Cyanide Sensitivity Research Articles

Interaction of phenols with δ9 terminal desaturase and other cyanide-sensitive factors in chicken liver microsomes

p-Cresol stimulates the oxidation of reduced cytochrome b 5 in chick embryo liver microsomes, which lack stearoyl-CoA desaturase activity, as well as in hepatic microsomes from neonatal chicks and fasted-refed adult chickens, both of which have greatly increased stearoyl-CoA desaturase activity. The K m value of p-cresol for the interaction in chick embryo microsomes is 2–4 mM, whereas that in adult liver microsomes is only 6 μM; the V value for p-cresol in the adult is 2-fold higher than that in chick embryo. During neonatal development the shift in the K m value of p-cresol from high to low occurs concomitantly with the formation of stearoyl-CoA desaturase activity in newly hatched chicks. Antibodies to Δ 9 desaturase completely inhibit stearoyl-CoA desaturation but inhibit only 50% of the p-cresol-stimulated oxidation of cytochrome b 5 in hepatic microsomes from adult chickens, indicating that the p-cresol effect in adult chicken microsomes is partly mediated by Δ 9 terminal desaturase. In chick embryo liver microsomes antibodies to Δ 9 desaturase do not inhibit p-cresol-dependent oxidation of cytochrome b 5. In chick embryo and adult microsomes p-cresol is converted to a more polar product whose formation is dependent on NADH, microsomes and is inhibited by KCN. The results support the conclusion that p-cresol interacts with several microsomal cyanide-sensitive factors including Δ 9 terminal desaturase. On the basis of cyanide sensitivity of the interaction of various phenols with hepatic microsomes, phenols can be classified into three groups; cyanide-sensitive phenols (e.g., p-cresol) which interact exclusively with cyanide-sensitive factors; cyanide-insensitive phenols (e.g., p-aminophenol, hydroquinone and catechol) which interact directly with cytochrome b 5 and partly cyanide-sensitive phenols (e.g., phenol) which interact with both cytochrome b 5 and cyanide-sensitive factors.

Stimulation of Tobacco Pollen Embryogenesis by Anaerobic Treatments

Summary Effects of various anaerobic environments surrounding tobacco anthers on pollen embryogenesis were studied. A short term (30 OR 60 min) treatment with 100% N2 stream at 1 bar had a stimulatory effect on the rate of anthers producing plantlets as well as on the number of plantlets formed per anther. The treatment with N2 containing 2.5 and 5% O2 also had a clear stimulatory effect on plantlet formation. The treatment with N2 containing 10% O2, however, gave a similar result as the control (21% O2). When the atmospheric pressure surrounding tobacco anthers was lowered, the cyanide sensitivity of pollen respiration increased. Possible roles of anaerobic pressure on tobacco pollen embryogenesis were discussed.

Involvement of cytochrome b5 and a cyanide-sensitive monooxygenase in the 4-demethylation of 4,4-dimethyl-zymosterol by yeast microsomes

According to Ohba et al. (Ohba M., Sato R., Yoshida Y., Nishino T. and Katsuki H. (1978) Biochem. Biophys. Res. Commun. 85, 21–27), yeast microsomes catalyze the removal of three methyl groups attached to the C-4 and C-14 positions of [1,7,15,22,26,30- 14C]lanosterol (4,4,14α-trimethyl-5α-cholesta-8, 24-dien-3β-ol) in the presence of NADPH, NAD + and molecular oxygen, concomitant with the liberation of 14CO 2 derived from C-30 (one of the two methyl groups at the C-4 position). In this process the methyl group at the C-14 position is first removed in a cyanide-insensitive reaction and then the two methyl groups at the C-4 position are removed by a cyanide-sensitive enzyme system. In this study it was found that the 14CO 2 formation from the 14C-labeled lanosterol was inhibited by antibodies to yeast cytochrome b 5 and by palmitoyl-CoA, a substrate of the cytochrome b 5-containing fatty acyl-CoA desaturase system of yeast microsomes. However, neither the antibodies nor palmitoyl-CoA inhibited the conversion of lanosterol to 4,4-dimethyl zymosterol (4,4-dimethyl-5α-cholesta-8,24-dien-3β-ol). It is concluded that cytochrome b 5 and a cyanide-sensitive enzyme are involved in the 4-demethylation of 4,4-dimethylzymosterol, but not the 14α-demethylation of lanosterol, by yeast microsomes. It is suggested that a cyanide-sensitive enzyme acts as the terminal 4-demethylase and cytochrome b 5 transfers reducing equivalents from NADPH to the terminal enzyme, as in the case of fatty acyl-CoA desaturation. The cyanide sensitivity of the 4-demethylation was, however, much greater than that of the desaturation.

