Regulation of electron transport in isolated chloroplasts by sequential binding of adenine nucleotides to the coupling factor protein

Changes in the ferricyanide-reducing activity in isolated spinach chloroplasts by adenine nucleotides were measured in the presence or absence of phosphate, arsenate or pyrophosphate at 15°C. The activity changes were analyzed and ascribed to the interaction between the nucleotides and the chloroplast coupling factor (CF₁).ADP and ATP (but not AMP) partially inhibited ferricyanide reduction through a 11 binding to the inhibition site on CF₁.When the ferricyanide reduction was coupled to either phosphorylation or arsenylation, the inhibition by ADP was released through a 11 binding of the second ADP molecule to the coupling site on the CF₁ with which the first ADP had been associated.The association constants of ADP for the inhibition and the coupling site were found to be approximately 5×10⁵ and 6×10⁴ M^–1, respectively. TheKm value of ADP for arsenylation (pH 8.3) was around 17 M.The ADP-regulated electron transport was defined based on these results. The ADP-regulated ferricyanide reduction, when coupled with phosphorylation, revealed a stoichiometry of P/e=1 between the amounts of esterified phosphate and reduced ferricyanide.Presented to the annual meeting of the Japanese Society of Plant Physiologists, Sendai, April 1974.     

Studies on cyst formation inPhysarum polycephalum myxamoebae

Encystment of myxamoebae ofPhysarum polycephalum was induced by transferring the amoebae to a high salt medium of 1/60 M Sørensen buffer (pH 6.0) containing 0.125 M NaCl, 1.6 mM MgCl₂ and 0.18 mM CaCl₂. The induction of cysts was blocked by inhibitors of protein synthesis, such as puromycin, cycloheximide and streptomycin. However, inhibitors of RNA synthesis, such as actinomycin D, proflavin and 8-azaguanine did not block the transformation. These results suggest that in the cyst formation,de novo RNA synthesis is not involved, whereas protein synthesis is required. Cyst formation was more strongly inhibited by inhibitors of oxidative phosphorylation than by other respiratory poisons. It seems that oxidative phosphorylation takes part in the energy supply of this differentiation.     

Development of glutathione peroxidase activity during dietary and genetic copper deficiency

Copper deficiency was produced in developing rodents to study a possible interaction between copper and the selenoenzyme, glutathione peroxidase (GSH-Px). Dietary copper deficiency was investigated in Sprague-Dawley rats and in three mouse strains (C57BL, C3H/HeJ, C58); genetic copper deficiency was studied in two of the mouse strains, C57BL and C3H/HeJ, using brindled mice. In certain cases it appeared that copper deficiency was associated with depressed liver GSH-Px activity; values from copper-deficient livers were 40–70% of control values. However, the decrease in liver GSH-Px in both rats and mice was only observed when body weight was also depressed and did not necessarily correlate with copper deficiency signs, such as lower serum ceruloplasmin or liver cytochrome oxidase activities. In weanling rats, serum GSH-Px activity was normal despite a 60% reduction in liver activity. Mouse liver GSH-Px activity rose fourfold during the first 3 weeks of life to 75% of the adult level. Rat liver GSH-Px also increased during the suckling period. When perinatal copper deficiency, nutritional or genetic, was severe enough to retard growth, liver GSH-Px activity was depressed. Unlike rats, adult murine liver GSH-Px was equivalent in males and females.     

Evidence for the in vivo regulation of glucose 6-phosphate dehydrogenase activity in Hydrogenomonas eutropha H 16 from measurements of the intracellular concentrations of metabolic intermediates

The inhibition of fructose utilization by whole cells of Hydrogenomonas eutropha H 16, following the addition of hydrogen to the gas phase, has been explained as an inhibition of glucose 6-phosphate dehydrogenase (Blackkolb and Schlegel, 1968a, b). The intracellular concentrations of glucose 6-phosphate, 6-phosphogluconate, three inhibitors of the enzyme (NADH, ATP and phosphoenolpyruvate) and some related metabolites were measured in cells incubated in the presence and absence of hydrogen. Inhibition of glucose 6-phosphate dehydrogenase was confirmed by an increase in the glucose 6-phosphate pool and a decrease in the 6-phosphogluconate concentration. The regulatory control is apparently due to a threefold increase in the NADH concentration while the concentrations of the other two inhibitors fell slightly. When the measured intracellular concentrations of intermediates were used in the in vitro assay of glucose 6-phosphate dehydrogenase activity, an almost total inhibition of the dehydrogenase was observed, therefore further regulatory factors must be considered.     

