Diffusion in immobilized-cell agar layers: influence of microbial burden and cell morphology on the diffusion coefficients ofl-malic acid and glucose

Summary The diffusivities ofl-malic acid and glucose in an agar membrane entrapping small amounts ofEscherichia coli orRhodospirillum rubrum whole cells were measured using time lag (TL) and steady state (SS) methods. Diffusivities were overestimated by the SS method. For concentrations of immobilizedR. rubrum cells ranging between 10⁴ and 10⁹ organisms cm^–3 agar (20 ng-2 mg dry weight cm^–3 agar), the diffusion coefficient ofl-malic acid, determined by both methods, was related to the logarithm of the membrane cell content by a decreasing linear relationship. The diffusion coefficient of glucose obtained by TL analysis was not significantly affected by the presence in the membrane of 3 ng-0.3 mg dry wt.E. coli cm^–3 agar. However, values arising from the SS method decreased linearly as a function of the amount of immobilized organisms. Membranes containingR. rubrum cells offered higher diffusional resistance tol-malic acid and glucose than those loaded with the same amount ofE. coli cells.     

The ultrastructure of chemolithoautotrophic Gallionella ferruginea and Thiobacillus ferrooxidans as revealed by chemical fixation and freeze-etching

By using sodium thioglycolate to dissolve the high amount of excreted stalk material in axenic cultures of the chemolithoautotrophic iron bacterium Gallionella ferruginea, the ultrastructure of Gallionella cells from pure cell suspensions could be studied without any loss of viability or disturbance by dense ferric stalk fibers, and compared with Thiobacillus ferrooxidans, also grown chemolithoautotrophically with ferrous iron as energy source. Both organisms were chemically fixed or freeze-etched. Particular structural differences between these iron-bacteria could be ascertained. G. ferruginea possesses intracytoplasmic membranes and soluble d-ribulose-1,5-bisphosphate-carboxylase, whereas T. ferrooxidans contains carboxysomes but no intracytoplasmic membranes; Gallionella forms poly--hydroxybutyrate and glycogen as storage material; T. ferrooxidans produces only glycogen. Both organisms also differ from each other with respect to the freeze fracture behaviour of the cell envelope layers. Whereas the cells of T. ferrooxidans exhibit a characteristic double cleavage, exposing the plasmic fracture face and exoplasmic fracture face of the outer membrane and cytoplasmic membrane, the exceptionally thin multilayered cell envelope of G. ferruginea revealed a particularly intimate association between the layers, resulting in a visualisation of the supramolecular organisation of only the inner fracture face of the cytoplasmic membrane. The results are discussed predominantly in relation to the extremely distinct environments of both organisms.     

Inhibition of Iron-oxidizing Activity by Bisulfite Ion in Thiobacillus ferrooxidans

Growth of Thiobacillus ferrooxidans on iron- and sulfur-salts media and iron oxidizing activity of this bacterium were strongly inhibited by bisulfite ion. The mechanism of inhibition by bisulfite ion of iron-oxidizing activity was studied with the plasma membrane of T. ferrooxidans AP19-3. The c-type cytochrome in the plasma membrane was reduced by ferrous ion and the cytochrome reduced by Fe²⁺ was oxidized by cytochrome c oxidase in the plasma membrane. In contrast, c-type cytochrome was reduced by bisulfite ion, but it was not oxidized by cytochrome c oxidase in the membrane. Cytochrome c-oxidizing activity was also inhibited by the ion when mammalian cytochrome c was used as an electron donor, suggesting that cytochrome c oxidase, one of the component of iron oxidase, is the site of inhibition by bisulfite ion.     

Volatilization of mercury byThiobacillus ferrooxidans

Thiobacillus ferrooxidans became significantly more tolerant to mercury stress after culturing in media of increasing mercury(II) concentrations. When mercuric chloride was added to the growth medium, the resistant organisms were found to volatilize elemental mercury (Hg⁰).T. ferrooxidans may be an important factor in the natural mercury cycle, since the environments whereT. ferrooxidans is found typically contain elevated levels of heavy metals, including mercury.     