Oxidized forms of ovine erythrocyte glutathione peroxidase cyanide inhibition of a 4-glutathione:4-selenoenzyme

[ 75Se]Glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) containing 4 mol selenium per mol was isolated in 33% yield using 10% ethanol to stabilize the purified enzyme. When reduced with GSH and rapidly separated from GSH by gel filtration chromatography, GSH peroxidase was eluted in a labile oxidized (iodiacetate-insensitive) form which was stable at 4°C but unstable at 25°C (form A). When GSH-reduced enzyme was allowed to oxidize in the course of dialysis a more stable oxidized form was obtained to oxidize in the course of dialysis a more stable oxidized form was obtained (form C) which was rapidly inactivated by cyanide. Using [ 35S]GSH, form C was shown to contain tightly bound glutathione in approx. equimolar ratio with selenium. The cyanide sensitivity of GSH peroxidase is therefore correlated with the presence of a glutathione moiety in the enzyme. The isolation of GSH peroxidase containing bound glutathione suggests that intermediates containing glutathione bound to selenium may be formed during the catalytic cycle.

Heat-induced changes in respiratory pathways and mitochondrial structure during microcycle conidiation of Neurospora crassa.

Changes in both respiratory pathways and mitochondrial structure of Neurospora crassa occurred under conditions of microcycle conidiation. Upon heat-treatment at 46 degrees C, conidia developed a highly cyanide-insensitive, hydroxamate-sensitive respiration associated with morphological alterations in mitochondrial membranes; such changes were time-dependent. When heat-treated conidia were shifted down to 25 degrees C, the alternate, hydroxamate-sensitive respiration decreased significantly, paralleling the recovery of well-cristated mitochondria with an electron-dense matrix in the germ tubes. The decrease in hydroxamate-sensitivity was associated with two periods of increase in cyanide sensitivity corresponding to the events of germination and precocious proconidial budding.

Hydrogen Cyanide Sensitivity in Bacterial Pathogens of Cyanogenic and Non-Cyanogenic Plants

Hydrogen Cyanide Sensitivity in Bacterial Pathogens of Cyanogenic and Non-Cyanogenic Plants

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl 1 II. Cyanide-insensitive respiration. Function and regulation

