EFFECT OF METABOLIC INHIBITORS ON NUCLEAR PORE FORMATION DURING THE HELA S3 CELL CYCLE

The effect of various antimetabolites on nuclear pore formation was studied in synchronized HeLa S₃ cells. The nuclear size was determined by light microscopy and the pore number per unit area of nuclear surface by the freeze-etching technique and electron microscopy. It was found that the inhibition of DNA replication or ribosomal RNA synthesis has no effect on nuclear size increase or pore formation. However, the inhibition of ATP synthesis effectively stops nuclear pore formation. Cycloheximide blocks nuclear pore formation at the same time during G₁ phase of the cell cycle when nuclear size increase is blocked by high concentrations of actinomycin D. This suggests that certain proteins or other factors leading to pore formation and nuclear size increase are transcribed and synthesized at about 3–4 h after mitosis, i.e., about 1–2 h before S phase begins.     

The relationship of cAMP levels and the rate of pool labelling of cAMP and related bases, nucleosides and nucleotides to the HeLa cell cycle.

Cellular cAMP levels as well as the rate of pool labelling of cAMP and related bases, nucleosides and nucleotides were determined in synchronized cultures of HeLa cells after pulse-labelling with [¹⁴C]adenine. The cAMP levels were found to be maximal in G 1 and minimal in G 2 and mitosis, as previously reported by others. The rate of labelling of the cAMP pools, however, was found to be maximal in G 2 and decreased to a minimum in G 1. This suggests that the rate of cAMP synthesis is highest when pool level is lowest and vice versa. A comparison of cAMP levels and the rate of 5′AMP pool labelling throughout the HeLa cell cycle indicated an inverse relationship. Such a relationship emphasizes the role of the cyclic 3′,5′-phosphodiesterase activity during the cell cycle. The kinetics of pool labelling of IMP, ATP, and hypoxanthine throughout the cell cycle suggested that the adenylate energy charge fluctuated as a function of the cell cycle. The apparent activation of the adenylate cyclase during G 2 and mitosis as reflected by the increased rate of cAMP pool labelling suggests that the super phosphorylation of H 1 histone during G 2-mitotic transition may be mediated by cAMP-dependent phosphokinases.     

CYTOLOGICAL STUDIES ON THE ANTIMETABOLITE ACTION OF 2,6-DIAMINOPURINE IN VICIA FABA ROOTS

At a concentration of 9.6 x 10^–5 M, 2,6-diaminopurine (DAP) completely inhibited cell enlargement, cell division, and DNA synthesis (determined by microphotometric measurement of Feulgen dye) in Vicia faba roots. Inhibition of cell enlargement was partially reversed by adenine, guanine, xanthine, adenosine, and desoxyadenosine. Guanine and the nucleosides gave the greatest reversal, suggesting that one point of DAP action upon cell enlargement is a disruption of nucleoside or nucleotide metabolism, possibly during pentosenucleic acid synthesis. DAP inhibited cell division by preventing onset of prophase. At the concentrations used it had no significant effect on the rate or appearance of mitoses in progress. Inhibition of entrance into prophase was not directly due to inhibition of DNA synthesis since approximately half of the inhibited nuclei had the doubled (4C) amount of DNA. Adenine competitively reversed DAP inhibition of cell division, giving an inhibition index of about 0.5. Guanine gave a slight reversal while xanthine, hypoxanthine, adenosine, and desoxyadenosine were inactive. A basic need for free adenine for the onset of mitosis was suggested by this reversal pattern. Meristems treated with DAP contained almost no nuclei with intermediate amounts of DNA, indicating that DAP prevented the onset of DNA synthesis while allowing that underway to reach completion. The inhibition of DNA synthesis was reversed by adenine, adenosine, and desoxyadenosine although synthesis appeared to proceed at a slower rate in reversals than in controls. Inhibition of DNA synthesis by DAP is probably through nucleoside or nucleotide metabolism. A small general depression of DNA content of nuclei in the reversal treatments was observed. This deviation from DNA "constancy" cannot be adequately explained at present although it may be a result of direct incorporation of DAP into DNA. The possible purine precursor, 4-amino-5-imidazolecarboxamide gave no reversal of DAP inhibition of cell elongation and cell division and only a slight possible reversal of inhibition of DNA synthesis.     

