SODIUM AND CHLORIDE FLUXES IN SYNAPTOSOMES IN VITRO

Synaptosomes incubated in a physiological saline extrude sodium and take up potassium. As would be expected this process is completely blocked by metabolic inhibitors such as cyanide and iodoacetate. However, when metabolic inhibitors are replaced by ouabain (100 μM) there is an increase in the steady state intrasynaptosomal sodium and chloride content even though there is no change in the potassium content. The increases are prevented when synaptosomes are incubated with metabolic inhibitors in addition to ouabain. There is therefore a ouabain-insensitive process that transports sodium, chloride and concomitantly water into synaptosomes. It appears not to function when the supply of metabolic energy is inhibited. The diuretic furosemide (1 mM) in the presence of ouabain inhibits the entry of sodium and chloride without affecting the intrasynaptosomal potassium concentration. Ethacrynic acid (1 mM) has a somewhat similar effect but in addition appears to damage the synaptosome membrane. Kinetic measurements were made of the uptake of sodium, potassium and chloride under conditions of metabolic inhibition and the permeability constants of the membrane determined. Values of 0.068, 0.117 and 0.032 × 10^-6 (cm s^-1) were found for the permeability constants of the membrane to (respectively) sodium, potassium and chloride. Measurements of the rate of uptake in the presence of ouabain revealed an inwardly directed sodium and chloride flux of 5-20 pmol cm^-2 s^-1. Calculation of the fluxes from the steady state ion concentrations also reveals an inwardly directed sodium and chloride flux, though of lesser magnitude. The influx of water is less than would be expected to preserve osmotic equality suggesting that the translocation of sodium and chloride is the primary event. Although its function remains uncertain the flux has a considerable effect on the ion content of synaptosomes.     

Effects of phenothiazines on inhibition of plasma membrane ATPase and hyperpolarization of cell membranes in the yeast Saccharomyces cerevisiae

The transmembranal potential, in Saccharomyces cerevisiae, has been calculated from the distribution of the lipophilic cation tetraphenylphosphonium (TPP⁺) between the intracellular and extracellular water. Trifluoperazine at concentrations of 10 to 50 μM, caused a substantial increase in the membrane potential (negative inside). This increase was observed only in the presence of a metabolic substrate and was eliminated by the addition of the protonophores 2,4-dinitrophenol and sodium azide, removal of glucose, replacement of glucose by the nonmetabolizable analog 3-O-methyl glucose, or by the addition of 100mM KCl. An increase in ⁴⁵CaCl₂ accumulation from solutions of low concentrations (1 μM) was observed under all conditions where membrane potential was increased. Proton ejection activity was monitored by measurements of the rates of the decrease in the pH of unbuffered cell suspensions in the presence of glucose. Trifluoperazine inhibited the changes in medium pH; this inhibition was not the result of an increase in the permeability of cell membranes to protons since in the absence of glucose, trifluoperazine did not cause a change in the rate of pH change generated by proton influx. The activity of plasma membrane ATPase was measured in crude membrane preparations in the presence of sodium azide to inhibit mitochondrial ATPase. Trifluoperazine strongly inhibited the activity of the plasma membrane ATPase. The effect of phenothiazines on transport and on membrane potential reported in this study and in the previous one (Eilam, Y. (1983) Biochim. Biophys. Acta 733, 242–248) were observed only in the presence of a metabolic substrate. The possibility that energy is required for the uptake of phenothiazines into the cells was eliminated by results showing energy-independent uptake of [³H]chlorpromazine. The results strongly suggest that phenothiazines activate energy-dependent K⁺-extrusion pumps, which lead to increased membrane potential. Increased influx of calcium seems to be energized by membrane potential, and therefore stimulated under all conditions where membrane potential is increased. The analog which does not bind to calmodulin, trifluoperazine sulfoxide, had no effect on the cells, but the involvement of calmodulin in the processes altered by trifluoperazine cannot as yet, be determined.     