1. The cyanide-insensitive respiration in Paramecium tetraurelia was found to be located in mitochondria. 2. Sensitivity of the mitochondrial respiration to cyanide depended on growth conditions. Under standard conditions of growth, 15–20% of respiration was insensitive to 1 mM cyanide. Full resistance to 1 mM cyanide was observed by growing cells in the presence of erythromycin (100–400 μg/ml) or 0.2 mM cyanide. The mitochondrial respiration of the mutant Cl 1 harvested during the exponential phase of growth was largely insensitive to cyanide (more than 80%). 3. Pyruvate was oxidized at the same rate by wild type mitochondria and mitochondria of the mutant Cl 1. In contrast, succinate oxidation was 2–3 times faster in mitochondria of the mutant Cl 1 than in wild type mitochondria. 4. The cyanide-insensitive respiration was inhibited by 1 mM salicyl-hydroxamic acid to nearly 100%. Other efficient respiratory inhibitors included amytal and heptylhydroxyquinoline. Antimycin was not inhibitory even at concentrations as high as 5 μg/mg protein, a finding consistent with the lack of antimycin binding sites. 5. The K m O 2 was less than 2 μM for the cyanide-sensitive respiration and 4–5 μM for the cyanide-insensitive respiration. 6. ADP stimulated the oxidation of pyruvate and succinate by mitochondria of the wild type and the mutant Cl 1 by 2–4-fold. AMP selectively stimulated the cyanide-insensitive respiration. Preincubation of Paramecium mitochondria with trace of ADP increased their cyanide sensitivity. This effect of ADP was prevented by uncouplers. 7. P O ratios in wild type mitochondria were close to 2 for pyruvate oxidation, between 1 and 2 for succinate oxidation and between 0.5 and 1 for ascorbate oxidation. In mitochondria of the mutant Cl 1, P O ratios for pyruvate and succinate oxidation were lower by about one third in comparison to those of wild type mitochondria. Addition of cyanide decreased P O ratios with pyruvate and succinate as substrates for both types of mitochondria (30% for the mutant, 40–70% for the wild type). Ascorbate oxidation by mitochondria of the mutant was not coupled to ATP synthesis and did not generate a membrane potential. 8. H + efflux dependent on O 2 pulses was measured in the presence of increasing concentrations of cyanide. Using succinate as substrate, the H + O ratio was 4 for wild type mitochondria and 1.5 for mitochondria of the mutant. The curve relating H + efflux to added cyanide was biphasic in both cases. There was a rapid drop of H + O to 30–50% of the original value when cyanide was added to 0.1 mM. Upon further addition of cyanide up to 1 mM, the H + O ratio decreased slowly towards zero. 9. Under steady-state conditions of respiration, the percentage of oxidation of the respiratory carriers increased from ubiquinone to cytochrome aa 3, except for the b-type cytochromes which remained more oxidized than cytochromes cc 1. 10. The CO-inhibited respiration in mitochondria of the mutant Cl 1 was partially photorestored by white light. As the only CO-reactive pigment in these mitochondria is a b-type cytochrome, the photorestoration data suggest that the CO-reactive cytochrome b may be autooxidizable. 11. The above data suggest that the respiratory chain of Paramecium mitochondria possesses three terminal oxidases, namely cytochrome aa 3 (absent in mitochondria of the mutant Cl 1), a CO-sensitive b-type cytochrome and a salicylic hydroxamic acid-sensitive oxidase.

Characteristics of proline transport in normal and starved cells of Candida albicans

1. 1. There was no apparent correlation between the rate of respiration and rate of accumulation of proline in Candida albicans cells. 2. 2. In contrast to normal cells, the respiration in the starved cells became completely cyanide insensitive. The starvation of cells in the presence of cycloheximide prevented the cells from becoming cyanide insensitive. The addition of Fe(III), however, accelerated the process. 3. 3. Oxidizable substrates e.g. NADH, acetate and glucose, when added to cyanide-insensitive starved cells, exhibited 40–280% stimulation in respiration rate. However, this enhancement in oxidation by various substrates was not coupled to a simultaneous increase in the proline uptake or in intracellular ATP levels. 4. 4. There was 6-fold stimulation in proline uptake when cyanide-insensitive cells were preincubated with 50 mM glucose. The preincubation of starved cells resulted in a partial restoration of cyanide sensitivity and increased intracellular ATP levels. The preincubation of starved cells with other oxidizable substrates resulted in a partial restoration of cyanide sensitivity but had no stimulatory effect on intracellular ATP levels and proline accumulation. 5. 5. Both the enhanced uptake and ATP levels in glucose preincubated cells were found to be completely abolished by iodoacetate. 6. 6. It is proposed that the increased proline uptake in cells preincubated with glucose was mainly due to the production of glycolytic energy.