Energy Generation from the CO Oxidation-Hydrogen Production Pathway in Rubrivivax gelatinosus

When incubated in the presence of CO gas, Rubrivivax gelatinosus CBS induces a CO oxidation-H₂ production pathway according to the stoichiometry CO + H₂O → CO₂ + H₂. Once induced, this pathway proceeds equally well in both light and darkness. When light is not present, CO can serve as the sole carbon source, supporting cell growth anaerobically with a cell doubling time of nearly 2 days. This observation suggests that the CO oxidation reaction yields energy. Indeed, new ATP synthesis was detected in darkness following CO additions to the gas phase of the culture, in contrast to the case for a control that received an inert gas such as argon. When the CO-to-H₂ activity was determined in the presence of the electron transport uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), the rate of H₂ production from CO oxidation was enhanced nearly 40% compared to that of the control. Upon the addition of the ATP synthase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), we observed an inhibition of H₂ production from CO oxidation which could be reversed upon the addition of CCCP. Collectively, these data strongly suggest that the CO-to-H₂ reaction yields ATP driven by a transmembrane proton gradient, but the detailed mechanism of this reaction is not yet known. These findings encourage additional research aimed at long-term H₂ production from gas streams containing CO.     

LIMITED DNA SYNTHESIS IN THE ABSENCE OF PROTEIN SYNTHESIS IN PHYSARUM POLYCEPHALUM

Actidione (cycloheximide), an antibiotic inhibitor of protein synthesis, blocked the incorporation of leucine and lysine during the S phase of Physarum polycephalum. Actidione added during the early prophase period in which mitosis is blocked totally inhibited the initiation of DNA synthesis. Actidione treatment in late prophase, which permitted mitosis in the absence of protein synthesis, permitted initiation of a round of DNA replication making up between 20 and 30% of the unreplicated nuclear DNA. Actidione treatment during the S phase permitted a round of replication similar to the effect at the beginning of S. The DNA synthesized in the presence of actidione was replicated semiconservatively and was stable through at least the mitosis following antibiotic removal. Experiments in which fluorodeoxyuridine inhibition was followed by thymidine reversal in the presence of actidione suggest that the early rounds of DNA replication must be completed before later rounds are initiated.     

Adenine nucleotide metabolism of blood platelets. IX. Time course of secretion and changes in energy metabolism in thrombin-treated platelets.

Changes in the energy metabolism of washed human platelets were compared with the kinetics of secretion induced by thrombin (5 units/ml). A 50% decrease in the level of metabolic ATP (3H-labelled), which was essentially complete in 30s, was matched in rate by adenine nucleotide secretion from storage in dense granules. Incubation of platelets with antimycin before thrombin addition increased the rate of fall in metabolic ATP, but did not affect the rate of adenine nucleotide secretion. beta-N-Acetylglucosaminidase secretion, which was slower than adenine nucleotide secretion in control platelets, was noticeably inhibited by antimycin, confirming previous reports that different regulatory mechanisms exist for dense and alpha-granule secretion. The rates of rephosphorylation of metabolic ADP to ATP via glycolysis and oxidative phosphorylation were estimated by measuring lactate production and O2 consumption in resting and thrombin-stimulated platelets and compared to the level of metabolic ATP (9-10 nmol/mg of platelet protein in the resting state). The rate of ATP production was stimulated at least two fold from 12 nmol to 24 nmol/min/mg within seconds of thrombin addition. This increased rate was maintained over the observed period of 5 min although the level of metabolic ATP had decreased to 4-5 nmol/mg within 30 s; the turnover of the remaining metabolic ATP thus increased four fold over the resting state although the actual stimulation of energy production was only two fold.     

EFFECTS OF IRRADIATION ON THE GENERATIVE CYCLE OF THE ESTROGEN STIMULATED VAGINAL EPITHELIUM

The effects of irradiation (300, 500 and 1500 rads) on mitosis and DNA synthesis in the estrogen primed vaginal epithelium have been studied. Dose-effect relations and the time sequence of effects on the two processes were investigated. The technique of tritiated thymidine labeling of DNA with autoradiography was used, in conjunction with the mitotic count, to study alterations in the generative cycle. Prior to irradiation, ovariectomized female rats were treated daily with diethylstilbestrol for a period of 2 weeks to create a steady state in the vaginal cell population. It was observed that:

• 1 Within 1 hr following ionizing radiation, mitotic figures disappear from the population and reappear at a time that is dose dependent. Those cells that have begun mitosis at the time of irradiation were able to complete that phase but no cells which were in G₂ were able to begin mitosis. Therefore, a G₂ block occurs within 1 hr post-irradiation.
• 2 Radiation reduces the rate of DNA synthesis thus prolonging the S phase. There is no evidence of a radiation-induced G₁ to S block in this system.