Inhibition of Sulfur Use by Sulfite Ion in Thiobacillus ferrooxidans

In Thiobacillus ferrooxidans AP19-3, elemental sulfur is oxidized by the cooperation of three enzymes, namely, hydrogen sulfide: ferric ion oxidoreductase (SFORase), sulfite: ferric ion oxidoreductase, and iron oxidase. Sulfite ions are one of the products when elemental sulfur is oxidized by SFORase. Under the conditions in which sulfite ions are accumulated in the cells, use of sulfur as an energy source by this strain was strongly inhibited. So the mechanism of inhibition by sulfite ions in T. ferrooxidans AP19-3 was studied. The activities of SFORase and iron oxidase were completely inhibited by 0.8 mm and 1.5 mm NaHSO₃, respectively. ¹⁴CO₂ uptake into washed intact cells was also completely inhibited by 1mm NaHSO₃ when ferrous ion or elemental sulfur was used as an energy source. However, the activities of ribulose-1,5-bisphosphate carboxylase, phosphoribulokinase, and ribosephosphate isomerase measured with a cell-free extract were not inhibited by NaHSO₃ at 1 mm, indicating that sulfite ions didn’t inhibit key enzymes of the Calvin cycle. Since the activity of CO₂ uptake into washed intact cells was absolutely dependent on Fe^{2 +} - or S0-oxidation, mechanism of inhibition of sulfur use by sulfite ions is proposed as follows: sulfite ions inhibit SFORase and iron oxidase, as a result T. ferrooxidans AP19-3 can not obtain a carbon source for CO₂ fixation and stops cell growth on sulfur-salts medium.     

Growth Kinetics of Three Species of Tetrahymena On Solid Agar*

A nutrient-agar method without liquid overlay has been developed for cultivation of ciliates. Three species of Tetrahymena-T. pyriformis strain W, T. rostrata strain UNI, and T. vorax strain V₂S, representing the 3 main groups of Tetrahymena species, were used; however the method should apply to other ciliates. Growth on the surface of the agar was facilitated by an optimal surface-to-volume ratio yielding a high density of ciliates (5.8 × 10₅ cells/cm² for T. pyriformis at 25 C) and short generation times (3 h for T. pyriformis at 30 C). At the highest density achieved, the cells became irregularly hexagonal and formed a monolayer “tissue” on the agar. Ciliates grown on agar were like those in liquid culture, typical oral ciliature, food-vacuole formation, and typical cortical patterns being retained. Advantages of this method include high cell density, easy recovery, and optimal O₂ supply. the organisms can also be cultivated on the surface of sterile cellulose-nitrate filters, facilitating in situ fixation and staining as well as transfer into different media by transfer of filters with cells, without prior centrifugation and resuspension.     

Pyrite oxidation by Thiobacillus ferrooxidans with special reference to the sulphur moiety of the mineral

Available cultures of Thiobacillus ferrooxidans were found to be contaminated with bacteria very similar to Thiobacillus acidophilus. The experiments described were performed with a homogeneous culture of Thiobacillus ferrooxidans.Pyrite (FeS₂) was oxidized by Thiobacillus ferrooxidans grown on iron (Fe²⁺), elemental sulphur (S^o) or FeS₂.Evidence for the direct utilization of the sulphur moiety of pyrite by Thiobacillus ferrooxidans was derived from the following observations: a. Known inhibitors of Fe²⁺ and S^o oxidation, NaN₃ and NEM, respectively, partially abolished FeS₂ oxidation. b. A b-type cytochrome was detectable in FeS₂-and S^o-grown cells but not in Fe²⁺-grown cells. c. FeS₂ and S^o reduced b-type cytochromes in whole cells grown on S^o. d. CO₂ fixation at pH 4.0 per mole of oxygen consumed was the highest with S^o, lowest with Fe²⁺ and medium with FeS₂ as substrate. e. Bacterial Fe²⁺ oxidation was found to be negligible at pH 5.0 whereas both FeS₂ and S^o oxidation was still appreciable above this pH. f. Separation of pyrite and bacteria by means of a dialysis bag caused a pronounced drop of the oxidation rate which was similar to the reduction of pyrite oxidation by NEM; indirect oxidation of the sulphur moiety by Fe³⁺ was not affected by separation of pyrite and bacteria.Bacterial oxidation and utilization of the sulphur moiety of pyrite were relatively more important with increasing pH.     