The relationship between dissolved oxygen concentration, ATP and embryogenesis in carrot ( Daucus carota) tissue cultures

Changing the level of dissolved oxygen in cell suspension cultures of carrot tissue to below a critical level led to an increase in both the cyanide sensitivity of respiration (CN - sensitivity) and the cellular concentration of adenosine-5′-triphosphate (ATP). Simultaneously the onset of adventive ambryogenesis was observed. Alternatively the level of ATP in the cells could be raised by adding adenosine to the medium. This led to a sharper rise in the level of cellular ATP but a fall in the CN - sensitivity of respiration. The ATP level rose to a maximum of approx. 1.8 μg/mg dry wt. Later the ATP concentration decreased accompanied by a rise in CN - sensitivity of respiration whether adenosine remained in the medium or not. Both dissolved oxygen and adenosine treatments produced more adventive embryogenesis than in control cultures. There was a relationship between increasing adenosine concentrations supplied in the medium and increased embryogenesis up to an optimum concentration of approx. 30 μg/ml of culture. Above this figure a relationship still held but the yield of embryoids was reduced.

Cyanide-resistant electron transport in sporulating Bacillus megaterium KM

The NADH oxidase activity of stage V mother-cell membranes, isolated from sporulating Bacillus megaterium KM, shows a greater inhibition by cyanide and displays this response at lower concentrations of cyanide than the stage V forespore inner membrane. Comparison of the effects of various respiratory inhibitors reveals that the difference in cyanide sensitivity between these membranes is located on the oxidase side of the 2-heptyl-4-hydroxyquinoline N-oxide-sensitive step. Both membranes contain cytochromes a+ a 3, b-562, b-555, c and d, with three potential oxidases: cytochromes a+ a 3, o and d. Cyanide difference spectra suggest that cytochromes b-562 and d may be the components involved in the cyanide-resistant electron transport pathway. Membrane ascorbate- N, N, N′, N′-tetramethylphenylenediamine and ascorbate 2,6-dichlorophenolindophenol oxidase activities are highly sensitive to cyanide. Evidence is presented for terminal branching of the respiratory chain with branches differing in cyanide sensitivity. The cyanide sensitivity of the NADH oxidase of membranes prepared from various stages of sporulation is compared. Morphogenesis of the mother-cell plasma membrane to a cyanide-sensitive form during stages II and III of sporulation is postulated.

Cyanide sensitivity and induction of the microsomal oleoyl-CoA desaturase of potato tuber

1. 1.The properties of oleoyl-CoA desaturase, induced in microsomal fractions by the ‘ageing’ treatment of potato tuber slices (aeration of slices for 3–18 h), were investigated to study the effect of cyanide on desaturation and of cycloheximide on the induction of the desaturase. 2. 2. The electrons needed for the desaturation can be supplied either by NADH or NADPH, but ascorbate can also drive the reaction; experiments with CO suggested that cytochrome P-450 was not involved in the desaturation. 3. 3. A strong inhibition of the desaturation by potassium cyanide was observed with each of the electron donors; total inhibition was noticed with 1 mM KCN; low concentrations (0.1 mM) caused 50% inhibition of the desaturation. 4. 5. The variation of the oleoyl-CoA desaturase activity during the ageing process and the drop in this activity in aged slices treated by cycloheximide indicate that the enzyme undergoes an active turnover.

Activities of superoxide dismutase and glutathione peroxidase in the red cells of Japanese acatalasemia blood.

The activities of superoxide dismutase and glutathione peroxidase in the red cells of normal and acatalasemia bloods were compared. Superoxide dismutase activity in the red cells of Japanese acatalasemia blood was higher than in normal control, while glutathione peroxidase activity remained within normal limits. Cyanide sensitivity of superoxide dismutase in the red cells of acatalasemia blood was similar to that in normal control.

Superoxide dismutases in mitochondria from Helianthls tuberosus and Neurospora crassa.