Characterization of mitochondrial bioenergetic functions between two forms of Leishmania donovani – a comparative analysis

Leishmaniasis is a growing health problem in many parts of the world partly due to drug resistance of the parasite. This study reports on the fisibility of studying mitochondrial properties of two forms of wild-type L. donovani through the use of selective inhibitors. Amastigote forms of L. donovani exhibited a wide range of sensitivities to these inhibitors. Mitochondrial complex II inhibitor thenoyltrifluoroacetone and F_oF₁-ATP synthase inhibitors oligomycin and dicyclohexylcarbodiimide were refractory to growth inhibition of amastigote forms, whereas they strongly inhibited the growth of promastigote forms. This result indicated that complex II and F_oF₁-ATP synthase were not functional in amastigote forms suggesting the presence of attenuated oxidative phosphorylation in the mitochondria of amastigote forms. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited cellular multiplication and substrate level phosphorylation in amastigote forms, suggesting the role of complex I and complex III for the survival of amastigote forms. Further we studied the mitochondrial activities of both forms by measuring oxygen consumption and ATP production. In amastigote form, substantial ATP formation by substrate level phosphorylation was observed in NADPH-fumarate, NADH-fumarate, NADPH-pyruvate and NADH-pyruvate redox couples. None of the redox couple generated ATP formation was inhibited by F_oF₁-ATP synthase inhibitor oligomycin. Therefore, we may conclude that there are significant differences between these two forms of L. donovani in respect of mitochondrial bioenergetics. Our results demonstrated bioenergetic disfunction of amastigote mitochondria. Therefore, these alterations of metabolic functions might be a potential chemotherapeutic target.     

Mass-spectrometric analysis of proteasome subunits exhibiting endoribonuclease activity

Proteasomes function as the main nonlysosomal machinery of intracellular proteolysis and are involved in the regulation of the majority of important cellular processes. Despite the considerable progress that has been made in understanding the functioning of proteasomes, some issues (in particular, the RNase activity of these ribonucleoprotein complexes and its regulation) remain poorly investigated. In this study, we found to several proteins with electrophoretic mobility that corresponds to that of 20S subunits of the core proteasome complex exhibit endoribonuclease activity with respect to the sense and antisense sequences of the c-myc mRNA 3′-UTR. Mass-spectrometric analysis of tryptic hydrolysates of these proteins showed that the samples contained 20S proteasome subunits—α1 (PSMA6), α5 (PSMA5), α6 (PSMA1), and α7 (PSMA3). A number of new phosphorylation sites of α1 (PSMA6) and α7 (PSMA3) subunits were found, and a form of α5 (PSMA5) subunit with a deletion of 20 N-terminal amino-acid residues was identified. The observed differences in the manifestation of endonuclease activity by individual subunits are apparently due to posttranslational modifications of these proteins (in particular, phosphorylation). It was shown that the specificity of RNase activity changes upon proteasome dephosphorylation and under the influence of Ca²⁺ and Mg²⁺ cations. It is concluded that posttranslational modifications of proteasome subunits affect the specificity of their RNase activity.     

Phosphorylative fumarate reduction in Vibrio succinogenes: Stoichiometry of ATP synthesis

1. Cells of Vibrio succinogenes, treated with EDTA at pH 8, catalyze the phosphorylation of their endogenous ADP and AMP as a function of the electron transport from formate to fumarate. The P/fumarate ratio obtained from the initial velocity of the phosphorylation on initiation of the electron transport and from the activity of fumarate reduction in the steady state was 0.90. The phosphorylation was prevented by 10μmol/g protein carbonylcyanide-3-chlorophenylhydrazone.
2. The esterification of external phosphate in the presence of ADP, hexokinase and glucose is catalysed by a membrane preparation of V. succinogenes in the steady state of fumarate reduction by H₂. The phosphorylation was fully abolished by either 5μmol/g protein carbonylcyanide-4-trifluoromethoxyphenylhydrazone or 30μmol/g protein carbonylcyanide-3-chlorphenylhydrazone. Phosphorylation was blocked also by dicyclohexylcarbodiimide, an inhibitor of the Mg²⁺-dependent membrane bound ATP synthase, and by low concentrations of the inhibitors of electron transport 2-(n-nonyl)-4-hydroxyquinoline-N-oxide or 4-chloromercuriphenylsulfonate.
3. The P/fumarate ratios, measured with the membrane preparation, were found to increase with progressive inhibition of the electron transport from hydrogen to fumarate by means of 4-chloromercuriphenylsulfonate. The extrapolated ratio at vanishing electron transport activity was 0.47.
4. About 50% of the membrane preparation was found to consist of inverted vesicles with the hydrogenase and formate dehydrogenase oriented to the inside. The residual part is considered as being incapable of performing energy transduction. The extrapolated P/fumarate ratio valid for the inverted vesicles was 0.94.