Based on these observations, it was further hypothesized that:

• 1 Cells in G₁ at the time of irradiation are relatively insensitive and continue to progress through the generative cycle at a rate primarily determined by the level of estrogen stimulation.
• 2 Radiation may interfere with the estrogen priming mechanism in this hormonedependent system thereby reducing the effective level of estrogen stimulation. This is seen in the behavior of cells which were in G₁ at the time of irradiation. The extent of the blockage of estrogen increases with radiation dose and after 1500 rads, estrogen stimulation is essentially at castrate level.

     

Rifampicin Inhibition of Ribonucleic Acid and Protein Synthesis in Normal and Ethylenediaminetetraacetic Acid-Treated Escherichia coli

The kinetics of ribonucleic acid (RNA) and protein synthesis in rifampicin-inhibited normal and ethylenediaminetetraacetic acid (EDTA)-treated Escherichia coli was measured. Approximately 200-fold higher external concentrations of rifampicin were needed to produce a level of inhibition in normal cells comparable to that observed in EDTA-treated cells. The rates of RNA and protein synthesis in both kinds of cells decreased exponentially, after an initial lag phase, at all rifampicin concentrations tested. The lag phase was longer and the final exponential slope less for protein synthesis than for RNA synthesis at a given rifampicin concentration. Below certain rifampicin concentrations, both the lag phase and the subsequent exponential decrease in the rates of RNA and protein synthesis were found to be rifampicin concentration dependent. At greater concentrations only the time of the lag phase was decreased by higher rifampicin concentrations, whereas the slope of the exponential decrease in the rates of RNA and protein synthesis was unaffected. In all cases, the exponential decrease continued to at least a 99.8% inhibition of the original rate of synthesis. These in vivo results are consistent with the mode of rifampicin action determined from in vitro studies; rifampicin prevents initiations of RNA polymerase on deoxyribonucleic acid, but not its propagation, by binding the enzyme essentially irreversibly. The results also indicate the size distribution of messenger RNA molecules in E. coli under our conditions.     

Biochemical lesions in copper-deficient rats caused by secondary iron deficiency. Derangement of protein synthesis and impairment of energy metabolism

Severe copper deficiency was induced in rats by rearing nursing dams and their offsprings on a semisynthetic diet comprising all the requisite nutrients and trace metals except copper. The copper-deprived rats exhibited growth retardation, severe anaemia, loss of caeruloplasmin, decrease of cytochrome oxidase, accumulation of salt-soluble collagen and a drastic decrease in iron in plasma and liver. Apart from these characteristic signs of deficiency, a marked inhibition of protein synthesis was found to occur both in vivo and in cell-free liver preparations. The curtailed ability to carry out endogenously coded amino acid incorporation into protein contrasted with the unimpaired poly(U)-acid-directed phenylalanine polymerization. This inhibition pattern, as well as the attendant disaggregation of the liver polyribosomes, suggested that the primary biosynthetic lesion was located at the stage of peptide-chain initiation. Concurrently with this alteration there was a pronounced depletion of the hepatic ATP content, associated with a parallel depression of mitochondrial respiration and an enhancement of ATPase activity. Supplementation of the copper-deficient diet with a 2–4-fold excess of iron (relative to the standard diet) prevented growth retardation and anaemia and restored normal energy metabolism, as well as unimpaired protein-synthesizing capacity. The conclusion that these disturbances were primarily determined by the secondary iron deficiency was also borne out by the finding that similar alterations occurred in rats maintained on a copper-sufficient but iron-deficient diet. On the other hand, the iron-fortified diet failed to reverse the other signs of copper deficiency, namely the loss of caeruloplasmin, the diminished rate of cytochrome oxidase and the increase of soluble collagen. The interrelations between the various biochemical lesions induced by deprivation of copper or iron are discussed and the possible role of ATP depletion in determining the derangement of protein synthesis is considered.     

