INCORPORATION OF PHENYLALANINE AS A SINGLE UNIT INTO RAT BRAIN PROTEIN: RECIPROCAL INHIBITION BY PHENYLALANINE AND TYROSINE OF THEIR RESPECTIVE INCORPORATIONS

Abstract— Of seven amino acids studied, glutamic acid and phenylalanine were incorporated in highest amounts into the hot-TCA-insoluble material of the 100,000 g supernatant fraction of rat brain homogenate. The system for incorporation of phenylalanine was RNase-insensitive and required ATP (apparent K_m = 0.64 m m ), KC1 (apparent K_m = 14 m m ) and MgCl₂ (optimal concentration range 4-15 m m ). The apparent K_m for phenylalanine was 2.9 m m . [¹⁴C]Phenylalanine did not undergo modification before incorporation. Tyrosine and phenylalanine inhibited the incorporation, respectively, of [¹⁴C]phenylalanine and [¹⁴C]tyrosine when incubated simultaneously or successively. The K_m and K_t (3.3 m m ) values for phenylalanine in the incorporation reaction and as inhibitor of the incorporation of [¹⁴C]tyrosine were similar. We suggest that both the enzyme and the acceptor for the incorporation of these two amino acids are the same. [¹⁴C]Phenylalanine and [¹⁴C]tyrosine entered into COOH-terminal positions in the reactions described. Brain exhibited a 25- to 100-fold higher capacity to incorporate phenylalanine than that of liver, kidney or thyroid. The acceptor capacity in rat brain rapidly decreased from day 5 to day 15 of postnatal age and then slowly until age 150 days.     

Physiological effects of Na2SO4 and NaCl on callus cultures of Brassica campestris (Chinese cabbage)

Hypocotyl-derived callus cultures of Brassica campestris L. ssp. pekinensis cv. Kim-jung (Chinese cabbage) were grown on Murashige and Skoog medium containing no additional salt, NaCl or Na₂SO₄. Na₂SO₄ was more than twice as inhibitory in comparison to the same concentration of NaCl when growth and fresh:dry weight ratios of established callus were measured. Levels of protein, starch, sucrose and α-amino nitrogen were not significantly altered in salt-grown callus. Concentrations of reducing sugars and chlorophyll were 2–3 times greater in callus grown on either salt. Proline concentration increased 15–20 fold on the highest levels of salt. Final concentrations (reached in 20–24 days) were closely correlated to the initial Na⁺ concentration of the medium, regardless of salt type. The osmotic potential in callus transferred to NaCl or Na₂SO₄ reached a maximum negative value after 16 days. For both salts, subsequent increases were correlated to increases in fresh:dry weight and growth. On both salts, turgor remained relatively constant (0. 6–0.75 MPa). Changes in Na⁺, K⁺, Mg²⁺ and Ca²⁺ content were correlated to initial Na⁺ concentration in the medium, not salt type. Accumulation of Na⁺ was accompanied by loss of K⁺ and Mg²⁺. Six to seven times less sulfate was measured in callus grown on Na₂SO₄ than chloride in callus grown on similar concentrations of NaCl.     

Cell Surface Sialoglycoproteins of Cultured Rat Cerebellar Interneurons

Abstract: The sialoglycoproteins of cultures of relatively pure rat cerebellar interneurons were labelled by NaIO₄ oxidation/NaB ³H₄ reduction. The labelled molecules were analysed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate followed by fluorography. Faint labelling could be detected in three components if cells were labelled without any oxidation. In young cultures, oxidation by galactose oxidase alone failed to reveal any additional bands. After oxidation by NaIO₄ or galactose oxidase in the presence of neuraminidase, many more components were labelled. After NaIO₄ oxidation, about 80% of the cell-associated radioactivity could be removed by treating the cells with neuraminidase, which left the cells more than 95% viable. The majority of the bands seen after neuraminidase treatment were substantially reduced when compared with untreated controls, supporting a surface localisation of these molecules. Reproducible developmental changes were seen in the profiles of bands labelled by NaIO₄/NaB ³H₄ in time course studies of cultures up to 8 days in vitro . Some bands became more prominent, and others disappeared. The gel profiles of the neuron cultures were quite distinct from those of cerebellar astrocyte cultures, which contain all the cell types likely to be contaminants of the neuron cultures.     