     

Tyrosine kinase inhibitor Thiotanib targets Bcr-Abl and induces apoptosis and autophagy in human chronic myeloid leukemia cells

Chronic myeloid leukemia (CML) is characterized by abnormal Bcr and Abl genes and enhanced tyrosine kinase activity. Anti-CML therapy has been much improved along with the applications of tyrosine kinase inhibitors (TKIs) which selectively target Bcr-Abl and have a cytotoxic effect on CML. Recently, four-membered heterocycles as “compact modules” have attracted much interest in drug discovery. Grafting these small four-membered heterocycles onto a molecular scaffold could probably provide compounds that retain notable activity and populate chemical space otherwise not previously accessed. Accordingly, a novel TKI, Thiotanib, has been designed and synthesized. It selectively targets Bcr-Abl, inducing growth inhibition, cell cycle arrest, and apoptosis of CML cells. Meanwhile, the compound Thiotanib could also induce autophagy in CML cells. Interestingly, inhibition of autophagy promotes Thiotanib-induced apoptosis with no further activation of caspase 3, while inhibition of caspases did not affect the cell survival of CML cells. Moreover, the compound Thiotanib could inhibit phosphorylation of Akt and mTOR, increase beclin-1 and Vps34, and block the formation of the Bcl-2 and Beclin-1 complex. This indicates the probable pathway of autophagy initiation. Our results highlight a new approach for TKI reforming and further provide an indication of the efficacy enhancement of TKIs in combination with autophagy inhibitors.     

Effect of High Cation Concentrations on Photosystem II Activities

The effects of wide concentration ranges of NaCl, KCl, and MgCl₂ on ferricyanide reduction and the fluorescence induction curve of isolated spinach (Spinacia oleracea) chloroplasts were investigated. Concentrations of the monovalent salts above 100 mm and MgCl₂ above 25 mm produced a decrease in the rate of ferricyanide reduction by thylakoids uncoupled with 2.5 mm NH₄Cl which cannot be attributed to changes in the primary photochemical capacity of photosystem II. Salt-induced decreases in the effective concentration of the secondary electron acceptor of photosystem II, plastoquinone, reduce the capacity for secondary photochemistry of photosystem II and this could contribute to the reduction in ferricyanide reduction by uncoupled thylakoids at high salinities. The rate of ferricyanide reduction by coupled thylakoids is little affected by salinity changes, indicating that the rate-limiting phosphorylation mechanism in electron flow from water to ferricyanide in coupled thylakoids is salt-tolerant, whereas the rate-limiting reaction in uncoupled ferricyanide reduction is considerably affected by salinity changes. Salt-induced changes in the fluorescence induction curve are interpreted in terms of changes in the rate constants for excitation decay by radiationless transitions, exciton transfer from photosystem II chlorophylls to other associated chlorophyll species, and photochemistry.     

A prepared anti-MSTN polyclonal antibody reverses insulin resistance of diet-induced obese rats via regulation of PI3K/Akt/mTOR&FoxO1 signal pathways

Suppression of myostatin (MSTN) is associated with skeletal muscle atrophy and insulin resistance. However, the mechanisms by which MSTN regulates insulin resistance are not well known. We have explored the signaling pathways through which MSTN regulates insulin resistance in diet-induced obese rats using a polyclonal antibody for MSTN. The anti-MSTN polyclonal antibody significantly improved insulin resistance and whole-body insulin sensitivity, decreased MSTN protein expression in muscle samples by 39 % in diet-induced obese rats. Furthermore, the anti-MSTN polyclonal antibody significantly enhanced PI3K activity (140 %), Akt phosphorylation (86 %), GLUT4 protein expression (23 %), the phosphorylation of mTOR (21 %), and inhibited the phosphorylation of FoxO1 (57 %), but did not affect the phosphorylation of GSK-3β. Thus, suppression of MSTN by the anti-MSTN polyclonal antibody reverses insulin resistance of diet-induced obesity via MSTN/PI3K/Akt/mTOR and MSTN/PI3K/Akt/FoxO1 signaling pathways.     