EFFECT OF OLIGOMYCIN ON DARK RESPIRATION IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE): IMPLICATIONS FOR DETERMINATION OF MAINTENANCE RESPIRATION

Oligomycin is an inhibitor of the mitochondrial ATP synthase. In nitrogen-replete cells of the marine diatom Phaeodactylum tricornutum Bohlin, the rate of dark respiration was high and markedly inhibited (62%–74%) in the presence of oligomycin. In contrast, the rate of dark respiration in nitrogen-deprived cells was about half that in nitrogen-replete cells but was only slightly inhibited (16%–30%) by oligomycin. Consistent with these effects on rates of dark respiration, oligomycin decreased the ATP level and the ATP:ADP ratio by about 40% in nitrogen-replete cells incubated in darkness but had a negligible effect on the ATP level and ATP:ADP ratio in nitrogen-deprived cells. In sodium and nitrogen-deprived cells, the rate of dark respiration was greater than that in nitrogen-replete cells, but there was little effect of oligomycin on the rate of dark respiration. In light-limited cells, the rate of dark respiration was similar to that in nitrogen-deprived cells, but the inhibition (57%) in the presence of oligomycin was greater. These results suggest that most of the O₂ consumption by nitrogen-replete cells was linked to mitochondrial ATP synthesis and that the rate of mitochondrial ATP synthesis in nitrogen-deprived and sodium and nitrogen-deprived cells was low. The potential implications of these results for our understanding of maintenance respiration are discussed.     

NON-IDENTICAL MECHANISMS OF MITOTIC ARREST BY RESPIRATORY INHIBITORS IN PEA ROOT TIPS AND SEA URCHIN EGGS

The adenosine triphosphate (ATP) content of pea root tips is about 0.40 mmole/kg fresh weight. The effects of partial and complete anaerobiosis, and of carbon monoxide and hydrogen cyanide, on the ATP level are described. The ATP content was shown to correspond closely to the oxygen uptake under these conditions. However, there was no relation between the ATP level and the rate of mitosis, a situation which is in contrast with that in sea urchin eggs. In anaerobic conditions, mitoses in pea root tips could continue at a reduced rate, even though the ATP content had fallen to 1.5 per cent of the normal value in air The ATP level in anaerobic conditions corresponded closely to the expected rate of ATP regeneration from known anaerobic sources of energy. Calculations show that even this severely restricted supply of energy would be more than adequate to drive the anaphase chromosome movements, so it is suggested that the concept of a mitotic energy reservoir is superfluous in root tips. No evidence could be found for the involvement during mitosis in sea urchin eggs of a non-respiratory ferrous complex such as occurs in pea root tips. Hence the dilemma remains, that whereas mitoses in both sea urchin eggs and pea root tips are arrested by respiratory inhibitors, yet the biochemical mechanisms of the arrest in the two types of cell are totally distinct.     

Modifications in photosystem II photochemistry in senescent leaves of maize plants

Analyses of CO2 exchange and chlorophyll fluorescence were carried out to assess photosynthetic performance during senescence of maize leaves. Senescent leaves displayed a significant decrease in CO2 assimilatory capacity accompanied by a decrease in stomatal conductance and an increase in intercellular CO2 concentration. The analyses of fluorescence quenching under steady-state photosynthesis showed that senescence resulted in an increase in non-photochemical quenching and a decrease in photo-chemical quenching. It also resulted in a decrease in the efficiency of excitation energy capture by open PSII reaction centres and the quantum yield of PSII electron transport, but had very little effect on the maximal efficiency of PSII photochemistry. The results determined from the fast fluorescence induction kinetics indicated an increase in the proportion of QB-non-reducing PSII reaction centres and a decrease in the rate of QA reduction in senescent leaves. Theoretical analyses of fluorescence parameters under steady-state photosynthesis suggest that the increase in the non-photochemical quenching was due to an increase in the rate constant to thermal dissipation of excitation energy by PSII and that the decrease in the quantum yield of PSII electron transport was associated with a decrease in the rate constant of PSII photochemistry. Based on these results, it is suggested that the decrease in the quantum yield of PSII electron transport in senescent leaves was down-regulated by an increase in the proportion of QB-non-reducing PSII reaction centres and in the non-photochemical quenching. The photosynthetic electron transport would thus match the decreased demand for ATP and NADPH in carbon assimilation which was inhibited significantly in senescent leaves.Key words: Chlorophyll fluorescence, gas exchange, maize (Zea mays L.), photochemical and non-photochemical quenching, photosystem II photochemistry.      

