Purification and characterization of a dextranase from Sporothrix schenckii

A dextranase (EC 3.2.1.11) was purified and characterized from the IP-29 strain of Sporothrix schenckii, a dimorphic pathogenic fungus. Growing cells secreted the enzyme into a standard culture medium (20 °C) that supports the mycelial phase. Soluble bacterial dextrans substituted for glucose as substrate with a small decrease in cellular yield but a tenfold increase in the production of dextranase. This enzyme is a monomeric protein with a molecular mass of 79 kDa, a pH optimum of 5.0, and an action pattern against a soluble 170-kDa bacterial dextran that leads to a final mixture of glucose (38%), isomaltose (38%), and branched oligosaccharides (24%). In the presence of 200 mM sodium acetate buffer (pH 5.0), the K _m for soluble dextran was 0.067 ± 0.003% (w/v). Salts of Hg²⁺, (UO₂)²⁺, Pb²⁺, Cu²⁺, and Zn²⁺ inhibited by affecting both V _max and K _m. The enzyme was most stable between pH values of 4.50 and 4.75, where the half-life at 55 °C was 18 min and the energy of activation for heat denaturation was 99 kcal/mol. S. schenckii dextranase catalyzed the degradation of cross-linked dextran chains in Sephadex G-50 to G-200, and the latter was a good substrate for cell growth at 20 °C. Highly cross-linked grades (i.e., G-10 and G-25) were refractory to hydrolysis. Most strains of S. schenckii from Europe and North America tested positive for dextranase when grown at 20 °C. All of these isolates grew on glucose at 35 °C, a condition that is typically associated with the yeast phase, but they did not express dextranase and were incapable of using dextran as a carbon source at the higher temperature. Received: 29 December 1997 / Accepted: 4 March 1998     

Isolation and characteristics ofTreponema zuelzerae nov. spec., an anaerobic,free-living spirochete

Summary An anaerobic, free-living spirochete was isolated from mud. The organism can be cultivated in ordinary nutrient media, e.g. yeast extract-glucose. End products of glucose fermentation are: lactic, acetic, and succinic acids, CO₂, and H₂. In cultures of this organism spheroid bodies are formed, especially during the stationary growth phase. Studies of slide cultures showed that these bodies, when inoculated in fresh medium, do not give rise to spiral cells whereas a rapid multiplication of normal cells, also present in the inoculum, was observed. Since the organism is serologically related toTreponema pallidum, it has been assigned to the genusTreponema, and is here described asTreponema zuelzerae nov. spec. Part of this work was carried out at the Hopkins Marine Station of Stanford University, Pacific Grove, U.S.A., under a Rockefeller Foundation fellowship. Present address: Laboratorium voor Microbiologie, Wageningen, the Netherlands.     

A Novel Phospholipase C Inhibitor,S-PLI Produced by Streptomyces Sp. Strain No. A-6288

Abstract

S-PLI, an inhibitor of phospholipase C (PLC) produced by Strepromyces sp. strain No. 6288, was purified from the culture filtrate by salting-out with solid ammonium sulfate, column chromatography on CM-cellulose and gel filtration on Sephadex G-75. The molecular weight of S-PLI was estimated to be 65,000 by SDS-polyacrylamide gel electrophoresis. The inhibitor was found to be a glycoprotein with a composition of 609 amino acids and 19 glucose residues having an isoelectric point at 7.8. S-PLI was stable from pH 3 to 10 at 37°C and up to 40° at pH 6.0. The inhibitory activity showed pH-and temperature-dependence with a maximum around pH 7.0 at 50°C. S-PLI inhibited phospholipase C in a competitive manner (K_i value; 9.5 × 10-⁶ mM), but did not inhibit S-Hemolysin, phospholipase A₂, phospholipase B, phospholipase D and phosphatases. S-PLI is the first reported example of a glycoproteinaceous inhibitor of microbial origin which is able to specifically inhibit phospholipase C.     