Promotion of respiration by auxin in the induction of cell division in suspension culture

Auxins (IAA, NAA, p-CPA) caused 20–30% promotion of respiration of auxin-dependent cell cultures ofNicotiana tabacum, Glycine max, Taraxacum mongolica, andAtriplex sp. and had small if any effect on respiration of auxin-independent cell cultures ofRubus sp. andScorzonera hispanica. Antiauxin (p-CPIBA) did not affect the respiration. The auxin effect on the respiration of tobacco cells was revealed 10 min after its addition to the suspension and reached a maximum value in 60 min. This stimulation preceded the induction of cell division by auxin. Mitochondria isolated from auxin-treated tobacco cells had greater oxidative and phosphorylative activity than mitochondria from untreated cells. However, isolated mitochondria did not respond to auxin. The inhibitors of respiration (cyanide, monoiodoacetate, malonate, and 2,4-dinitrophenol) eliminated auxin effect on the respiration and cell division. It is concluded that the promotion of respiration is a common event for the auxin effects both on cell extension and on cell division. This promotion is necessary for the induction of cell division and is exerted via direct activation of mitochondriain situ.     

Effects of purine nucleotides on photosynthetic electron transport in isolated chloroplasts

The effects of guanylates and inosinates (and adenylates) on phosphorylation, ferricyanide reduction, and light-induced H⁺ uptake in spinach chloroplasts were studied. GDP, GTP, IDP, and ITP (but not GMP and IMP) stimulated the light-induced H⁺ uptake and partially inhibited ferricyanide reduction. Phosphate, arsenate, and phlorizin increased the extent of inhibition by these nucleotides and decreased the values of their apparent dissociation constants for the inhibition process. In the presence of phosphate (or arsenate), restoration of ferricyanide reduction from the level inhibited by guanylates and inosinates was observed as phosphorylation (or arsenylation) proceeded. These results suggest that phosphorylation of GDP and IDP as well as ADP takes place after two steps of nucleotide binding to the chloroplast coupling factor 1. The apparent dissociation constants of GDP and IDP for these two binding steps were estimated to be about 34 and 38 µM for the first and 110 and 160 µM for the second step, respectively (at pH 8.3, 15°C). Above pH 9, the ratio (P/e) of the extent of phosphorylation to the increment of electron transport from the basal level measured in the presence of [ATP + Pi] or [ADP + Pi + phlorizin], became increasingly large. When the electron transport level inhibited by dicyclohexylcarbodiimide was taken to be the basal activity, the P/e ratio remained almost constant ( 1) from pH 7.0 up to 10.     

Nonspecificity of some commonly used inhibitors of DNA synthesis in cultures ofStreptococcus faecalis

The specificity of three commonly used inhibitors of DNA synthesis were tested in the batch culture ofStreptococcus faecalis ATCC 8043 in rich broth medium. It was shown that nalidixic acid, mitomycin C and 6-(4-hydroxyphenylazo)uracil inhibit the cell mass as much as they decrease net DNA synthesis. Hence the drugs tested are highly unspecific inhibitors of DNA synthesis inS. faecalis; i.e. they all interfere with other processes as well as with DNA synthesis.     