Source of Energy for Gliding Motility in Flexibacter polymorphus: Effects of Metabolic and Respiratory Inhibitors on Gliding Movement

The effects of selected metabolic and respiratory inhibitors on the gliding motility of Flexibacter polymorphus were examined. Motility and oxygen consumption were quantitatively inhibited in a reversible manner by specific respiratory poisons, suggesting that gliding velocity was linked to electron transport activity. Arsenate had little influence on the number or rate of gliding filaments, despite a 95% decrease in the concentration of intracellular adenosine 5′-triphosphate (ATP). At concentrations of cyanide or azide that abolished gliding movement, cells possessed a level of ATP that should have been sufficient to allow motility. Proton-conducting uncouplers of oxidative phosphorylation, such as carbonylcyanide m-chlorophenylhydrazone (CCCP) and tetrachlorosalicylanilide, strongly inhibited locomotion yet did not suppress respiratory activity or intracellular ATP sufficiently to account for their effect on movement. Inhibition of motility by CCCP (but not by tetrachlorosalicylanilide) was partially reversed by sulfhydryl compounds. However, unlike CCCP, inhibition of motility by p-chloromercuribenzoate, a known sulfhydryl-blocking reagent, was associated with a corresponding reduction in respiratory activity and ATP content of cells. Protein synthesis was not blocked by concentrations of CCCP inhibitory for motility, indicating that utilization of existing ATP in this energy-requiring process was not impaired. These data suggest (but do not unequivocally prove) that ATP may not function as the sole energy donor for the gliding mechanism, but that some additional product of electron transport is required (e.g., the intermediate of oxidative phosphorylation).     

Adenine nucleotide metabolism of blood platelets IX. Time course of secretion and changes in energy metabolism in thrombin-treated platelets

Changes in the energy metabolism of washed human platelets were compared with the kinetics of secretion induced by thrombin (5 units/ml). A 50% decrease in the level of metabolic ATP (³H-labelled), which was essentially complete in 30 s, was matched in rate by adenine nucleotide secretion from storage in dense granules. Incubation of platelets with antimycin before thrombin addition increased the rate of fall in metabolic ATP, but did not affect the rate of adenine nucleotide secretion. β-N-Acetylglucosaminidase secretion, which was slower than adenine nucleotide secretion in control platelets, was noticeably inhibited by antimycin, confirming previous reports that different regulatory mechanisms exist for dense and α-granule secretion. The rates of rephosphorylation of metabolic ADP to ATP via glycolysis and oxidative phosphorylation were estimated by measuring lactate production and O₂ consumption in resting and thrombin-stimulated platelets and compared to the level of metabolic ATP (9–10 nmol/mg of platelet protein in the resting state). The rate of ATP production was stimulated at least two fold from 12 nmol to 24 nmol/min/mg within seconds of thrombin addition. This increased rate was maintained over the observed period of 5 min although the level of metabolic ATP had decreased to 4–5 nmol/mg within 30 s; the turnover of the remaining metabolic ATP thus increased four fold over the resting state although the actual stimulation of energy production was only two fold.     

腺苷三磷酸和环腺苷单磷酸对丝状真菌纤维素酶合成的调节

以虫荧光素酶法检验了四株丝状真菌在葡萄糖—无机盐液体培养过程中的胞内ATP含量。结果表明,只有当胞内ATP浓度低于10~(-S)mg/ml时,真菌才开始合成胞外纤维素酶(FPA)。以不同浓度的各种碳源培养时,菌体胞内ATP含量只要超过10~(-1)mg/ml,FPA的合成即发生阻遏。菌体胞内ATP含量与FPA合成呈显著负相关。以高效液相色谱(HPLC)法检测了菌体培养液中的cAMP含量。在非阻遏条件下,外源cAMP可以提高FPA的合成水平。但外源cAMP不能解除已经发生的酶合成阻遏。菌体ATP和cAMP水平是调节真菌纤维素酶合成的重要因子。     

