Induction of the d-Amino Acid Oxidase from Trigonopsis variabilis

Induction of the d-amino acid oxidase (EC. 1.4.3.3) from the yeast Trigonopsis variabilis was investigated by using a minimal medium containing glucose as the carbon and energy source, (NH(inf4))(inf2)SO(inf4) as the nitrogen source, and various d- and dl-amino acid derivatives as inducers. The best new inducers found were N-carbamoyl-d-alanine, N-acetyl-d-tryptophan, and N-chloroacetyl-d-(alpha)-aminobutyric acid; when the induction effects of these compounds were compared with the effects of d-alanine as the nitrogen source and inducer, the resulting activities of d-amino acid oxidase per gram of dried yeast were 4.2, 2.1, and 1.5 times higher, respectively. The optimum concentration of the best inducer, N-carbamoyl-d-alanine, was 5 mM. This inducer could also be used in its racemic form. The induction was pH dependent. After cultivation of the yeast in a 50-liter bioreactor, d-amino acid oxidase activity of about 3,850 (mu)kat (231,000 U) was obtained. In addition, production of the d-amino acid oxidase was found to be significantly dependent on the metal salt composition of the medium. Addition of zinc ions was required to obtain high d-amino acid oxidase levels in the cells. The optimum concentration of ZnSO(inf4) was about 140 (mu)M.     

Induction and peroxisomal appearance of gulonolactone oxidase upon clofibrate treatment in mouse liver

Various antihyperlipemic peroxisome proliferators are known to be carcinogenic in rodents but not in human, other primates and guinea pig, which species lost their ability to synthesize ascorbate due to mutations in the gulonolactone oxidase gene. Ascorbate synthesis is accompanied by H2O2 production, consequently its induction can be potentially harmful; therefore, the in vivo effect of the peroxisome proliferator clofibrate was investigated on gulonolactone oxidase expression in mouse liver. Liver weights and peroxisomal protein contents were increased upon clofibrate treatment. Elevated plasma ascorbate concentrations were found in clofibrate-treated mice due to the higher microsomal gulonolactone oxidase activities. Remarkable gulonolactone oxidase activity appeared in the peroxisomal fraction upon the treatment. Increased activity of the enzyme was associated with an elevation of its mRNA level. According to the present results the evolutionary loss of gulonolactone oxidase may contribute to the explanation of the missing carcinogenic effect of peroxisome proliferators in humans.     

Regulation of derepressed synthesis of arylsulfatase by tyramine oxidase in Salmonella typhimurium.

The participation of tyramine oxidase in the regulation of arylsulfatase synthesis in Salmonella typhimurium was studied. Arylsulfatase synthesis was repressed by inorganic sulfate, cysteine, methionine, or taurine. This repression was relieved by tyramine, octopamine, or dopamine, which induced tyramine oxidase synthesis, although the level of arylsulfatase activity was very low. The induction of tyramine oxidase and derepression of arylsulfatase by tyramine were strongly inhibited by glucose and ammonium chloride, and the repression of both enzymes was relieved by use of xylose as a carbon source after consumption of glucose or by use of tyramine as the sole source of nitrogen, irrespective of the carbon source used. The initial rates of tyramine uptake by cells grown with glucose and xylose were similar. Results with tyramine oxidase-constitutive mutants showed that constitutive expression of the tyramine oxidase gene resulted in derepression of arylsulfatase synthesis in the absence of tyramine. Thus, catabolite and ammonium repressions of arylsulfatase synthesis and the induction of the enzyme by tyramine seem to reflect the levels of tyramine oxidase synthesis. These results in S. typhimurium support our previous finding that the specific regulation system of arylsulfatase synthesis by tyramine oxidase is conserved in enteric bacteria.     

Catabolite repression of the formation of precursors of electron transfer complexes III and IV in adapting bakers' yeast: dependence upon a mitochondrially translated repressor.