Cation-stimulated H+ efflux by intact roots of barley

Abstract. Rates of proton extrusion and potassium (⁸⁶Rb) influx by intact roots of barley ( Hordeum vulgare cvs . Fergus, Conquest and Betzes) plants were simultaneously measured in short-term (15min) experiments. The nature and extent of apparent coupling between these ion fluxes was explored by manipulating conditions of temperature, pH and cation composition and concentration during flux determinations. In addition, the influence of salt status upon these fluxes was examined. At low K⁺ concentrations (0.01 to 1 mol m⁻³), H⁺ efflux and K⁺ influx were strongly correlated in both low- and high-K⁺ roots, although K⁺: H⁺ exchange stoichiometries were almost consistently greater than 2:1. At higher concentrations (1 to 5 mol m⁻³), H⁺ efflux was either reduced or remained unchanged while K⁺ influxes increased. In the presence of Na₂SO₄, rates of H⁺ extrusion demonstrated similar cation dependence, although below 10 mol m⁻³ Na₂SO₄, H⁺ fluxes were generally 50% lower than in equivalent concentrations of K₂SO₄. These observations are considered in the context of current hypotheses regarding the mechanisms of k⁺/H⁺ exchange.     

Mechanism of the inhibition of phloem loading by sodium sulfite: Effect of the pollutant on the transmembrane potential difference

Sodium sulfite (Na₂SO₃) decreased uptake from 1 m M sucrose by the parenchyma and by the veins of leaves of broadbean ( Vicia faba L. cv. Aguadulce). The decrease depended on the concentration of the pollutant and the duration of pretreatment. The inhibition was non-competitive. Sulfite affected the transmembrane potential difference (PD) of the leaf tissues. The short-term response obeyed an 'all or nothing' law. At 0.1 m M and above, sulfite led to a quick depolarization of one-third of the initial potential after a lag phase of about 5 min; for concentrations lower than 0.1 m M , sulfite did not affect the potential. By contrast, the long-term effect of Na₂SO₃ on the transmembrane PD strongly depended on its concentration. After 2–12 h of pretreatmemt there was no effect at 10 μ M , a weak effect at 0.1 m M , and then increasing depolarization as the pollutant concentration increased. The inhibitory effect of Na₂SO₃ on sucrose uptake is thus, at least partly, due to its effect on a component of the proton-motive force. ΔΨ. However, the lack of correlation noticed with 0.1 m M Na₂SO₃ between the effect on sucrose uptake and the long-term effect on transmembrane PD suggests numerous sites of sulfite action.     

Protein Synthesis during Photocontrolled Progression of the Cell Cycle in Single-celled Protonemata of the Fern Adiantum Capillus-veneris

Protein synthesis during photoinduced, synchronous progression of the cell cycle in single-celled protonemata of the fern Adiantum capillus-veneris was studied by tracer techniques. Nuclei of the protonemata were labelled with ³H-thymidine during spore germination so that the amount of ³H incorporated into the TCA-insoluble fraction of the cells could be used as a measure of the cell number in each sample. The rate of the incorporation of ¹⁴C-amino acids into TCA-insoluble materials was not significantly varied at different stages of the cell cycle or by treatment with blue light. Extracts of cells labelled with ³⁵S-methionine at various times after the transfer from red light condition (G₀) to darkness (G₁ to S) were analyzed by two-dimensional gel electrophoresis. At least 3 of about 200 spots showed significant changes in intensity on fluorograms. Spot A (molecular weight 20,000, isoelectric point 6.3) was detectable only in early G₁, whereas spot B (molecular weight 19,500, isoelectric point 6.3) was found only in the late G₁ and S phases. When the cells were exposed to blue light before the dark incubation, the times of disappearance of spot A and appearance of spot B were advanced depending upon the progression of the cell cycle but not upon the clock time.     