Reversal of Na+-dependent glycine transport in sheep reticulocyte membrane vesicles

Inside-out membrane vesicles have been prepared from sheep reticulocytes. With these vesicles, Na⁺-dependent glycine uptake and net accumulation have been demonstrated to occur in reverse, i.e., from extravesicular (normal cytoplasmic) to intravesicular (normal extravesicular) surface. Uptake and accumulation are inhibited by energization of the sodium pump by ATP whereby the Na⁺ electrochemical gradient is dissipated. Glycine-dependent Na⁺ uptake was also observed, providing evidence that Na⁺-dependent glycine influx into these vesicles, equivalent to normal efflux, is characterized by Na⁺-glycine co-transport.     

Sink Metabolism in Tomato Fruit : II. Phloem Unloading and Sugar Uptake

Analysis of [³H]-(fructosyl)-sucrose translocation in tomato (Lycopersicon esculentum Mill.) indicates that phloem unloading in the fruit occurs, at least in part, to the apoplast followed by extracellular hydrolysis. Apoplastic sucrose, glucose, and fructose concentrations were estimated as 1 to 7, 12 to 49, and 8 to 63 millimolar, respectively in the tomato fruit pericarp tissue. Hexose concentrations were at least four-fold greater than sucrose at all developmental stages. Short-term uptake of [¹⁴C]sucrose, -glucose, and -fructose in tomato pericarp disks showed first order kinetics over the physiologically relevant concentration range. The uptake rate of [¹⁴C]-(glucosyl)-1′-fluorosucrose was identical to the rate of [¹⁴C]sucrose uptake, suggesting sucrose may be taken up directly without prior extracellular hydrolysis. Short-term uptake of all three sugars was insensitive to 10 micromolar carbonyl cyanide m-chlorophenylhydrazone and to 10 micromolar p-chloromercuribenzene sulfonic acid. However, long-term accumulation of glucose was sensitive to carbonyl cyanide m-chlorophenylhydrazone. Together these results suggest that although sucrose is at least partially hydrolyzed in the apoplast, sucrose may enter the metabolic carbohydrate pool directly. In addition, sugar uptake across the plasma membrane does not appear to be energy dependent, suggesting that sugar accumulation in the tomato fruit is driven by subsequent intracellular metabolism and/or active uptake at the tonoplast.     

Potassium activation of Na+-dependent leucine transport in brush-border membrane vesicles from rat jejunum

Na⁺-dependent leucine uptake was greater in potassium loaded brush-border membrane vesicles compared with controls. This effect was not mediated by an electrical potential difference, since it was still present in voltage-clamped conditions. Inhibition experiments indicate the same Na⁺-dependent leucine transport activity in the presence or in the absence of potassium. The affinity of sodium for the cotransporter was identical at 10 or 100 mM potassium. Leucine kinetics at different potassium concentrations showed a maximum 2.4-fold increase in V_max, while K_m was unaffected. The secondary plots of the kinetic results were not linear. This kinetic behaviour suggests that K⁺ acts as a non-essential activator of Na⁺-dependent leucine cotransport. A charge compensation of sodium-leucine influx is most probably a component of the potassium effect in the presence of valinomycin.     

Does endogenous cyclic GMP inhibit potassium stimulated 45Ca uptake by P2 fraction from rat brain?

The effects of sodium azide (NaN₃), hydroxylamine (NH₂OH) and sodium nitroprusside on potassium-stimulated ⁴⁵Ca uptake (K-stimulated ⁴⁵Ca uptake) by P₂ fraction of Gray and Whittaker from rat brain were investigated. During preincubation with these reagents, the contents of cyclic GMP in synaptosomes increased, reaching maximum levels within 2 min. On preincubation for 2 min, NaN₃, an activator of membrane bound guanylate cyclase, inhibited K-stimulated ⁴⁵Ca uptake, but NH₂OH and sodium nitroprusside did not affect it. Sodium cyanide, another metabolic inhibitor, had no effect on K-stimulated ⁴⁵Ca uptake. There was a correlation between inhibition of K-stimulated ⁴⁵Ca uptake and increase in the cGMP level on preincubation with NaN₃ for various periods. Based on these results role of cGMP in or around the membrane was discussed in relation to the K-stimulated ⁴⁵Ca uptake by P₂ fraction.     

