasmic hydrogenase.     

THE EFFECTS OF CURRENT FLOW ON BIOELECTRIC POTENTIAL : III. NITELLA

String galvanometer records show the effect of current flow upon the bioelectric potential of Nitella cells. Three classes of effects are distinguished. 1. Counter E.M.F''S, due either to static or polarization capacity, probably the latter. These account for the high effective resistance of the cells. They record as symmetrical charge and discharge curves, which are similar for currents passing inward or outward across the protoplasm, and increase in magnitude with increasing current density. The normal positive bioelectric potential may be increased by inward currents some 100 or 200 mv., or to a total of 300 to 400 mv. The regular decrease with outward current flow is much less (40 to 50 mv.) since larger outward currents produce the next characteristic effect. 2. Stimulation. This occurs with outward currents of a density which varies somewhat from cell to cell, but is often between 1 and 2 µa/cm.² of cell surface. At this threshold a regular counter E.M.F. starts to develop but passes over with an inflection into a rapid decrease or even disappearance of positive P.D., in a sigmoid curve with a cusp near its apex. If the current is stopped early in the curve regular depolarization occurs, but if continued a little longer beyond the first inflection, stimulation goes on to completion even though the current is then stopped. This is the "action current" or negative variation which is self propagated down the cell. During the most profound depression of P.D. in stimulation, current flow produces little or no counter E.M.F., the resistance of the cell being purely ohmic and very low. Then as the P.D. begins to recover, after a second or two, counter E.M.F. also reappears, both becoming nearly normal in 10 or 15 seconds. The threshold for further stimulation remains enhanced for some time, successively larger current densities being needed to stimulate after each action current. The recovery process is also powerful enough to occur even though the original stimulating outward current continues to flow during the entire negative variation; recovery is slightly slower in this case however. Stimulation may be produced at the break of large inward currents, doubtless by discharge of the enhanced positive P.D. (polarization). 3. Restorative Effects.—The flow of inward current during a negative variation somewhat speeds up recovery. This effect is still more strikingly shown in cells exposed to KCl solutions, which may be regarded as causing "permanent stimulation" by inhibiting recovery from a negative variation. Small currents in either direction now produce no counter E.M.F., so that the effective resistance of the cells is very low. With inward currents at a threshold density of some 10 to 20 µa/cm.², however, there is a counter E.M.F. produced, which builds up in a sigmoid curve to some 100 to 200 mv. positive P.D. This usually shows a marked cusp and then fluctuates irregularly during current flow, falling off abruptly when the current is stopped. Further increases of current density produce this P.D. more rapidly, while decreased densities again cease to be effective below a certain threshold. The effects in Nitella are compared with those in Valonia and Halicystis, which display many of the same phenomena under proper conditions. It is suggested that the regular counter E.M.F.''S (polarizations) are due to the presence of an intact surface film or other structure offering differential hindrance to ionic passage. Small currents do not affect this structure, but it is possibly altered or destroyed by large outward currents, restored by large inward currents. Mechanisms which might accomplish the destruction and restoration are discussed. These include changes of acidity by differential migration of H ion (membrane "electrolysis"); movement of inorganic ions such as potassium; movement of organic ions, (such as Osterhout''s substance R), or the radicals (such as fatty acid) of the surface film itself. Although no decision can be yet made between these, much evidence indicates that inward currents increase acidity in some critical part of the protoplasm, while outward ones decrease acidity.     

THE RELATIONS OF TEMPERATURE TO THE POTASSIUM EFFECT AND THE BIOELECTRIC POTENTIAL OF VALONIA

The effect of temperature upon the bioelectric potential across the protoplasm of impaled Valonia cells is described. Over the ordinary tolerated range, the P.D. is lowest around 25°C., rising both toward 15° and 35°. The time curves are characteristic also. The magnitude of the temperature effect can be controlled by changing the KCl content of the sea water (normally 0.012 M): the magnitude is greatly reduced at 0.006 M KCl, enhanced at 0.024 M, and greatly exaggerated at 0.1 M KCl. Conversely, temperature controls the magnitude of the potassium effect, which is smallest at 25°, with a cusped time course. It is increased, with a smoothly rising course, at 15°, and considerably enhanced, with only a small cusp, at 35°. A temporary "alteration" of the protoplasmic surface by the potassium is suggested to account for the time courses. This alteration does not occur at 15°; the protoplasm recovers only slowly and incompletely at 25°, but rapidly at 35°, in such fashion as to make the P.D. more negative than at 15°. This would account for the temperature effects observed in ordinary sea water.