Synthesis of RNA,Protein, Cellulose,and Mucopolysaccharide and Changes in the Chemical Composition of Hartmannella culbertsoni During Encystment under Axenic Conditions*

Hartmannella culbertsoni trophozoites are transformed into viable cysts on exposure to a non-nutrient agar medium containing 15 mM MgCl₂ and 20 mM taurine. Amebae differentiating in this encystment medium incorporate more uracil-2-¹⁴C into RNA and more leucine-1-¹⁴C or valine-1-¹⁴C into proteins than controls. Encysting organisms incorporate significantly more glucose-U-¹⁴C into cellulose and glucosamine-1-¹⁴C into mucopolysaccharides. Incorporation of glucose-U-¹⁴C into cellulose and of glucosamine-1-¹⁴C into mucopolysaccharides are inhibited by actinomycin D or cycloheximide.     

Oxidation of cysteine to cystine by membrane fractions of Chlorella fusca

Isolated membrane fractions of Chlorella fusca 211-8b obtained by french-press treatment and sonication catalyzed the oxidation of l-cysteine to l-cystine. The pH-optimum of this reaction was determined to be around 8–8.5 and a stoichiometry of 4 SH-groups oxidized for one O₂ consumed was obtained. This thiol-oxidation system was specific for D-and l-cysteine; Dl-homocysteine and cysteamine were oxidized at about half the rate whereas all other thiols tested including glutathione, mercaptoethanol, mercaptopropionic acid and dithioerythritol were not oxidized by these membrane fractions. The apparent K_m for l-cysteine was determined as 3.3 mmol l^-1. Rates of 200 mol cysteine oxidized mg^-1 chlorophyll h^-1 were normally obtained. Extremely high rates of oxygen uptake were measured using l-cysteine methyl ester and l-cysteine ethyl ester. This thioloxidation system was not inhibited by mitochondrial electron-transport inhibitors such as rotenone or antimycin A, nor by the chloroplast electron-transport inhibitors 2,5-dibromothymochinone and 2,4-dinitrophenylether of iodonitrothymol. The cysteine oxidation catalyzed by C. fusca membranes was inhibited, however, by salicylhydroxamic acid, o-phenanthrolin, N,N-disalicyliden-1,3-diaminopropane 5,5-disulfonic acid, ethylenediaminetetraacetic acid, high KCN levels and by the buffers, N-[2-hydroxyl-1,1-bis(hydroxymethyl) ethyl] glycine and phosphate. This cysteine-oxidation system seems to function as a counterpart of thioredoxin-mediated light activation of enzymes, allowing reduced thiol groups to be oxidized again by O₂ (dark inactivation).Abbreviation DTNB 5,5-dithio-bis(-2-nitrobenzoic acid). Ellmann reagent     

Organic acids and prolonged nitrogenase activity by non-growing,free-living Rhizobium japonicum

Rhizobium japonicum 61-A-101 grew and fixed nitrogen more effectively on media containing an organic acid and a pentose sugar than on media containing only one of these carbon sources. Peak specific activities in the range 10–15 nmol C₂H₄ · h^-1 · mg protein^-1 were found for these organisms in a spot of growth about 1 cm diameter on agar surfaces exposed to air. Increasing concentrations of the organic acids (succinate or malonate) in a medium containing arabinose resulted in longer lasting activity. The inclusion of a third carbon source, glycerol, gave activity which remained at the maximum from about the 8 to the 18 day after inoculation although no growth of the bacteria occurs during the last 8 or 10 days. At low concentration of organic acid l-arabinose was a much better carbon source for supporting nitrogenase activity of these organisms that the d-form. Both organic acids affected the morphology of the bacteria. Higher concentrations, especially of malonate, gave swollen and distorted cells. When bacteria growing on organic acid-containing agar plates were suspended and plated after appropriate dilution on yeast extract — mannitolglycerol agar there was heterogeneity of colony form, with up to 90% microcolonies after growth on high malonate concentrations. The effects of malonate may be correlated with characteristics of the bacteroid form inside the nodule which contains relatively high concentrations of organic acids, especially malonate.     

Efficient cycloreversion of cis,syn-thymine photodimer by a Zn2+–1,4,7,10-tetraazacyclododecane complex bearing a lumiflavin and tryptophan by chemical reduction and photoreduction of a lumiflavin unit