When bakers' yeast cells which had been grown anaerobically in galactose were aerated in the presence of 10% glucose, they showed a 40% decrease in $\text{in}\text{vivo}$ [¹⁴C]-leucine incorporation into a washed mitochondrial membrane fraction compared with cells which had been aerated in a low glucose medium. The observed catabolite repression of membrane protein synthesis was primarily due to a decrease in cytoplasmic translational activity, but this repression was entirely dependent upon concomitant mitochondrial translation. The inductions of reduced coenzyme Q cytochrome $\text{c}$ reductase (complex III) and of cytochrome $\text{c}$ oxidase (complex IV) activities were repressed 30 and 60%, respectively, by aeration of the cells for 8 hours in 10% glucose. The catabolite repression of the formation of these two inner membrane complexes was again shown to be dependent upon concomitant mitochondrial translation. Both the amino acid incorporation and enzyme induction data suggest that catabolite repression of both cytoplasmically and mitochondrially translated mitochondrial membrane proteins is mediated through a mitochondrially translated repressor.     

STUDIES IN VIVO ON THE RELATIONSHIP BETWEEN BRAIN TRYPTOPHAN, BRAIN 5-HT SYNTHESIS AND HYPERACTIVITY IN RATS TREATED WITH A MONOAMINE OXIDASE INHIBITOR AND L-TRYPTOPHAN

Abstract— The effect of l -tryptophan loading upon the amount of 5-HT accumulating in the brains of rats pretreated with a monoamine oxidase inhibitor was studied. The amount of brain 5-HT accumulated increased with increasing tryptophan dosages and brain tryptophan concentrations up to a tryptophan dose of 120 mg/kg body wt. and a brain tryptophan of about 70 μg/g brain. Above this dose and concentration no further increase in brain 5-HT accumulation occurred. After monoamine oxidase inhibition and tryptophan loading gross hyperactivity and hyperpyrexia occurred. Monoamine oxidase inhibition, tryptophan administration and intact aromatic amino acid decarboxylase activity were all collectively essential for the production of hyperactivity and hyperpyrexia. DL-Parachlorophenyl-alanine prevented both the occurrence of hyperactivity and the increased accumulation of, brain 5-HT. Indices of hyperactivity correlated with the amount of brain 5-HT accumulating in 1 h after tryptophan loading but not with the overall concentration of brain 5-HT, suggesting that hyperactivity was dependent upon the rate of 5-HT synthesis. Reserpine and tetra-benazine pretreatment speeded the onset and rate of development of the hyperactive state without altering the synthesis of brain 5-HT. It is suggested that when monoamine oxidase is inhibited and the rate of 5-HT synthesis is increased, granular uptake and storage of 5-HT and other rate-limiting mechanisms for 5-HT inactivation are unable to prevent 5-HT 'spilling over’to produce hyperactivity. The crucial dependence of 5-HT synthesis upon brain tryptophan concentration and the ability of intraneuronal metabolism, when monoamine oxidase activity is intact, to cope with increased 5-HT synthesis and prevent ‘spillover’, raise the possibility that brain 5-HT synthesis is normally in excess of functional needs, and suggest that intraneuronal metabolism and the intraneuronal organization of 5-HT pools are of more importance than synthesis in regulating the amount of 5-HT available for functional activity.     

Tetramethyl-p-phenylenediamine oxidase reaction in Azotobacter vinelandii.

It was possible to quantitate the tetramethyl-p-phenylenediamine (TMPD) oxidase reaction in Azotobacter vinelandii strain O using turbidimetrically standarized resting cell suspensions. The Q(O2) value obtained for whole cell oxidation of ascorbate-TMPD appeared to reflect the full measure of the high respiratory oxidative capability usually exhibited by this genera of organisms. The Q(O2) value for the TMPD oxidase reaction ranged from 1,700 to 2,000 and this value was equivalent to that obtained for the oxidation of the growth substrate, e.g., acetate. The kinetic analyses for TMPD oxidation by whole cells was similar to that obtained for the "particulate" A. vinelandii electron transport particle, that fraction which TMPD oxidase activity is exclusively associated with. Under the conditions used, there appeared to be no permeability problems; TMPD (reduced by ascorbate) readily penetrated the cell and oxidized at a rate comparable to that of the growth substrate. This, however, was not true for the oxidation of another electron donor, 2,6-dichloroindophenol, whose whole cell Q(O2) values, under comparable conditions, were twofold lower. The TMPD oxidase activity in A. vinelandii whole cells was found to be affected by the physiological growth conditions, and resting cells obtained from cells grown on sucrose, either under nitrogen-fixing conditions or on nitrate as the combined nitrogen source, exhibited low TMPD oxidase rates. Such low TMPD oxidase rates were also noted for chemically induced pleomorphic A. vinelandii cells, which suggests that modified growth conditions can (i) alter the nature of the intracellular terminal oxidase formed (or induced), or (ii) alter surface permeability, depending upon the growth conditions used. Preliminary studies on the quantitative TMPD oxidation reaction in mutant whole cells of both Azotobacter and a well-known Mucor bacilliformis strain AY1, deficient in cytochrome oxidase activity, showed this assay can be very useful for detecting respiratory deficiencies in the metabolism of whole cells.     