STUDY OF RNA IN SUBCELLULAR FRACTIONS FROM RAT BRAIN: SIMULTANEOUS INCORPORATION OF [14C]URIDINE AND [3H]METHYL-l-METHIONINE

Abstract— By using a combination of subcutaneous and intraventricular injections of [¹⁴C]uridine and [³H]methyl- l -methionine we have obtained maximum incorporation in about 40 min of both radioactive precursors into nuclear RNA from rat brain. In this nuclear fraction we found at least two different types of RNA that were rapidly labelled. One of them incorporated both [¹⁴C]uridine and [³H]methyl groups and seemed to correspond to species of rRNA and their precursors. The other RNA fraction was less methylated or non-methylated and exhibited sedimentation coefficients distributed along a continuous 8–30 % sucrose density gradient. At least part of the latter type of RNA very probably was mRNA, but much of it must conespond to a different RNA similar to that recently described in HeLa cells by P enman , V esco and P enman (1968).
We also found that labelled 185 and 285 rRNA components began leaving the nucleus for the cytoplasm within 24 to 33 min after the radioactive precursors had been injected, and, in the cytoplasmic fraction, the patterns of incorporation for [¹⁴C]uridine and [³H]-methyl groups were similar for the 18S and 28S rRNA components. We estimate that in this fraction of rat brain the 18S rRNA component was 1·4 times more methylated than the 28S component. We also detected a lower sedimentation coefficient for the non- or slightly methylated, species of soluble RNA found in the cytoplasmic fraction.     

Maintenance of the Adult Rat Superior Cervical Ganglion In Vitro: Comparison of Organ and Explant Culture Systems

Abstract: Concentrations of selected intermediates of energy metabolism whole rat superior cervical ganglia maintained in vitro by an organ culture technique were compared with values measured in small slices of this maintained under essentially the same conditions. Rates of incorporation [³H]leucine into trichloroacetic acid-precipitable material in whole ganglia mained constant for at least 48 h: however, the oxidation-reduction state tissue as indexed by (NAD):(NADH) ratios calculated from measured amounts of lactate and pyruvate decreased more than 50% within ³h in vitro . Ganglion explants prepared by cutting the tissue into ³⁰⁰-pm transverse sections played (NAD):(NADH) ratios that were about three times greater than noted in whole ganglia maintained in vitro for the same period of time. explants contained significantly higher concentrations of pyruvate and α-ketoglutarate than whole ganglia maintained in culture. Maintenance of vorable metabolic state may support the extensive growth of neurites seen explant cultures of superior cervical ganglia. Outgrowth of processes containing catecholamines could be detected readily in explant cultures of ganglia adult rats; however, this was somewhat slower and less consistent than growth observed in explants from neonatal rats. Outgrowth of neurites adult ganglia was minimal without the addition of Nerve Growth Factor.     

THE EFFECT OF DIABETES, INSULIN AND WALLERIAN DEGENERATION ON LEUCINE METABOLISM OF ISOLATED RAT SCIATIC NERVES

Abstract– ¹⁴CO₂ production and ¹⁴C incorporation into proteins was studied in isolated rat sciatic nerves during incubation with 0.1 mM-[1-¹⁴C]leucine. Rats were made diabetic with streptozotocin. Nerves from diabetic rats incubated with glucose oxidized more [¹⁴C]leucine than controls. This difference was abolished in the presence of insulin (1 mU/ml). The effects of diabetes and insulin on leucine oxidation could not be demonstrated in the absence of glucose. Insulin stimulated the incorporation of [¹⁴C] from leucine into proteins by nerves from controls and diabetic rats.
Nerves undergoing Wallerian degeneration showed a marked increase in DNA content and stimulated incorporation of [¹⁴C]leucine into proteins. ¹⁴CO₂ production from leucine proceeded at 75% of the rate observed in intact nerves. Neither insulin nor diabetes affected leucine metabolism in degenerating nerves.
Neither the extracellular space nor the concentration of free amino acids were significantly different in nerves obtained from control and diabetic rats, except for lower glutamine content in the latter.
In vitro leucine metabolism of nerves is affected by diabetes, insulin and the integrity of the axon. The Schwann cell is suggested as a possible site of the observed changes in leucine metabolism.     