Nonosmotic Effects of Polyethylene Glycols upon Sodium Transport and Sodium-Potassium Selectivity by Rice Roots

Addition of polyethylene glycol (PEG) as an osmotic agent (at −230 kilopascals) dramatically lessened the toxicity of NaCl (at 50 moles per cubic meter) to rice (Oryza sativa L.) seedlings. This was explained by a reduction in the uptake of NaCl. This reduction was much greater than could be accounted for by the lowered transpiration rate resulting from the solute potential changes due to the PEG.

Low concentrations of PEG (−33 kilopascals and less) had no effect upon transpiration rate but reduced sodium uptake (from 10-50 moles per cubic meter NaCl) by up to 80%. PEG (at −33 kilopascals) also reduced chloride uptake but had no effect upon the uptake of potassium from low (0.5-2.0 moles per cubic meter) external concentrations. However, the increased uptake of potassium occurring between 2 and 10 moles per cubic meter external concentration was abolished by PEG. Similar concentrations of mannitol had no effect upon sodium uptake in rice. PEG, in similar conditions, had much less effect upon sodium uptake by the more salt-resistant species, barley.

²²Na studies showed that PEG reduced the transport of sodium from root to shoot, but had a long half time for maximal effect (several days).

¹⁴C-labeled PEG was shown to bind to microsomal membranes isolated from rice roots; it is suggested that this is due to multipoint attachment of the complex ions of PEG which exist in aqueous solutions. It is argued that this reduces passive membrane permeability, which accounts for the large effect of PEG on sodium influx in rice and the different effects on sodium influx and (carrier-dependent) potassium influx.

     

Uptake of N-methyl-4-phenylpyridinium ion (MPP+) into PC12h pheochromocytoma cells

The uptake and accumulation of N-methyl-4-phenylpyridinium ion (MPP⁺), a neurotoxin produced by oxidation of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), into PC12h pheochromocytoma cells were examined. Concentration gradients of MPP⁺ were established at its low concentrations of 10 to 100 nM. Uptake of MPP⁺ into PC12h cells was mediated by saturable, carrier mediated transport systems with two different kinetic properties; a high-affinity and low-capacity system and a low-affinity and high-capacity system. The apparent K_m values of these two systems were obtained to be 254.4 ± 96.5 nM and 23.1 ± 6.9 μM, respectively, and the maximal uptake velocity was obtained to be 8.47 ± 1.72 and 28.6 ± 5.2 pmol/min/mg protein, respectively. The uptake by a high-affinity system was mediated by a carrier system common to dopamine and noradrenalin and MPTP itself proved to be taken up by this system, which was further confirmed by the inhibition of the MPP⁺ uptake by nomifensine and mazindol. The uptake was inhibited by metabolic inhibitors, such as carbonyl cyanide m-chlorophenyl hydrazone, sodium cyanide and 2,4-dinitrophenol, and the uptake was inhibited by ouabain and nigercin. By subcellular fractionation, MPP⁺ taken up was found to be localized mainly in cytosol fraction, but a definite amount of MPP⁺ was found also in mitochondrial fraction.     

Icreased drug permeability in Chinese hamster ovary cells in the presence of cyanide

In this report we investigated whether the modulation of drug permeability in Chinese hamster ovary (CHO) cells was an energy-dependent process. We observed that (1) in the absence of glucose, metabolic inhibitors such as cyanide, azide, and dinitrophenol stimulated the uptake of [³H]colchicine and other drug; (2) cyanide-induced stimulation of drug uptake could be prevented by the presence of metabolizable sugars such as glucose and ribose; (3) cyanide-treated cells were fully viable; (4) on the addition of cyanide and glucose the kinetics of drug permeability changes were very rapid. These data are consistent with the hypothesis that an energy-dependent membrane barrier against the uptake of a variety of drugs was operative in CHO cells.The nature of this energy-dependent membrane barrier was examined in colchicine-resistant mutants (CH^RC4 and CH^RC5 cells) previously characterized as membrane mutants with greatly reduced drug permeability (Ling and Thompson, (1974) J. Cell Physiol. 83, 103–116). The mutants were more refractile to the cyanide-induced stimulation of drug permeability but more sensitive to the glucose prevention cyanide-induction. In the presence of cyadine, the uptake rate of [³H] colchicine by CH^RC4 cells increased by about 100-fold and approached a rate similar to that of wild-type cells. These results suggest that the colchicine-resistant mutants may be altered in their energy-dependent modulation of drug permeability.     