Superoxide dismutase has been studied in a wide range of organisms (Fridovich, 1975). Early observations suggested that superoxide dismutases isolated from eukaryotes (McCord & Fridovich, 1969; Misra & Fridovich, 1972; Beauchamp & Fridovich, 1973) showed little diversity in their properties, having mol.wts. near 32000 and containing two copper and two zinc atoms per molecule. The activity of these enzymes was inhibited by cyanide. In contrast, two different cyanide-insensitive enzymes could be isolated from prokaryotes, e.g. Escherichia coli, which contained either two manganese atoms or one iron atom per molecule (Keele et al., 1970; Yost & Fridovich, 1973). From these original observations it would appear that there was a distinction between thesuperoxide dismutases in eukaryotes and prokaryotes. However, more recent observations suggest that this may not be correct (Puget & Michelson, 1974; Lumsden & Hall, 1975). Weisiger & Fridovich (19736) have reported that mitochondria isolated from chicken liver contained both manganese and cupro-zinc superoxide dismutases. The manganese enzyme was located in the matrix space and the cupro-zinc enzyme in the intermembrane space. A similar spatial distribution of these two enzymes has been reported in mitochondria isolated from rat liver by Peeters-Joris et al. (1975). Misra & Fridovich (1972) found that soluble extracts of Neurospora crassa contained only the cupro-zinc enzyme; however, the isolation procedure used by these workers involved extraction with an ethanol/chloroform mixture, which is known to inactivate the manganese enzyme (Weisiger & Fridovich, 1973~). The distribution of superoxide dismutase within the mitochondrion is of interest with respect to the endosymbiotic theory of mitochondrial evolution (Fridovich, 1975; Lumsden & Hall, 1975). Tyler (1975) reported that superoxide ions, formed during respiratory-chain activity, inhibited NADH oxidation in heart muscle submitochondrial particles and concluded that mitochondrial superoxide dismutase protected the NADH dehydrogenase against the harmful effects of superoxide ions. Superoxide dismutase may have a similar role in plant and fungal mitochondria, although there have been no reports of superoxide dismutase distribution and cyanide sensitivity in plant mitochondria. Misra & Fridovich (1972) reported a cupro-zinc superoxide dismutase in soluble extracts of N . crassu hyphae, but did not isolate mitochondria from this organism. In the present work we have studied the localization of superoxide dismutase in mitochondria isolated from Jerusalem artichoke (Helianthus tuberosus) and N. crassa. We provide evidence that in both these types of mitochondria the matrix contains a cyanide-insensitive enzyme (probably manganese dismutase) and the intermembrane space contains a cyanide-sensitive enzyme (cupro-zinc superoxide dismutase). Mitochondria were isolated from tubers of Jerusalem artichoke by the method of Palmer & Arron (1976) and from hyphae of N. crassa, which had been grown in submerged culture for 24h, by a modification of the method of Hall & Baltscheffsky (1968). The inner and outer membranes of the mitochondria were separated by osmotic swelling and contraction and then differential centrifugation as described by Moreau & Lance (1972). The different types of superoxide dismutase were separated and assayed on polyacrylamide gels by the method of Beauchamp & Fridovich (1971). The mitochondria from Jerusalem artichoke gave gels with three bands of superoxide dismutase activity (Fig. l), one of which was insensitive to cyanide and the remainder were sensitive. The cyanide-insensitive enzyme activity appeared to be confined to the matrix, whereas the cyanide-sensitive enzyme activities appeared to be

Manganese-dependent NADPH oxidation by granulocyte particles. The role of superoxide and the nonphysiological nature of the manganese requirement.

Recent work has indicated that superoxide is involved in the manganese-stimulated oxidation of NADPH by crude granule preparations of guinea pig neutrophils. The characteristics of a model manganese-requiring NADPH-oxidizing system that employs a defined O2-generator have now been compared to the original neutrophil-granule system. With respect to pH dependence, cyanide sensitivity, and reduced pyridine nucleotide specificity, the properties of the two systems are very similar. Additional information has been obtained concerning cation specificity and the kinetics of the metal-catalyzed NADPH oxidation. From the similarities between the properties of the model and neutrophil particle systems, we postulate that the manganese-dependent NADPH oxidation observed in the presence of neutrophil granules represents in large part of nonenzymatic free radical chain involving the oxidation of NADPH to NADP, with O2- as both the chain initiator and one of the propagating species. In this reaction, the neutrophil particles serve only as a source of O2-. Further, the same changes in kinetics (decrease in apparent Km for NADPH) observed previously when granules from phagocytizing rather than resting cells were employed could be mimicked by varying the rate of O2-generation by the model system. We conclude from these results that it is unnecessary to invoke a manganese-requiring enzyme as a component of the phagocytically stimulated respiratory system of the neutrophil.