Influence of Growth Hormone and Thyroxine On Cell Kinetics In the Proximal Tibial Growth Plate of Snell Dwarf Mice

Abstract. In Snell dwarf mice, the influence of short-term treatment with human growth hormone (hGH) or thyroxine on the proliferative and sulphation activity of the proximal tibial growth plate was studied. By autoradiographic methods, the [³H]methylthymidine incorporation after a single injection was measured, after 2 hr incorporation time. the labelling index was calculated and the number of labelled mitoses was counted. In addition, the distribution of the labelled nuclei over the proliferating and degenerating zones was determined by continuous labelling for 25 and 73 hr.
In untreated dwarf mice after [³H]-methylthymidine administration, the number of labelled nuclei in the growth plate is low. Labelling occurs, as expected, mainly in the cells of the proliferative zones. the number of labelled nuclei in control dwarf mice was similar after 25 and 73 hr continuous labelling. This suggests that many cells are in a resting G_o or prolonged G₁ phase. Both hGH and T₄ treatment induce a significant increase of the number of labelled nuclei per growth plate and of the number of mitoses. Since hormonal treatment induces a small number of mitoses after 2 hr incorporation of the label, the minimal G₂ phase of the cell cycle is less than 2 hr. In addition, treatment with hGH and T₄ stimulates chondrocytes in the zone of proliferative and hypertrophic cells to actively incorporate [³⁵S]-sulphate.     

Gas exchange and growth responses of ectomycorrhizal Picea mariana, Picea glauca, and Pinus banksiana seedlings to NaCl and Na2SO4

Abstract: Black spruce (Picea mariana), white spruce (Picea glauca), and jack pine (Pinus banksiana) seedlings were inoculated with Hebeloma crustuliniforme or Laccaria bicolor and subjected to NaCl and Na₂SO₄ treatments. The effects of ectomycorrhizas on salt uptake, growth, gas exchange, and needle necrosis varied depending on the tree and fungal species. In jack pine seedlings, ectomycorrhizal (ECM) fungi reduced shoot and root dry weights and in the ECM white spruce, there was a small increase in dry weights. Sodium chloride treatment reduced net photosynthesis and transpiration rates in the three studied tree species. However, NaCl-treated black spruce and jack pine colonized by H. crustuliniforme maintained relatively high photosynthetic and transpiration rates and needle necrosis of NaCl-treated black spruce seedlings was reduced by the ECM fungi. Higher concentrations of Na+ were found in shoots compared with roots of the three examined conifer species. ECM fungi reduced the concentrations of Na+ mainly in the shoots and this reduction was greater in plants treated with NaCl compared with Na₂SO₄. Shoots contained generally higher concentrations of Cl^- compared with roots. In the NaCl-treated black spruce and white spruce, both ECM species significantly reduced Cl^- concentrations. Our results point to overall greater phytotoxicity of NaCl compared with Na₂SO₄ and support our earlier findings which demonstrated beneficial effects of ECM fungi for woody plants exposed to NaCl stress.     

Metabolism of 14C-aspartate during shoot bud formation in cultured cotyledon explants of radiata pine

Aspartate metabolism was investigated in excised cotyledons of radiata pine ( Pinus radiata D. Don). These cotyledons were cultured under shoot-forming (plus N⁶-benzyladenine, SF), non-shoot-forming (minus N⁶-benzyladenine, NSF) and unresponsive (plus N⁶-benzyladenine, OLD) conditions, then incubated with [¹⁴C]-aspartate for 3-h pulse treatments followed by 3-h chase treatments with cold aspartate. The majority of label was recovered in the CO₂, amino acid, organic acid and pellet fractions. Uptake was greatest in all tissue types early in culture. Most (over 80%) of the [¹⁴C]-aspartate taken up by the tissues was converted to CO₂ at day 0 in SF and NSF tissues, CO₂ accounted for less than 50% of the total radioactivity in other tissues. Greater incorporation into fractions was observed in SF tissues during promeristemoid formation, while in NSF tissues the greatest incorporation was observed during a period of rapid elongation. Generally, less incorporation was observed in OLD cotyledons than in SF and NSF cotyledons. Analysis of the amino acid fraction showed that labelled aspartate was converted to other amino acids, mainly glutamate, glutamine, asparagine and 4-aminobutyric acid.     