Biogenesis of mitochondria 20 the effects of altered membrane lipid composition on mitochondrial oxidative phosphorylation inSaccharomyces cerevisiae

1. The lipid composition of mitochondria isolated from a fatty acid desaturase mutant ofSaccharomyces cerevisiae may be extensively manipulated by growing the organism on defined supplements of unsaturated fatty acid (UFA).
2. The fatty acid composition of the mitochondrial lipids closely follows that of the whole cells from which the mitochondria are isolated. UFA-depleted mitochondria contain normal levels of sterols, neutral lipids and total phospholipids, but have much lower levels of phosphatidyl inositides.
3. UFA-depleted mitochondria possess a full complement of cytochromes, oxidase both NAD-linked and flavoprotein-linked substrates at normal rates, and have levels of succinate and malate dehydrogenases similar to those of UFA-supplemented mitochondria. However, UFA-depletion has a marked effect on the ability of cytochromec to reactivate the NADH oxidase activity of cytochromec-depleted mitochondria.
4. The efficiency of oxidative phosphorylation decreases progressively with the UFA content of the mitochondria, and oxidative phosphorylation is completely lost in mitochondria containing approximately 20% UFA.
5. The incorporation of UFA into the lipids of UFA-depleted mitochondriain vivo results in a recoupling of oxidative phosphorylation. Recoupling is insensitive to both chloramphenicol and cycloheximide, indicating that all the proteins necessary for oxidative phosphorylation are present in UFA-depleted mitochondria, and that the less of oxidative phosphorylation is a purely lipid lesion.
6. ATPase activity is apparently unaffected by UFA-depletion, but³²P_i-ATP exchange activity is lost in mitochondria which have been extensively depleted in UFA.
7. Valinomycin stimulates the respiration of UFA-supplemented mitochondria in media containing potassium, but has no effect on the respiration of UFA-depleted mitochondria, suggesting that active transport of potassium is lost as a result of UFA-depletion.

     

Effects of salt stress on nitrogenase activity and growth of four legumes

The effects of salt stress on nitrogenase (N2-ase) activity, growth and nitrogen content ofVicia faba (L.),Medicago sativa (L.) Merrill,Glycine max andVigna sinensis (L.) were investigated. Four levels of salinity were applied and salt treatments were imposed on inoculated and N-fertilized plants.M. sativa tolerated mild levels of salinity but higher salt concentrations depressed N₂-ase activity of this species. The other three legumes were considerably affected by salt treatments, and N₂-ase activity was significantly reduced by salinity. Vicia faba, carrying elongate nodules, could restore a partial N₂-ase activity upon recovery from salt stress whereasG. max andV. sinesis, both with spherical nodules, could not regain significant activity when salinity was removed. Salt stress retarded growth of both inoculated and N-fertilized plants. The nitrogen content of both treatments was also affected by salinity and the effect was more severe for inoculated than N-fertilized plants.     

Knockdown of LYRM1 Rescues Insulin Resistance and Mitochondrial Dysfunction Induced by FCCP in 3T3-L1 Adipocytes

LYR motif-containing 1 (LYRM1) was recently discovered to be involved in adipose tissue homeostasis and obesity-associated insulin resistance. We previously demonstrated that LYRM1 overexpression might contribute to insulin resistance and mitochondrial dysfunction. Additionally, knockdown of LYRM1 enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We investigated whether knockdown of LYRM1 in 3T3-L1 adipocytes could rescue insulin resistance and mitochondrial dysfunction induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to further ascertain the mechanism by which LYRM1 is involved in obesity-associated insulin resistance. Incubation of 3T3-L1 adipocytes with 1 µM FCCP for 12 h decreased insulin-stimulated glucose uptake, reduced intracellular ATP synthesis, increased intracellular reactive oxygen species (ROS) production, impaired insulin-stimulated Glucose transporter type 4 (GLUT4) translocation, and diminished insulin-stimulated tyrosine phosphorylation of Insulin receptor substrate-1 (IRS-1) and serine phosphorylation of Protein Kinase B (Akt). Knockdown of LYRM1 restored insulin-stimulated glucose uptake, rescued intracellular ATP synthesis, reduced intracellular ROS production, restored insulin-stimulated GLUT4 translocation, and rescued insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt in FCCP-treated 3T3-L1 adipocytes. This study indicates that FCCP-induced mitochondrial dysfunction and insulin resistance are ameliorated by knockdown of LYRM1.     