Mannitol Biosynthesis in Pyrenochaeta terrestris II. D-Mannitol-l-phosphatase

Cell-free extracts of mycelial mats of Pyrenochaeta terrestris contained an enzyme which hydrolyzed mannitol-l-phosphate to mannitol and inorganic phosphate. Greatest mannitol-1-phosphatase activity occurred early in the growth period when the mannitol content of the mats was at a maximum. The enzyme was active over a broad pH range with optimum activity between pH 6.5–7.0 in 0.05 M Tris-maleate buffer. Maiinitnl-1-phosphatase was inhibited by reagents known to inhibit enzymes containing -SH groups. A 10-fold purification was attained by a combination of (NII₄)₂ SO₄ fractionation and gel filtration on Sephadex G-100. The partially purified enzyme required Mg^?2 for activity and did not hydrolyze a number of sugar phosphates. Km values for mannitol-l-phosphate and Mg^?2 with the partially purified extract were 3 × 10^?3 M and 1 × 10^?4 M respectively.     

Biodegradation of phenanthrene by Arthrobacter polychromogenes isolated from a contaminated soil

Summary Degradation of phenanthrene by Arthrobacter polychromogenes isolated from a contaminated soil was investigated. In experiments in which [9-¹⁴C]-phenanthrene was incubated with cultures of A. polychromogenes containing 150 mg phenanthrene/l it was shown that after 26 h of incubation 47.7% of the recovered radiolabelled carbon originally present was metabolized to ¹⁴CO₂, 47.8% was recovered from the aqueous fraction, and 4.5% remained in the dichloromethane fraction. Increasing phenanthrene concentration in the culture medium resulted in improved growth and degradation rates, probably due to the higher amount of phenanthrene crystals in the medium. Shifting the temperature from 30°C to 35°C did not influence phenanthrene degradation significantly but inhibited cell division of A. polychromogenes. Medium supplementation with glucose led to stimulation of phenanthrene degradation at low amounts of glucose (0.45 g/l) whereas at higher concentrations (3 g/l) phenanthrene mineralization decreased.Professor Dr. D. Behrens dedicated to his 65th birthdayOffprint requests to: H.-J. Rehm     

Transport of an anionic substrate by the H+/monosaccharide symport inRhodotorula gracilis: Only the protonated form of the carrier is catalytically active

Summary The yeastRhodotorula gracilis accumulated glucuronate by an H⁺/symport. The transport was electroneutral, driven by the chemical gradient of protons pH. The observed stoichiometry amounted to 1 proton per molecule glucuronate. At pH 4, the half-saturation constantK _T was at its lowest value (K _T =8mm), whereas the maximal velocityV _T reached a maximum (V _T =15 nmol/min×mg dry wt). Monosaccharides competitively inhibited the uptake of glucuronate and vice versa. Hence, the two substrates share the same transport system. The steady-state accumulation of glucuronate reflected the course of the pH gradient. It is concluded that glucuronate is transported as an anionic substrate by the protonated carrier, the driving force being the chemical gradient of the H⁺ (pH). The ternary carrier/H⁺/glc-COOO-complex is electroneutral and independent of the membrane potential. Simultaneous uptake of organic acids (acetic or propionic acid) which is also energized by the pH gradient led to a noncompetitive inhibition of glucuronate transport. Thus, manipulation of the driving force, pH, reducedV _T without affectingK _T . Kinetic and energetic arguments are presented which stronly suggest that only the protonated carrier is catalytically active inR. gracilis.     

Enzyme kinetics in mammalian cells. 3. Regulation of activities of galactokinase, galactose-1-phosphate uridyl transferase and uridine diphosphogalactose-4-epimerase in human erythrocytes

The sequential enzyme assay as previously described has been used to study various effects on the three enzymes in human red cells involved in the phosphorylation of galactose: galactokinase, galactose-1-phosphate uridyl transferase and uridine diphospho-galactose-4-epimerase.