The Role of Calcium in Mediating Smut Disease Severity and Salt Tolerance in Corn under Chloride and Sulphate Salinity

The effect of sahne irrigation water containing NaCl or Na₂SO₄ with and without CaSO₄ application on the gram yield, smut index and plant nutrient contents of two intermediate smut-susceptible corn cultivars was investigated in cemented plots containing sandy loam soil. The plants were artificially infected and the disease severity was rated. At the end of silking stage, the leaf C1, Na and Ca contents were detected and Ca/Na ratio was calculated. At harvest, the grain yield of smut-free and smutted plants was recorded. The results showed that Na₂SO₄-treated plants had a greater yield advantage than those exposed to NaCl-treatments. The inclusion of CaSO₄ was beneficial to both salinity types and improved the yield potential. Similarly, smut susceptibility was reduced by 10.8% for the Na₂SO₄-stressed plants, compared with 22.7% for NaCl treatment. The comparable reductions in disease severity associated with Ca supply dropped to 33.0 and 17.0%, respectively, indicating the beneficial contribution of a Ca supply on the mediating disease reaction. Although the smut index was markedly decreased as the leaf C1 content increased in Ca-deficient plants subjected to saline irrigation water without Ca supply, the increase in the Ca/Na ratio in plant tissue, associated with supplemental CaSO₄ application to each salinity type, appeared to have an additive effect on smut disease control and salt tolerance.     

The Effects of Electroshock Convulsions on Calcium Transport within Synaptic Terminals

Abstract: Calcium transport was assessed within synaptic terminals isolated from cerebral cortices of rats which experienced one maximal electroshock (ES) convulsion daily. No significant change in calcium content [(Ca₁)] of synaptosomes was present after 2 consecutive days of maximal convulsions. After 4 and 6 days of maximal seizures, (Ca₁) rose 20% and 37%, respectively. ¹⁵Ca²⁺ influx within synaptosomes in vitro increased after 6 days of ES convulsions (1.94 ± 0.4 μmol/g protein/min in ES convulsions versus 1.54 ± .03 μmol/g protein/min in controls). The higher rate of ⁴⁵Ca²⁺ influx in convulsed animals was accounted for by elevated internal sodium [(Na₁)] values. Maximal ⁴⁵Ca²⁺ efflux decreased after ES convulsions (0.48 μmol/g protei/min in ES convulsions versus 0.8 μmol/g protei/min in controls). The slower rate of ⁴⁵Ca²⁺ efflux after convulsions was also accounted for by elevated (Na₁). Our results suggest that (Ca₁) increased within synapses after in vivo ES convulsions secondary to a primary ionic event, namely, elevated (Na₁).     

Untersuchungen über den Stickstoffstoffwechsel von Pansenprotozoen (Entodinium) mittels 14C-Karbonatinkorporation

SYNOPSIS. Isolated rumen protozoan species (Entodinium) were incubated in vitro with starch, antibiotics and Na₂¹⁴CO₃. ¹⁴C-carbonate was incorporated into amino acids of the Entodinium protein. Arginine, aspartic acid, alanine, and glutamic acid had the highest specific activities. Carbonate was incorporated into the TCA-soluble fraction, into cell polysaccharides, and into ether and acetone extracts of TCA-precipitates.     

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [¹⁴C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[¹⁴C]Tyrosine was released from assembled and non-assembled [¹⁴C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[¹⁴C]tyrosine preparation in the presence of CaCl₂ at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [¹⁴C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[³H]Phenylalanine was released from a preparation of tubulinyl-[³H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[¹⁴C]tyrosine and tubulinyl-[³H]phenylalanine was partially assembled, the ratio of ¹⁴C/³H found in the microtubules was the same as in the non-assembled tubulin fraction.     