Relationship between Sodium Influx and Salt Tolerance of Nitrogen-Fixing Cyanobacteria

The relationship between sodium uptake and cyanobacterial salt (NaCl) tolerance has been examined in two filamentous, heterocystous, nitrogen-fixing species of Anabaena. During diazotrophic growth at neutral pH of the growth medium, Anabaena sp. strain L-31, a freshwater strain, showed threefold higher uptake of Na⁺ than Anabaena torulosa, a brackish-water strain, and was considerably less salt tolerant (50% lethal dose of NaCl, 55 mM) than the latter (50% lethal dose of NaCl, 170 mM). Alkaline pH or excess K⁺ (>25 mM) in the medium causes membrane depolarization and inhibits Na⁺ influx in both cyanobacteria (S. K. Apte and J. Thomas, Eur. J. Biochem. 154:395-401, 1986). The presence of nitrate or ammonium in the medium caused inhibition of Na⁺ influx accompanied by membrane depolarization. These experimental manipulations affecting Na⁺ uptake demonstrated a good negative correlation between Na⁺ influx and salt tolerance. All treatments which inhibited Na⁺ influx (such as alkaline pH, K⁺ above 25 mM, NO₃⁻, and NH₄⁺), enhanced salt tolerance of not only the brackish-water but also the freshwater cyanobacterium. The results indicate that curtailment of Na⁺ influx, whether inherent or effected by certain environmental factors (e.g., combined nitrogen, alkaline pH), is a major mechanism of salt tolerance in cyanobacteria.     

The oxidation of glutathione by nitroprusside: Changes in glutathione in intact erythrocytes during incubation with sodium nitroprusside as detected by 1H spin echo NMR spectroscopy

Spin echo ¹H NMR spectroscopy showed that when the hypotensive agent sodium nitroprusside, Na₂[Fe(CN)₅NO]·2H₂O, was incubated with intact erythrocytes in ²H₂O saline, glutathione in the erythrocytes was oxidised to diglutathione. This was confirmed by ¹H FT NMR for the in vitro reaction. ¹³C FT NMR showed that the stoichiometry of the glutathione nitroprusside reaction was 1:1; the inorganic products were nitric oxide and hexacyanoferrate(II), [Fe(CN)₆]⁻. At no stage was free cyanide liberated. The reaction of nitroprusside with glutathione, which occurs after the nitroprusside has crossed the erythrocyte membrane, is compared with the reaction of nitroprusside with haemoglobin. In neither of these reactions with major erythrocyte components was any free cyanide liberated by sodium nitroprusside.     

l-leucine, l-methionine, and l-phenylalanine share a Na+/K+-dependent amino acid transporter in shrimp hepatopancreas

Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of ³H-l-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. ³H-l-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na⁺- and K⁺-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. ³H-l-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, l-methionine, at 20 mM significantly (p < 0.0001) inhibited the 2-min uptakes of 1 mM ³H-l-leucine in both Na⁺- and K⁺-containing incubation media. The residual ³H-l-leucine uptake in the two media were significantly greater than zero (p < 0.001), but not significantly different from each other (p > 0.05) and may represent an l-methionine- and cation-independent transport system. ³H-l-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [l-leucine], following the carrier-mediated Michaelis–Menten equation. In NaCl, ³H-l-leucine influx displayed a low apparent K _M (high affinity) and low apparent J _max, while in KCl the transport exhibited a high apparent K _M (low affinity) and high apparent J _max. l-methionine or l-phenylalanine (7 and 20 mM) were competitive inhibitors of ³H-l-leucine influxes in both NaCl and KCl media, producing a significant (p < 0.01) increase in ³H-l-leucine influx K _M, but no significant response in ³H-l-leucine influx J _max. Potassium was a competitive inhibitor of sodium co-transport with ³H-l-leucine, significantly (p < 0.01) increasing ³H-l-leucine influx K _M in the presence of sodium, but having negligible effect on ³H-l-leucine influx J _max in the same medium. These results suggest that shrimp BBMV transport ³H-l-leucine by a single l-methionine- and l-phenylalanine-shared carrier system that is enhanced by acidic pH and can be stimulated by either Na⁺ or K⁺ acting as co-transport drivers binding to shared activator sites.     