• 1 Enzyme activities in undiluted lysates appear to reflect the respective activities in whole cells.
• 2 Added extracellular Gal-1-P, G-1-P, UDPGal and UPDG do not affect enzyme activities in whole cells.
• 3 The kinase and transferase enzymes do not appear to be associated with the membrane fraction of the red cells.
• 4 Galactokinase activity is inhibited by G-6-P and Gal-1-P, but not by glucose, G-1-P, UDPG, UDPGal, UTP or NAD⁺. It is inhibited by ATP and ADP in high concentration.
• 5 Galactose-1-phosphate uridyl transferase activity is inhibited by G-1-P, G-6-P, UDPG, UDPGal, ATP, and ADP. It is not affected by UTP, NAD⁺, or galactose.
• 6 Uridine diphospho-galactose-4-epimerase activity is inhibited by UDPG, ATP, ADP, UTP and NADH. It is stimulated by NAD⁺ and possibly by Gal-1-P. It is unaffected by G-1-P, G-6-P.
• 7 The rates of the three reactions decrease with decreasing temperature. The activities of transferase and epimerase are inactivated at the same rate, the kinase activity is inactivated more slowly.
• 8 Dilution experiments indicate the presence in lysates of a pool of UDPG (or, possibly UDPGal) which regulates the activities transferase and the epimerase enzymes.
• 9 Results of dilution experiments suggest that the radioactive product of the transferase enzyme is different from commercially available UDPGal-u-¹⁴C.
• 10 ATP, UTP and UDPG interact with some substance(s) in the red cell lysate to cause a time dependent inactivation of the epimerase. These interactions are the result of glucose metabolism.

     

Purification and properties of UDP-glucose: coniferyl alcohol glucosyltransferase from suspension culturesof Paul's scarlet rose.

UDP-glucose:coniferyl alcohol glucosyltransferase was isolated from 10-day-old, darkgrown cell suspension cultures of Paul's scarlet rose. The enzyme was purified 120-fold by (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-100. The enzyme has a pH optimum of 7.5 in Tris-HCl buffer, required an -SH group for activity, and is inhibited by ?-chloromercuribenzoate and EDTA. Its molecular weight is estimated to be 52,000. The enzyme is specific for the glucosylation of coniferyl alcohol (K_m 3.3 × 10^?6 M) and sinapyl alcohol (K_m 5.6 × 10^?6 M). With coniferyl alcohol as substrate the apparent K_m value for UDP-glucose is 2 × 10^?6m. The enzyme activity can be detected in a number of callus-tissue and cell-suspension cultures. The role of this enzyme is believed to be to catalyze the transfer of glucose from UDPG to coniferyl (or sinapyl) alcohol as storage intermediates in lignin biosynthesis.     

The role of nitric oxide in the metabolism of labeled glucose in isolated rat uterus. Influence of 17β-estradiol

The influence of nitric oxide (NO) on the production of ¹⁴CO₂ from labeled glucose in uteri isolated from ovariectomized-estrogenized rats was studied. Nitroprusside, an NO donor (NP), 200 μM increased the formation of labeled CO₂ from [U-¹⁴C]glucose. This effect was blunted by hemoglobin (Hb) 20 μg/mL, an NO scavenger. The addition of N-monomethyl arginine (NMMA), an inhibitor of NO synthase decreased the stimulatory action of NP at 400 mM. Incubation of uterine strips in the presence of NP plus acetylsalicylic acid (ASA) 10⁻⁴ M (a cyclooxygenase inhibitor), inhibited the stimulatory action of NP on glucose metabolism. PGE₂ (10⁻⁷ M) added to the incubation medium containing NP and ASA reversed the effect of the inhibitor. Neither NP nor Hb nor NMMA modified the ¹⁴CO₂ production from labeled glucose in uterine strips from ovariectomized rats. The addition of NP to the incubating medium increased PGE accumulation by uterine strips from rats treated with estradiol, but not in ovariectomized animals. These results suggest that NO exerts a positive influence on glucose metabolism and PGE synthesis in isolated rat uteri from estrogenized animals.     