DNA photolyases (EC 4.1.99.3) are enzymes that catalyze photoreversion of cis,syn-thymine photodimer (T[c,s]T), which is one of major photolesion products in DNA, by utilizing UV light. In this work, we have designed and synthesized Zn²⁺–1,4,7,10-tetraazacyclododecane complexes bearing a lumiflavin and l-tryptophan (ZnL³) or l-phenylalanine (ZnL⁴) as artificial DNA photolyases. We have found that (ZnL³)_red, whose flavin unit was reduced in situ by Na₂S₂O₄, accelerates the photoreversion of T[c,s]T utilizing near-UV light in aqueous solution at pH 7.6 and 11. Interestingly, more efficient photoreversion of T[c,s]T was achieved by UV irradiation of an oxidized form of ZnL³ [(ZnL³)_ox] in the presence of an excess amount of Et₃N at pH 11. UV–vis and fluorescence measurements and action spectra showed that an oxidized form of flavin of (ZnL³)_ox was photoreduced by Et₃N into its reduced form (ZnL³)_red, which promoted the photoreduction of T[c,s]T. Comparison of the photochemical properties of ZnL³ with those of ZnL⁴ suggested that a tryptophan unit in ZnL³ contributed to the stabilities of the flavin through intramolecular photoinduced electron transfer.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Microbial leaching of arsenic from low‐sulfide gold mine material

Drainages from high‐sulfide tailings near abandoned lode deposits in Alaska, U.S.A., and Yukon, Canada, were found to be acidic, to contain large numbers of Thiobacillus ferrooxidans, and to have high concentrations of dissolved arsenic. Drainages from active placer gold mines are not acidic, but T. ferrooxidans and concentrations of dissolved arsenic exceeding 10 μg/L are found in some streams affected by placer mine drainage. Placer mine material containing low amounts of sulfides (326 (μg/g) and moderately high amounts of arsenic (700 μg/g) was leached with growing cultures of T. ferrooxidans, T. ferrooxidans‐spent filtrate, and acid ferric sulfate. The results showed that while more arsenic was released from this material by growing cultures of T. ferrooxidans than by abiotic controls, acid ferric sulfate released much more arsenic than did either growing cultures of T. ferrooxidans or spent culture filtrate containing oxidized iron. Cation exchange chromatography showed that oxidized iron from T. ferrooxidans culture filtrate is chemically less reactive than the iron in aqueous solutions of ferric sulfate salt. These results indicate that arsenic release from both high‐ and low‐sulfide mine wastes is enhanced biologically, but that rates and amounts of arsenic release are primarily controlled by iron species.     

Expression,purification and characterization of an iron-sulfur cluster assembly protein,IscU, from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

An iron-sulfur cluster assembly protein, IscU, is encoded by the operon iscSUA in Acidithiobacillus ferrooxidans. The gene of IscU was cloned and expressed in Escherichia coli. The protein was purified by one-step affinity chromatography to homogeneity. The protein was in apo-form, the [Fe₂S₂] cluster could be assembled in apoIscU with Fe²⁺ and sulfide in vitro, and in the presence of IscA and IscS, the IscU could utilize l-cysteine and Fe²⁺ to synthesize [Fe₂S₂] cluster in the protein. Site-directed mutagenesis for the protein revealed that Cys37, Asp39, Cys63 and Cys106 were involved in ligating with the [Fe₂S₂] cluster.     

Molecular cloning and expression ofThiobacillus ferrooxidans chromosomal ribulose bisphosphate carboxylase genes inEscherichia coli

The genes coding ford-ribulose-1,5-bisphosphate carboxylase (RuBPCase) from an iron-oxidizing bacterium,Thiobacillus ferrooxidans, were cloned into anEscherichia coli plasmid, pUC18. The recombinant plasmid, termed pTR11, contained a 4.0-kb PstI fragment including the entire coding regions for both large and small subunits of RuBPCase.Escherichia coli carrying pTR11 did not show any CO₂-fixing activity. However, a derivative plasmid with an appropriate deletion, which was placed under the control of atac promoter, conferred ribulose bisphosphate-dependent CO₂-fixing activity on the host cell. Analysis of gel-filtration chromatography of the RuBPCase synthesized inE. coli revealed that it had a hexadecameric form like the native enzyme ofT. ferrooxidans.     