Transient Activation of Plasmalemma K Efflux and H Influx in Tobacco by a Pectate Lyase Isozyme from Erwinia chrysanthemi

A purified pectate lyase isozyme derived from Erwinia chrysanthemi induced rapid net K⁺ efflux and H⁺ influx in suspension-cultured tobacco cells. Comparable fluxes of other ions (Na⁺, Cl⁻) were not observed. The K⁺ efflux/H⁺ influx response began within 15 minutes after addition of enzyme to cell suspensions and continued for approximately 1 hour after which cells resumed the net H⁺ efflux exhibited prior to enzyme treatment. The response was not prolonged by a second enzyme dose 1 hour after the first. The K⁺/H⁺ response was characterized by saturation at low enzymic activity (2 × 10⁻³ units per milliliter), and inhibition by the protonophore, carbonyl cyanide m-chlorophenylhydrazone, and was not associated with membrane leakiness caused by structural cell wall damage. The total K⁺ loss and H⁺ uptake induced by enzyme was one-fourth to one-third that induced by Pseudomonas syringae pv. pisi and did not reduce cell viability. These results indicate that pectate lyase induces a K⁺ efflux/H⁺ influx response in tobacco similar to but of shorter duration than that induced by P. syringae pv. pisi during the hypersensitive response. Pectate lyase or other cell wall degrading enzymes may therefore influence the induction of hypersensitivity.     

In Vivo pH Regulation by a Na/H Antiporter in the Halotolerant Alga Dunaliella salina

Na⁺/H⁺ exchange activity in whole cells of the halotolerant alga Dunaliella salina can be elicited by intracellular acidification due to addition of weak acids at appropriate external pH. The changes in both intracellular pH and Na⁺ were followed. Following a mild intracellular acidification, intracellular Na⁺ content increased dramatically and then decreased. We interpret the phase of Na⁺ influx as due to the activation of the plasma membrane Na⁺/H⁺ antiporter and the phase of Na⁺ efflux as due to an active Na⁺ extrusion process. The following observations are in agreement with this interpretation: (a) the Na⁺ influx phase was sensitive to Li⁺, which is an inhibitor of the Na⁺/H⁺ antiporter, did not require energy, and was insensitive to vanadate; (b) the Na⁺ efflux phase is energy-dependent and sensitive to the plasma membrane ATPase inhibitor, vanadate. Following intracellular acidification, a drastic decrease in the intracellular ATP content is observed that is reversed when the cells regain their neutral pH value. We suggest that the intracellular acidification-induced change in the internal Na⁺ concentration is due to a combination of Na⁺ uptake via the Na⁺/H⁺ antiporter and an active, ATPase-dependent, Na⁺ extrusion. The Na⁺/H⁺ antiporter seems, therefore, to play a principal role in internal pH regulation in Dunaliella.     

Alkali Cation/Sucrose Co-transport in the Root Sink of Sugar Beet

The mechanism of sucrose transport into the vacuole of root parenchyma cells of sugar beet was investigated using discs of intact tissue. Active sucrose uptake was evident only at the tonoplast. Sucrose caused a transient 8.3 millivolts depolarization of the membrane potential, suggesting an ion co-transport mechanism. Sucrose also stimulated net proton efflux. Active (net) uptake of sucrose was strongly affected by factors that influence the alkali cation and proton gradients across biological membranes. Alkali cations (Na⁺ and K⁺) at 95 millimolar activity stimulated active uptake of sucrose 2.1- to 4-fold, whereas membrane-permeating anions inhibited active sucrose uptake. The pH optima for uptake was between 6.5 and 7.0, pH values slightly higher than those of the vacuole. The ionophores valinomycin, gramicidin D, and carbonyl cyanide m-chlorophenylhydrazone at 10 micromolar concentrations strongly inhibited active sucrose uptake. These data are consistent with the hypothesis that an alkali cation influx/proton efflux reaction is coupled to the active uptake of sucrose into the vacuole of parenchyma cells in the root sink of sugar beets.     