Superoxide dismutase of bovine and frog rod outer segments

Bovine rod outer segments (ROS) contain soluble superoxide dismutase (SOD) which from cyanide sensitivity and electrophoretic mobility appears identical to CuZn SOD of erythrocytes. Enzyme activity of ROS extracts is 200–400 times as much as remainder of retina. Frog ROS also contains a cyanide-sensitive SOD which is not due to erythrocyte contamination since the retina is avascular. SOD in ROS may inhibit free radical oxidation of polyunsaturated fatty acids. In light, high oxygen concentrations in developing retina may activate lipid peroxidation leading to retrolental fibroplasia. High concentrations of ascorbic acid in the retina may act as a protective mechanism against superoxide.

Molar growth yields, respiration and cytochrome patterns of Beneckea natriegens when grown at different medium dissolved-oxygen tensions.

The effect of medium dissolved-oxygen tension on the molar growth yield, respiration and cytochrome content of Beneckea natriegens in chemostat culture (D 0-37 H-1) was examined. The molar growth yield (Y), the specific rate of oxygen (qo2) and glucose consumption, and the specific rate of carbon dioxide evolution were independent of the dissolved-oxygen tension above a critical value (greatest than 2 mmHg). However, the potential respiration rate increased with reduction in the dissolved-oxygen tension at values of the dissolved-oxygen tension well above the critical value. Changes in the cytochrome content occurred at dissolved-oxygen tensions well above the critical value. An increase in cytochrome c relative to cytochrome b was observed as the dissolved-oxygen tension was decreased. Reduction of the dissolved-oxygen tension to less than I mmHg caused a switch to fermentative metabolism shown by the apparent rise in YO2 and decrease in the molar growth yield from glucose. At this point the potential respiration rate (qO2) increased to its highest value, while the cytochrome pattern reverted to that observed at dissolved-oxygen tensions above 96 mmHg. There appeared to be no correlation between cytochrome content, potential qO2, in situ qO2, and cyanide sensitivity of the organism at various dissolved-oxygen tensions.

Effect of nicotinamide adenine dinucleotide on the membrane-associated reduced nicotinamide adenine dinucleotide oxidase of Mycobacterium phlei.

NAD+ had a biphasic effect on the NADH oxidase activity in electron transport particles from Mycobacterium phlei. The oxidase was inhibited competitively by NAD+ at concentrations above 0.05 mM. NAD+ in concentrations from 0.02 to 0.05 mM resulted in maximum stimulation of both NADH oxidation and oxygen uptake with concentrations of substrate both above and below the apparent K-M. Oxygen uptake and cyanide sensitivity indicated that the NAD+ stimulatory effect was linked to the terminal respiratory chain. The stimulatory effect was specific for NAD+. NAD+ was also specific in protecting the oxidase during heating at 50 degrees and against inactivation during storage at 0 degrees. NAD+ glycohydrolase did not affect stimulation nor heat protection of the NADH oxidase activity if the particles were previously preincubated with NAD+. Binding studies revealed that the particles bound approximately 3.6 pmol of [14C1NAD+ per mg of electron transport particle protein. Although bound NAD+ represented only a small fraction of the total added NAD+ necessary for maximal stimulation, removal of the apparently unbound NAD+ by Sephadex chromatography revealed that particles retained the stimulated state for at least 48 hours. Further addition of NAD+ to stimulated washed particles resulted in competitive inhibition of oxidase activity. Desensitization of the oxidase to the stimulatory effect of NAD+ was achieved by heating the particles at 50 degrees for 2 min without appreciable loss of enzymatic activity. Kinetic studies indicated that addition of NADH to electron transport particles prior to preincubation with NAD+ inhibited stimulation. In addition, NADH inhibited binding of [14C]NAD+. The utilization of artificial electron acceptors, which act as a shunt of the respiratory chain at or near the flavoprotein component, indicated that NAD+ acts as at the level of the NADH dehydrogenase at a site other than the catalytic one resulting in a conformational change which causes restoration as well as protection of oxidase activity.