Purification and characterization of an intracellular β-glucosidase from the protoplast fusant of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed using 35% polyethylene glycol 4,000 with 0.01 mM CaCl₂. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular (β-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 5.4 and temperature of 65°C, respectively. This enzyme showed relatively high stability against pH and temperature and was stable in the pH range of 3.0–6.6. Na⁺, K⁺, Ca²⁺, Mg²⁺ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe³⁺. The enzyme activity was strongly inhibited by glucose, the end product of glucoside hydrolysis. The K _m and V _max values against salicin as substrate were 0.035 mM and 1.7215 μmol min⁻¹, respectively.     

Ethylene formation by cultures of Escherichia coli

Growth of Escherichia coli strain B SPAO on a medium containing glucose, NH₄Cl and methionine resulted in production of ethylene into the culture headspace. When methionine was excluded from the medium there was little formation of ethylene. Ethylene formation in methionine-containing medium occurred for a brief period at the end of exponential growth. Ethylene formation was stimulated by increasing the medium concentration of Fe³⁺ when it was chelated to EDTA. Lowering the medium phosphate concentration also appeared to stimulate ethylene formation. Ethylene formation was inhibited in cultures where NH₄Cl remained in the stationary phase. Synthesis of the ethylene-forming enzyme system was determined by harvesting bacteria at various stages of growth and assaying the capacity of the bacteria to form ethylene from methionine. Ethylene forming capacity was greatest in cultures harvested immediately before and during the period of optimal ethylene formation. It is concluded that ethylene production by E. coli exhibits the typical properties of secondary metabolism.Abbreviations HMBA 2-Hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - KMBA 2-keto-4-methylthiobutyric acid - MOPS 3-[N-morpholino] propanesulphonic acid     

Beneckea gazogenes sp. nov., a red,facultatively anaerobic,marine bacterium

A red, facultatively anaerobic marine bacterium was isolated from samples of saltwater marsh mud. The Gram-negative microorganism was rod-shaped and possessed a single sheathed polar flagellum. The red pigment that the cells produced was identified as prodigiosin. THe bacteria grew readily on synthetic seawater media containing carbohydrates, amino acids, organic acids, or tricarboxylic acid cycle intermediates as sole sources of carbon and energy. Na⁺ was required for growth. Cells fermented glucose to CO₂, H₂, ethanol, acetate, succinate, and pyruvate. The guanine-plus-cytosine content of the strain’s DNA was 47.1 mole%. The morphological and physiological characteristics of this bacterium, together with the mole % G+C of its DNA, place it in the genusBeneckea. The main features of this strain are sufficiently different from previously described members of the genus to place it in a new species for which the nameBeneckea gazogenes is proposed.     

(+)-Abscisic Acid and Two Compounds Showing Chlorophyll Degradation Activity in Cuscuta pentagona Engelm

The purified polyethylene glycol (PEG) dehydrogenase from cells of a synergistic mixed culture of Flavobacterium and Pseudomonas species showed a similar absorption spectrum to those of other quinoproteins reported so far. The prosthetic group of the PEG dehydrogenase after extraction with cold methanol and purification by DEAE-Sephadex A-25 column chromatography and Sephadex G-25 gel filtration showed the same elution profiles as those of authentic pyrrolo-quinoline quinone (PQQ). Absorption and fluorescence spectra of the purified prosthetic group and its prosthetic group capability for glucose dehydrogenase indicated that it was identical with authentic PQQ.

The enzyme was induced during bacterial cell growth on a medium containing PEG 6000 as a sole source of carbon. The purified enzyme oxidized primary alcohols of C₂-C₁₆ and the corresponding aldehydes of C₄-C₇. The enzyme also reacted with nonionic surfactants containing PEG residues. The enzyme reduced 2,6-dichlorophenolindophenol (DCIP) and the Km value for DCIP was calculated to be 1.4 × 10^?4m. The DCIP reductase activity was inhibited by carbonyl reagents like semicarbazide, hydrazine, hydroxylamine and 1,4-benzoquinone. 1,4-Benzoquinone inhibited the DCIP reductase activity competitively as to DCIP.     