The effect of the uncoupler carbonyl cyanide m-chlorophenylhydrazone on K+ transport,ATP level and intracellular pH of Chlorella fusca

1. Low concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced net K⁺ uptake by Chlorella fusca, optimal concentrations being 3 μM CCCP in the light and 1 μM CCCP in the dark. Higher concentrations increasingly stimulated K⁺ release. 2. Measurements of the unidirectional K⁺ fluxes showed that CCCP-induced net K⁺ uptake in the light was mainly a consequence of an inhibition of efflux. In the dark, influx was slightly stimulated in addition. 3. In conditions of CCCP-induced net K⁺ uptake, the ATP level was decreased by less than 10%. With higher CCCP concentrations it fell drastically. 4. By means of the 5,5-dimethyloxazolidine-2,4-dione distribution technique, an acidification of the cell interior on the addition of CCCP was found. 5. It is concluded that uncoupler-induced net K⁺ uptake is due to an enhanced proton leakage into the cell across the plasmalemma. Intracellular acidification by this process stimulates ATP-dependent K⁺/H⁺ exchange which, in itself, is not affected at low uncoupler concentrations.     

Asymmetry of the Na+-succinate cotransporter in rabbit renal brush-border membranes

We have investigated the symmetry of Na⁺-succinate cotransport in rabbit renal brush-border membrane vesicles. Succinate influx and efflux kinetics were measured under voltage-clamped conditions using [¹⁴C]succinate and a rapid filtration procedure. Both influx and efflux were Na⁺-dependent, saturable, temperature-sensitive, and influenced by the trans Na⁺ and succinate concentrations. The system was judged to be asymmetric, since the maximal velocity for influx was 3-fold higher than that for efflux, and trans Na⁺ inhibited influx more than efflux. This may be due to the asymmetrical insertion of the transporter in the brush-border membrane, which leads to differences in either the forward and backward translocation rates of the fully loaded carrier or the Na⁺ and succinate binding constants at the inner and outer faces of the membrane.     

Preparation of coupled mitochondria from Euglena by sonication

A fast method for obtaining mitochondria from Euglena gracilis Klebs with respiratory controls of 2.5–3 is described. The method consists, essentially, in sonication of the cells and differential centrifugation of the homogenate. This mitochondrial preparation is able to generate a membrane potential, even in presence of cyanide, and shows uptake of added Ca²⁺. ATP hydrolysis is increased by addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP).     

Solution NMR Structure and Functional Analysis of the Integral Membrane Protein YgaP from Escherichia coli

The solution NMR structure of the α-helical integral membrane protein YgaP from Escherichia coli in mixed 1,2-diheptanoyl-sn-glycerol-3-phosphocholine/1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) micelles is presented. In these micelles, YgaP forms a homodimer with the two transmembrane helices being the dimer interface, whereas the N-terminal cytoplasmic domain includes a rhodanese-fold in accordance to its sequence homology to the rhodanese family of sulfurtransferases. The enzymatic sulfur transfer activity of full-length YgaP as well as of the N-terminal rhodanese domain only was investigated performing a series of titrations with sodium thiosulfate and potassium cyanide monitored by NMR and EPR. The data indicate the thiosulfate concentration-dependent addition of several sulfur atoms to the catalytic Cys-63, which process can be reversed by the addition of potassium cyanide. The catalytic reaction induces thereby conformational changes within the rhodanese domain, as well as on the transmembrane α-helices of YgaP. These results provide insights into a potential mechanism of YgaP during the catalytic thiosulfate activity in vivo.     

Aerobic and anaerobic metabolism in Entamoeba histolytica

Respiration by Entamoeba histolylica is confirmed. A doubling of the rate of oxygen uptake was observed upon the addition of d-glucose to cells in which the glycogen reserve had been partially depleted. In cells metabolizing endogenous substrates the rate of oxygen uptake was not influenced by sodium cyanide or sodium succinate. It was slightly depressed when d-mannose was the added sugar. The end products, CO₂, ethanol, and acetate accounted for essentially all of the glucose carbon utilized in both aerobic and anaerobic experiments. The radioactivity from uniformly labelled ¹⁴C-glucose was found in these products. Three times as much ethanol as acetate was produced in the anaerobic experiments and in the aerobic experiments this ratio was approximately reversed.