Urocortin-2 suppression of p38-MAPK signaling as an additional mechanism for ischemic cardioprotection

Urocortin-2 (UCN2) is cardioprotective in ischemia/reperfusion injury (I/R) through short-lived activation of ERK1/2. Key factors involved in I/R, e.g. apoptosis, mitochondrial damage, p38 kinase, and Bcl-2 family, have not been well-investigated in UCN2-induced cardioprotection. We assessed the role of p38-MAPK in anti-apoptotic Bcl-2 signaling and mitochondrial stabilization as a putative mechanisms in UCN2-induced cardioprotection. Isolated hearts from adult Sprague–Dawley rats and cultured H9c2 cells were subjected to I/R protocols with or without 10 nM UCN2 treatment. The effect of a specific p38 inhibitor SB202190 was tested in H9c2 cells. Cardiac function, LDH release, and mitochondrial membrane potential (MMP) were used to assess the degree of myocardial injury in hearts and H9c2 cells. Post-perfusion, hearts were collected for Western blot analyses or mitochondria/cytosol isolation to analyze p38 activation and Bcl-2 family members. UCN2 treatment improved rate-pressure product (58 ± 5 vs. 31 ± 4 % of Baseline; P < 0.05) and decreased LDH release (20 ± 9 vs. 90 ± 40 mU/ml LDH, P < 0.01) at the end of 60 min reperfusion. UCN2 reduced phospho-p38 levels and Bax activation. UCN2 increased the expression of Bcl-2 and inhibited the accumulation of p-Bim. With additional experiments, it was confirmed that UCN2 increases the phosphorylation of ERK1/2 in the early phase of UCN2 treatment and increases the overshot recovery of ERK1/2 phosphorylation during reperfusion. UCN2 and SB202190 partially prevented the loss of MMP induced by I/R. However, combined treatment with UCN2 and SB202190 did not provide additive benefit. UCN2 is cardioprotective in I/R in association with reduced phosphorylation of p38 together with the increased ERK1/2 activation and increased Bcl-2 family member pro-survival signaling. These changes may stabilize cardiac mitochondria, similar to p38 inhibitors, as part of a pro-survival mechanism during I/R.     

Enzyme(s) responsible for tellurite reducing activity in a moderately halophilic bacterium,Salinicoccus iranensis strain QW6

Oxyanions of tellurium, like tellurate (TeO₄ ^2?) and tellurite (TeO₃ ^2?), are highly toxic for most microorganisms. There are a few reports on the bacterial tellurite resistance mechanism(s). Salinicoccus iranensis, a Gram-positive halophilic bacterium, shows high tellurite resistance and NADH-dependent tellurite reduction activity in vitro. Since little is known regarding TeO₃ ^2? resistance mechanisms in halophilic microorganisms, here one of the enzymatic reduction activities presented in this microorganism is investigated. To enhance the enzymatic activity during purification, the effect of different parameters including time, inoculation, different pHs, different tellurite concentrations and different salts were optimized. We also examined the tellurite removal rates by diethyldithiocarbamate (DDTC) during optimization. In the culture medium the optimum conditions obtained showed that at 30 h, 2 % inoculum, pH 7.5, without tellurite and with 5 % NaCl (w/v) the highest enzyme activity and tellurite removal were observed. Results of the purification procedure done by hydroxyapatite batch-mode, ammonium sulfate precipitation, followed by phenyl-Sepharose and Sephadex G-100 column chromatography, showed that the enzyme consisted of three subunits with molecular masses of 135, 63 and 57 kDa. In addition to tellurite reduction activity, the enzyme was able to reduce nitrate too. Our study extends the knowledge regarding this process in halophilic microorganisms. Besides, this approach may suggest an application for the organism or the enzyme itself to be used for bioremediation of polluted areas with different contaminants due to its nitrate reductase activity.     

Characteristics of (Na++K+)-ATPase inBoergesenia forbesii

(Na⁺+K⁺)-ATPase proved to be present in the vegetative thalli ofBoergesenia forbesii (Harvey) Feldmann. The ATPase was extracted with Triton X-100 and partially purified by Sephadex G-150 gel filtration. The enzyme was activated with Mg²⁺ and further stimulated by the addition of K⁺ and Na⁺. It was observed thatp-chloromercuribenzoate (PCMB),N-ethylmaleimide (NEM), iodoacetoamide, copper sulfate, zinc sulfate, lead nitrate and cadmium chloride inhibited the enzyme activity, but ouabain was ineffective, andN,N′-dicyclohexylcarbodiimide (DCCD) did not apparently inhibit the activity, but rather promoted it slightly. The ATPase activity was also shown in the isolated cell wall ofBoergesenia thalli, and the enzyme activity was detected in the wall itself by using electron microscopic methods.