Comparative metabolic effects of fructose and glucose in human fibroblast cultures

Summary The comparative metabolic effects of fructose and glucose were determined in human fibroblast cultures. Cells were grown in four different media containing 5.5 and 27.5 mM of glucose and fructose, respectively. For these two hexoses, we compared their uptake, consumption, and conversion into¹⁴CO₂ and¹⁴C-lipids. D-Fructose was taken up in fibroblasts by an unsaturable process and its consumption was much smaller than that ofD-glucose. Whatever the experimental procedure, the glycogen content of cells grown in fructose media was significantly lower than of those grown in glucose media. Labeling of fructose and glucose with¹⁴C showed that more carbon from fructose than from glucose was incorporated into CO₂ and glycerolipids. The relative distribution of¹⁴C in the different lipid fractions was similar for both hexoses. These results indicated that the pathways of intermediary metabolism in fibroblast cultures were influenced by the nature of the carbohydrate present in the culture medium and that fructose was a better lipogenic substrate than glucose in human fibroblast cultures. This work was supported by grants for the Institut National de la Santé et de la Recherche Medicale (ATP 82-79-114).     

Inhibitory effect of ethanol on growth and solute accumulation by Saccharomyces cerevisiae as affected by plasma-membrane lipid composition

Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rate, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing d-[6-³H] glucose, d-[1-¹⁴C] glucosamine, l-[U-¹⁴C] lysine or arginine, or KH₂ ³²PO₄ lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5–7 times greater than that in the cell-free medium.     

The Role of Ethylene Diisothiocyanate (EDI) in the Antifungal Action of Disodium Ethylene Bisdithiocarbamate (Nabam). I. The Mode of Action of EDI on the Metabolism of Yeast

With glucose as a substrate, the oxygen consumption in yeast in inhibited by 2· 10^-5M ethylene diisothiocyanate. The degree of inhibition was only to a small extent dependant on pH. Radiorespirometric experiments with uniformely labelled glucose showed that the CO₂-production from glucose increased, probably due to increased glycolytic activity. Conversion of C-1 to CO₂ was unaffected by the inhibitor, while the evolution of CO₂ from C-6 was strongly inhibited. The same was the case with CO₂ from C-1 in acetate. Respiration of ethanol was more strongly inhibited than that of glucose or acetate. Experiments with dual wavelength spectrophotometry showed the inhibition to be located on the Krebs cycle side of the respiratory flavoproteins. It is concluded that the action of ethylene diisothiocyanate on respiration must be located at the mitochondria.     

Glucose uptake in mouse brain regions under hypoxic hypoxia

Glucose uptake of individual structures within the brain was studied by dry-autoradiography with 2-deoxy-D-[¹⁴C(U)]glucose under mild hypoxic hypoxia (12% O₂88% N₂ or 9% O₂91% N₂ for 1 hr). Glucose consumption in the whole brain was estimated by combined gas chromatography-mass spectrometry (GC-MS). Mild hypoxia increased the optical density of the autoradiograph in all regions. The deuterated G-6-P (Glucose-6-phosphate) synthesized from deuterated glucose decreased significantly with 9% O₂ hypoxia (P<0.05). The ratio of the deuterated G-6-P to deuterated glucose, a more appropriate indicator of glucose utilization than the concentration of deuterated G-6-P, decreased significantly with 12% O₂ hypoxia (P<0.01). The hippocampus, white matter, colliculus superior, and corpus geniculatum laterale appeared to be particulary sensitive to hypoxia.     

Lactate Uptake and Release in the Presence of Glucose by Sympathetic Ganglia of Chicken Embryos and by Neuronal and Nonneuronal Cultures Prepared from These Ganglia