Characterization of K+ Channels in the Basolateral Membrane of Rat Tracheal Epithelia

To study K⁺ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 μg/ml) in the symmetrical high K⁺ (145 mm) Ringer solution. During measurement of the macroscopic K⁺ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types of K⁺ currents: one is an inwardly rectifying K⁺ current and the other is a slowly activating outwardly rectifying K⁺ current. The inwardly rectifying K⁺ current is inhibited by Ba²⁺. The slowly activating K⁺ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristae 13-acetate (PMA) and lowering temperature. This is consistent with the biophysical characteristics of I _SK channel. RT-PCR analysis revealed the presence of I _SK cDNA in the rat trachea epithelia. Although 0.1 mm Ba²⁺ only had minimal affect on short-circuit current (I _sc) induced by cAMP in intact epithelia, 0.1 mm clofilium strongly inhibited it. These results indicate that I _SK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia. Received: 1 March 1996/Revised: 5 August 1996     

Mechanisms of cation permeation across apical cell membrane ofNecturus gallbladder: Effects of luminal pH and divalent cations on K+ and Na+ permeability

Summary Conventional microelectrode techniques were combined with unilateral mucosal ionic substitutions to determine the effects of luminal pH and luminal alkali-earth cation concentrations on apical membrane cation permeability inNecturus gallbladder epithelium. Acidification of the mucosal solution caused reversible depolarization of both cell membranes and increase of transepithelial resistance. Low pH media also caused: (a) reduction of the apical membrane depolarization induced by high K, and (b) increase of the apical membrane hyperpolarization produced by Na replacement with Li or N-Methyl-d-glucamine. These results, in conjunction with estimates of cell membrane conductances, indicate that acidification of the luminal solution produces a reduction of apical membrane K permeability (P _K). Addition of alkali earth cations (Mg²⁺, Ca²⁺, Sr²⁺, or Ba²⁺) produced cell membrane depolarization, increase of relative resistance of the luminal membrane and reduction of the apical membrane potential change produced by a high-K mucosal medium. These results, as those produced by low pH, can be explained by a reduction of apical membraneP _K. The effects of Ba²⁺ on membrane potential and relative apical membraneP _K were larger than those of all other four cations at all concentrations tested (1–10mm). The effect of Sr²⁺ was significantly larger than those of Mg²⁺ and Ca²⁺ at 10mm, but not different at 5mm. The reduction ofP _K produced by mucosal acidification appears to be mediated by: (a) nonspecific titration of membrane fixed negative charges, and (b) an effect of luminal proton activity on the apical K channel. Divalent cations reduce apical membraneP _K probably by screening negative surface charges. The larger magnitude of the effects of Ba²⁺ and Sr²⁺ can be explained by binding to membrane sites, in the surface or in the K channel, in addition to their screening effect. We suggest that the action of luminal pH on K secretion in some segments of the renal tubule could be mediated in part by this pH-dependent K permeability of the luminal membrane.     

A New Enzyme Species of p-Hydroxybenzoate Hydroxylase during Oxygenating Process Observed by the Stopped-flow Method

Sulfite ion (HSO₃ ^-) is one of the products when elemental sulfur is oxidized by the hydrogen sulfide:ferric ion oxidoreductase of Thiobacillus ferrooxidans AP19-3. Under the conditions in which HSO₃ ^- is accumulated in the cells, the iron oxidase of this bacterium was strongly inhibited by HSO₃ ^-. Since cytochrome c oxidase is one of the most important components of the iron oxidase enzyme system in T. ferrooxidans, effects of HSO₃ ^- on cytochrome c oxidase activity were studied with the plasma membranes of HSO₃ ^--resistant and -sensitive strains of T. ferrooxidans, OK1-50 and AP19-3. The enzyme activity of AP19-3 compared with OK1-50 was strongly inhibited by HSO₃ ^-. To investigate the inhibition mechanism of HSO₃ ^- in T. ferrooxidans, cytochrome c oxidases were purified from both strains to an electrophoretically homogeneous state. Cytochrome c oxidase activity of a purified OK1-50 enzyme was not inhibited by 5 mM HSO₃ ^-. In contrast, the same concentration of HSO₃ ^- inhibited the enzyme activity of AP19-3 50%, indicating that the cytochrome c oxidase of OK1-50 was more resistant to HSO₃ ^- than that of AP19-3. Cytochrome c oxidases purified from both strains were composed of three subunits. However, the molecular weight of the largest subunit differed between OK1-50 and AP19-3. Apparent molecular weights of the three subunits of cytochrome c oxidases were 53,000, 24,000, and 19,000 for strain AP19-3 and 55,000, 24,000, and 19,000 for strain OK1-50, respectively.     