Changes in oxidative metabolism and ultrastructure accompanying differentiation of the mitochondrion in Trypanosoma brucei

A method is described for obtaining optimal growth and morphological transformation of Trypanosoma brucei 792G in a monophasic blood lysate medium starting from populations of bloodstream trypanosomes containing over 90 per cent intermediate and stumpy forms. Transformation was accompanied by: (1) an increase in length of the trypanosomes from 13·8 (± 3·0) μm to 23·0 (± 2·5) μm; (2) an increase in the kinetoplastic index from 1·0 to greater than 2·0; (3) development of the mitochondrion from a single abflagellar canal to a network of subpellicular canals, with outgrowth of the post-kineto-plastic region of the mitochondrion; (4) replacement of some of the tubular mitochondrial cristae by plate-like cristae; (5) the acquisition of succinoxidase, succinate-cytochrome c reductase, and glycerophosphate-cytochrome c reductase activities; (6) a marked increase in proline oxidase and NADH-cytochrome c reductase activities; (7) loss of the surface coat of the flagellate and concomitant reduction in the smooth-membrane systems lying between the nucleus and flagellar pocket; (8) reduction in infectivity of the trypanosomes to the mammalian host. Although growth in primary culture continued up to 100 h, transformation was completed in 48 h. All the respiratory enzyme activities tested were insensitive to cyanide throughout transformation. Division of trypanosomes appeared to be taking place throughout the transformation process. Cyanide sensitivity developed only after subculture of the trypanosomes into a biphasic medium.

Energetics of growth in a tyrothricin-producing strain of Bacillus brevis.

Summary: The particulate NADH oxidase activity of Bacillus brevis is inhibited by tyro-cidine and the kinetics of inhibition indicate that the NADH dehydrogenase region is the primary site of action. A decrease in NADH oxidase activity of electron transport particles and of NADH oxidase activity of whole organisms occurs in batch culture in the later stages of growth when production of tyrothricin increases. That respiration exhibited by growing cells takes place via the respiratory electron-transport system is indicated by the similarities in levels of activity and cyanide sensitivity of endogenous and NADH oxidation of growing cells. Exponential growth on glycerol asparagine media is diphasic, a slower growth rate commencing as the O2 tension of the culture approaches zero and tyrothricin production increases. Molar growth yield data based on overall measurements of growth related to O2 uptake in batch culture lead to low ATP/O ratios. In an attempt to determine Y 0 and ATP/O ratios under optimum conditions of growth a method is described which was based entirely on the growth and respiratory activity of exponentially growing cells. Such measurements led to higher values for Y 0 and to ATP/O ratios approaching 3, indicating the participation of three sites of phosphorylation during exponential growth. Tyrocidine and DNP inhibited growth, the tyrocidine at concentrations similar to those produced at the end of the exponential growth phase. Y 0 /Y ATP ratios (ATP/O ratios) on glycerol asparagine media were lower than on peptone yeast extract medium which yielded no tyrothricin, a possible indication that some form of uncoupling occurs in organisms grown on glycerol asparagine medium which does not yield tyrothricin.

Physiological studies of methane and methanol-oxidizing bacteria: oxidation of C-1 compounds by Methylococcus capsulatus.

Methylococcus capsulatus grows only on methane or methanol as its sole source of carbon and energy. Some amino acids serve as nitrogen sources and are converted to keto acids which accumulate in the culture medium. Cell suspensions oxidize methane, methanol, formaldehyde, and formate to carbon dioxide. Other primary alcohols are oxidized only to the corresponding aldehydes. Oxidation of formate by cell suspensions is more sensitive to inhibition by cyanide than is the oxidation of other one carbon compounds. This is due to the cyanide sensitivity of a soluble nicotinamide adenine dinucleotide-specific formate dehydrogenase. Oxidation of formaldehyde and methanol is catalyzed by a nonspecific primary alcohol dehydrogenase which is activated by ammonium ions and is independent of pyridine nucleotides. Some comparisons are made with a strain of Pseudomonas methanica.