Abstract: Uptake and output of lactate were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14–15 days old. The chains, typically containing 30–40 μg of protein, were incubated in Eagle's minimum essential medium containing bicarbonate buffer, 6–17 mM glucose, various concentrations of lactate, and either [U-¹⁴C]lactate, [1-¹⁴C]glucose, or [6-¹⁴C]glucose. The average rate of uptake of labeled lactate was measured with incubations of 5–6 h, starting with various external lactate concentrations. From these data the instantaneous relation between lactate uptake rate and concentration was deduced with a simple computerized model. The instantaneous uptake rate increased with the concentration according to a relation that fit the Michaelis-Menten equation, with V_max = 360 μmol/g protein/h and K_m = 4.8 mM. Substantial fractions of the lactate carbon were recovered from tissue constituents and in several nonvolatile products in the medium, as well as in CO₂. Glucose uptake averaged about 108 μmol/g protein/h and did not vary greatly with external lactate concentration, although the metabolic partitioning of glucose carbon was considerably affected. Regardless of initial concentration, the lactate concentration in the medium tended to change towards approximately 0.6 mM, showing that uptake equaled output at this level, with rates at about 40 μmol/g protein/h. With the steady-state concentration of 0.6 mM lactate, about 20% of the glucose carbon was shunted out into the medium before it was reabsorbed and metabolized into various products. Lactate uptakes by neuronal and nonneuronal cultures prepared from the ganglia did not differ consistently from one another or from uptake by undissociated ganglia. The neuronal cultures tended to oxidize a greater fraction of the consumed lactate to CO₂ and to convert a smaller fraction of the lactate to products in the medium than did the nonneuronal cultures. Computer modeling, using known parameters for blood-brain transport of lactate in the adult rat and data on uptake by the ganglia, suggests that lactate may supply substantial fuel to the brain, even in the presence of abundant glucose, when the lactate concentration in the blood is raised to levels commonly observed in exercising humans, such as 10–20 mM. This is in agreement with the findings of several investigators in hypoglycemic humans and in animals with intermediate blood lactate concentrations.     

NUTRITIONAL REQUIREMENTS FOR SEXUAL REPRODUCTION IN MESOTAENIUM KRAMSTAI (CHLOROPHYTA) 1

Optimum conditions for conjugation in the heterothallic saccoderm desmid Mesotaenium kramstai Lemmer-mann have been determined. In culture, cells acquired the ability to form gamete pairs just prior to the onset of stationary phase after sufficient nitrate had been depleted from the medium. The appearance of potential gametes was delayed by increasing the concentration of KNO₃ When cells of both mating types were harvested from 15 to 18 day old cultures, washed, resuspended in fresh medium, and mixed, approximately 50 percent of the cells paired (measured three days after mixing) in a medium containing 0.13 mM or less KNO₃. At greater concentrations, fewer pairs formed; no pairs formed in medium containing 0.5 mM KNO₃. Conjugation was not inhibited by other macronutrients. Calcium and magnesium were essential for maximum conjugation. Although Ca²⁺ and Mg²⁺ contentrations of 0.05 mM and 0. I mM, respectively, were sufficient for optimum growth, maximum conjugation required more than 10 times these values. Few gamete pairs formed when either Ca²⁺ or Mg²⁺ was omitted from the medium, no pairing occurred when both Ca²⁺ and Mg²⁺ were omitted.     

Inhibition of glucose utilization for acetylcholine synthesis by cerebrospinal fluid.

Abstract— Replacement of bicarbonate-Locke incubation medium with feline CSF reduced [¹⁴C]ACh formation from [U-¹⁴C]glucose by rat brain mince approx 30%. CSF was obtained from a cannula leading to the cisterna magna of freely moving cats. The component of CSF responsible for inhibition was characterized as a dialyzable heat-stable organic anion. Choline acetyltransferase activity was not altered by CSF. [¹⁴C]ACh synthesis and ¹⁴CO₂ production from [U-¹⁴C]glucose but not from [2-¹⁴C]-pyruvate were inhibited by CSF, suggesting inhibition in the metabolism of glucose to pyruvate. The anionic fraction of human CSF was as potent as that from feline CSF in inhibiting ¹⁴CO₂ production from [U-¹⁴C]glucose. Brain hexokinase was inhibited by the anionic fraction of feline CSF. The inhibition was non-competitive with respect to glucose and uncompetitive with respect to ATP. It is suggested that inhibition of hexokinase by CSF was responsible at least in part for the inhibition of glucose metabolism which resulted in decreased [¹⁴C]ACh synthesis and ¹⁴CO₂ production.