Influence of the oxygenation level on d-xylose fermentation by free and agar-entrapped cultures of Candida shehatae

This paper investigates the effects of the oxygenation level on the performance of d-xylose alcoholic fermentation by free- and immobilized-cell batch cultures of Candida shehatae (ATCC 22984). Yeast cells were immobilized in composite agar layer/microporous membrane structures. Fermentations were performed under varying oxygenation levels corresponding to different O₂ flow rates (OFRs). Low OFRs enhanced the fermentation performance of free and immobilized yeasts. The best ethanol yield coefficient, obtained at an OFR of 5 mmol O₂ h^–1 dm^–3 for both culture modes, was slightly higher (0.425 g g^–1) for immobilized cultures than for their free counterparts (0.39 g g^–1). More sustained aeration inhibited ethanol production by free and immobilized organisms. However, this inhibition was more pronounced for agar-entrapped cultures. Xylitol production of free cultures normally decreased as the OFR increased. At high OFR, however, immobilized organisms surprisingly produced more xylitol than at lower OFR or in anaerobiosis. This effect is discussed by referring to the mass transfer limitations that occur inside the immobilized-cell structures. Gel-entrapped cultures displayed higher specific and volumetric production rates of ethanol and xylitol than free-cell cultures.     

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles is modulated by membrane potential

Summary The ATP-dependent Ca²⁺ transport activity (T. Takuma, B.L. Kuyatt and B.J. Baum,Biochem. J. 227:239–245, 1985) exhibited by inverted basolateral membrane vesicles isolated from rat parotid gland was further characterized. The activity was dependent on Mg²⁺. Phosphate (5mm), but not oxalate (5mm), increased maximum Ca²⁺ accumulation by 50%. Half-maximal Ca²⁺ transport was achieved at 70nm Ca²⁺ in EGTA-buffered medium while maximal activity required >1 m Ca²⁺ (V _max=54 nmol/mg protein/min). Optimal rates of Ca²⁺ transport were obtained in the presence of KCl, while in a KCl-free medium (mannitol or sucrose) 40% of the total activity was achieved, which could not be stimulated by FCCP. The initial rate of Ca²⁺ transport could be significantly altered by preimposed membrane potentials generated by K⁺ gradients in the presence of valinomycin. Compared to the transport rate in the absence of membrane potential, a negative (interior) potential stimulated uptake by 30%, while a positive (interior) potential inhibited uptake. Initial rates of Ca²⁺ uptake could also be altered by imposing pH gradients, in the absence of KCl. When compared to the initial rate of Ca²⁺ transport in the absence of a pH gradient, pH _i =7.5/pH _o =7.5; the activity was 60% higher in the presence of an outwardly directed pH gradient, pH _i =7.5/pH _o =8.5; while it was 80% lower when an inwardly directed pH gradient was imposed, pH _i =7.5/pH _o =6.2. The data show that the ATP-dependent Ca²⁺ transport in BLMV can be modulated by the membrane potential, suggesting therefore that there is a transfer of charge into the vesicle during Ca²⁺ uptake, which could be compensated by other ion movements.     

Ultrastructural localization of adenosine triphosphatase activity in lymphocytes activated in vitro by phytohaemagglutinin

Summary The ultrastructural localization of Ca²⁺, Mg²⁺-activated ATPase was studied in phytohaemagglutinin activated lymphocytes and in normal unstimulated lymphocytes. Cells, fixed in paraformaldehyde-glutaraldehyde, were incubated in a medium containing 3mm ATP, 5mm CaCl₂ and 2.4mm Pb(NO₃)₂ in 0.1m tris buffer at pH 8.5, the optimum pH for histochemical demonstration of this enzyme. Reaction product was localized i the endoplasmic reticulum, nuclear membrane, Golgi apparatus and mitochondria and on the membrane surrounding large electron-dense bodies. Cytoplasmic vesicles and the plasma membrane were negative. Activity in unstimulated lymphocytes showed a similar localization but the amount of endoplasmic reticulum was much less than in activated lymphocytes.The pH of the medium was critical for the localization of the enzyme. At pH 7.5, the cytoplasmic reaction was almost completely inhibited but a dense precipitate was present on the outer surface of the plasma membrane. The reaction was stimulated by either Ca²⁺ or Mg²⁺ and was greatly decreased in the absence of these cations or in the presence ofp-chloromercuribenzoate orN-ethylmaleimide. Oligomycin inhibited selectively the reaction in mitochondria but not the reaction at other sites. While the reaction in mitochondria showed complete substrate specificity, a mild reaction was obtained at the other sites with uridine diphosphate or sodium -glycophosphate as substrate. ATP was, however, the preferential substate.