Effects of Monensin on Assembly of Po Protein into Peripheral Nerve Myelin

Abstract: The ionophore monensin has been used in a variety of systems to block secretion of glycoproteins or assembly of glycoproteins into membranes. We examined the effects of monensin on assembly of the Po glycoprotein into PNS myelin, and compared this agent with the glycosylation inhibitor tunicamycin in our system. Sciatic nerves from 9-day-old rat pups were sliced and incubated in vitro . Electron microscopy of the Schwann cells in slices incubated with monensin revealed extensive swelling of the Golgi complex. Incubation with 10⁻⁷ M monensin inhibited total protein synthesis by about 20% and fucose incorporation into protein about 35%. Following isolation of myelin, proteins were separated by sodium dodecyl sulfate gel electrophoresis. Monensin inhibited the appearance of Po in myelin, while causing its accumulation in a denser membrane fraction. In addition, a slightly faster-migrating species of P_o labeled with both [³H]fucose and [¹⁴C]glycine was observed in all fractions. Assembly of basic proteins into myelin was not affected. Preincubation with 10 μg/ml tunicamycin for 30 min prior to incubation with [³H]fucose and [¹⁴C]glycine for 2 h resulted in a 65% decrease in [³H]fucose incorporation into P_o, and the appearance of a new [¹⁴C]glycine-labeled peak that migrated in the region of the 23K protein reported by Smith and Sternberger. [³H]Fucose incorporation was inhibited earlier, and to a greater extent, than protein synthesis. Our results show that processing of the P_o glycoprotein is sensitive to both monensin and tunicamycin, and that monensin partially blocks assembly of P_o into myelin.     

EFFECT OF DISSOLVED OXYGEN PARTIAL PRESSURE ON THE ACCUMULATION OF ASTAXANTHIN IN CHEMOSTAT CULTURES OF HAEMATOCOCCUS LACUSTRIS (CHLOROPHYTA)

The effect of dissolved oxygen partial pressure on the accumulation of astaxanthin in the green alga Haematococcus lacustris ( Gir.) Rostaf (UTEX16) was studied in N-limited continuous chemostat cultures. The steady-state astaxanthin content measured against culture volume, cell number, and biomass dry weigh of Haematococcus cultures was proportional to the dissolved O₂ partial pressure in the culture medium, over the range of 0–50% O₂ The steady-state biomass dry weight concentrations remained at between 0.52 and 0.57 g. L^-1 over the range of dissolved O₂ partial pressure studied. Steady-state cell densities at dissolved O₂ partial pressures above the air saturation level (1.13–1.58 × 10⁵ cells.mL^-1) were about half of that measured at lower dissolved O₂ partial pressures (2.42–2.63 × 10⁵ cells.mL^-1). Both biflagellated zoospores and nonmotile aplanospores were found at steady state. The fraction of nonmotile cells was higher at dissolved O₂ partial pressures above the air saturation level (94.44–98.01%) than at dissolved O₂ partial pressure below the air level (79.64–86.12 and 91.75% ).     

TURNOVER OF PROTEIN PHOSPHORUS IN RESPIRING SLICES OF GUINEA PIG CEREBRAL CORTEX: CELLULAR LOCALIZATION OF PHOSPHOPROTEIN SENSITIVE TO ELECTRICAL STIMULATION

Abstract— In experiments designed to localize the increased turnover of phosphoprotein-P which occurs in respiring brain slices as a result of electrical stimulation, a cell separation procedure was used to prepare a fraction enriched in neuronal cell bodies from incubated slices labelled with [³²P]phosphate. Labelled phosphoprotein was found to be twice as concentrated in the neuron-enriched fraction as in other fractions. Electrical stimulation for 10 s increased the rate of incorporation of [³²P]phosphate into phosphoproteins in the neuron-enriched fraction by 25 per cent ( P < 0.05), but had no effect on incorporation into a partially purified glial fraction contaminated with neuropil and cell debris.     

Effect of some factors on determination of viable microbial cells by peroxyoxalate chemiluminescence method

The effects of physical and chemical factors on the production of H₂O₂ from Escherichia coli cells were studied. When 20 mmol 1^-1 Tris-HCl buffer was used for this purpose the electron transport system (ETS) showed the highest activity at pH 7.6-8.2. KCN promoted the production of H₂O₂ from E. coli cells, and the optimum concentration was changed in different reaction times and pH values. Glucose, 5 mg ml^-1, increased the ETS activity about twofold. The other substrates and surfactants did not increase the chemiluminescence intensity. NaNO₂ and Na₂SO₄ in inorganic salts significantly reduced the ETS activity above 70%. In addition, the optimum temperature for the production of H₂O₂ was 30°C in this study. When glucose (5 mg ml^-1) and KCN (0.2 mmol 1^-1) were added to the reaction buffer containing 0.5 mmol 1^-1 menadione, the detectable minimum cell densities (averages of triplicate assay) of E. coli, Enterobacter cloacae and Serratia marcescens were 5 times 10³ cells ml^-1, 10⁴ cells ml^-1 and 10⁴ cells ml^-1 respectively.