PARTICIPATION OF A NON-RESPIRATORY FERROUS COMPLEX DURING MITOSIS IN ROOTS

A systematic survey was undertaken, of the effects of carbon monoxide and hydrogen cyanide (in the presence of 20 per cent oxygen), in darkness and light, on the relative rates of respiration, mitosis, and interphase in pea root tips. The inhibition of respiration by carbon monoxide was light-sensitive, but the inhibition by hydrogen cyanide was light-stable. The inhibitions were presumably due to combination of the inhibitor with the iron of cytochrome oxidase, in its divalent and trivalent forms respectively. In contrast, the inhibitions of mitosis by both poisons proved to be light-sensitive. The light-sensitive inhibition of mitosis by carbon monoxide shows that an iron complex is responsible for the process. That the inhibition of mitosis by hydrogen cyanide is also light-reversible shows that, in contrast with cytochrome oxidase, the mitotic iron complex remains always in the divalent state. The relative affinities of the mitotic ferrous complex, in molar units, were 0.68 for CO/O₂, and 0.37 for HCN/O₂. The properties of the complex are analogous to, yet distinct from, Gastrophilus haemoglobin and reduced cytochrome oxidase. It is considered that the arrest of mitosis by oxygen lack, carbon monoxide, and hydrogen cyanide is definitely due to interference with this unidentified, non-respiratory ferrous complex.     

Proteomic Analysis of MG132-Treated Germinating Pollen Reveals Expression Signatures Associated with Proteasome Inhibition

Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa) germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.     

VITAMIN B12 AND THE MACROMOLECULAR COMPOSITION OF EUGLENA : III. Effect of Cycloheximide on the Recovery from B12-Induced Unbalanced Growth

When cycloheximide is added to (B12)-deficient cultures before or after replenishment of the cells with B₁₂, reversion of these cells is inhibited. This inhibition is not caused by interference of the inhibitor in the uptake of B₁₂ as measured by division kinetics. Cycloheximide does not inhibit the initial increase in the rate of DNA synthesis caused by B₁₂ replenishment, but within 30–45 min the rate decreases and DNA synthesis ceases. Cycloheximide added to replenished deficient cells after completion of DNA duplication inhibits cell division. The total cellular protein and RNA in replenished cells treated with cycloheximide does not change. B₁₂ added to deficient cells does not stimulate the incorporation of [¹⁴C]leucine into protein during resumption and completion of DNA duplication. However, there is a large increase in [¹⁴C]leucine incorporation into the protein of these cells soon after completion of DNA duplication and before resumption of cell division. The addition of cycloheximide to B₁₂-replenished or to nonreplenished deficient cells rapidly inhibits the incorporation. We suggest that the addition of B₁₂ accelerates the rate of DNA synthesis in the deficient cells and that possibly no new protein synthesis is required except for mitosis. However, protein synthesis is needed for continuous DNA synthesis.     

Effects of dinitrophenol and oligomycin on the coupling between anaerobic metabolism and anaerobic sodium transport by the isolated turtle bladder

Dinitrophenol (1 x 10^-5 M) has been found to inhibit anaerobic sodium transport by the isolated urinary bladder of the fresh water turtle. Concurrently, anaerobic glycolysis was stimulated markedly. However, tissue ATP levels diminished only modestly, remaining at approximately 75% of values observed under anaerobic conditions without DNP. The utilization of glucose (from endogenous glycogen) corresponded closely to that predicted from the molar quantities of lactate formed. Thus the glycolytic pathway was completed in the presence of DNP and if ATP were synthesized normally during glycolysis, synthesis should have been increased. On the other hand, the decrease in Na transport should have decreased ATP utilization. Oligomycin did not block sodium transport either aerobically or anaerobically, but ATP concentrations did decrease. When anaerobic glycolysis was blocked by iodoacetate, pyruvate did not sustain sodium transport thus suggesting that no electron acceptors were available in the system. Two explanations are entertained for the anaerobic effect of DNP: (a) Stimulation by DNP of plasma membrane as well as mitochondrial ATPase activity; (b) inhibition of a high energy intermediate derived from glycolytic ATP or from glycolysis per se. The arguments relevant to each possibility are presented in the text. Although definitive resolution is not possible, we believe that the data favor the hypothesis that there was a high energy intermediate in the anaerobic system and that this intermediate, rather than ATP, served as the immediate source of energy for the sodium pump.