Regulation of phenylalanine oxidase synthesis in Proteus mirabilis.

Cells of Proteus mirabilis could oxidize L-phenylalanine to phenylpyruvate only when grown in the presence of a number of amino acids, particularly, L-alanine, L-asparagine, L-glutamate, and L-glutamine. Production of phenylalanine oxidase was slowly lost upon growth in a minimal medium containing ammonium ions as a nitrogen source but was reversed by the addition of casein hydrolysate. Oxidase activity as well as a phenylalanine-dichlorophenolindophenol (DCIP) reductase activity increased in P. mirabilis only during cell multiplication. Both rifampin and nalidixic acid caused inhibition of oxidase synthesis. A phenylalanine-active transport was found to be operative when bacteria were grown in the absence of added amino acids. After anaerobic growth, cells of P. mirabilis had lost their ability to carry the phenylalanine oxidase reaction when assayed in the presence of air, and nitrate could not be used as an electron acceptor for the oxidation of phenylalanine. However, some phenylalanine-dichlorophenolindophenol reductase activity was still present in anaerobic bacteria at the early stage of cell multiplication.     

Sulfur mustards induce neurite extension and acetylcholinesterase synthesis in cultured neuroblastoma cells

When neuroblastoma cells (N18) in vitro were exposed to the bifunctional alkylating agent di-2-chloroethyl sulfide (HS), the specific activity of acetylcholinesterase began to rise rapidly after an initial lag period of 1 to 2 days. The five-fold increase in enzyme activity at 4 days after exposure to 0.5 μg/ml of HS was accompanied by a 25-fold rise in the rate of reappearance of acetylcholinesterase activity following essentially irreversible inhibition. Based on previous experience with acetylcholinesterase synthesis in serum deprived neuroblastoma cells, this behavior indicates induction of the enzyme. Vinblastine blocked the concomitant large increase in neurite extension which was stimulated by HS, but left acetylcholinesterase induction unaffected. Since enzyme activity was inversely related to the ability of the monolayer cells to form microcolonies, we conclude that acetylcholinesterase induction is dependent upon inhibition of cell division and independent of neurite extension. The monofunctional analogue of HS, 2-chloroethyl ethyl sulfide (CEES), produced similar effects, but much higher concentrations were required.     

Mitochondrial biogenesis during fungal spore germination: respiration and cytochrome c oxidase in Neurospora crassa.

The germination of conidiospores of wild-type Neurospora crassa was found to be dependent upon the function of the cytochrome-mediated electron transport pathway. The cyanide-insensitive alternate oxidase did not contribute significantly to the respiration of these germinating spores. The dormant spores contained all of the cytochrome components and a catalytically active cytochrome c oxidase required for the activity of the standard respiratory pathway, and these preserved components were responsible for the accelerating rates of oxygen uptake which began immediately upon suspension of the spores in an incubation medium. Mitochondria of the dormant spores contained all of the subunit peptides of the functional cytochrome c oxidase; nevertheless, de novo synthesis of these subunits began at low rates in the first stages of germination. Reactivation of the respiratory system of germinating N. crassa spores seems not to be dependent initially upon the function of either the mitochondrial or cytoplasmic protein-synthesizing systems. The respiratory activity of spores of three mutant cytochrome c oxidase-deficient strains of N. crassa also was found to depend upon the function of the cytochrome electron transport pathway; the dormant and germinating spores of these strains contained a catalytically active cytochrome c oxidase. Cytochrome c oxidase may be present in the dormant and germinating spores of these strains as the result of a developmental-phase-specific synthesis of and requirement for the enzyme.     

Glucocorticoid induction of proline oxidase in LLC-RK1 cells

Dexamethasone induced proline oxidase in cultured LLC-RK1 cells, an epithelial cell line derived from rabbit kidney. The dexamethasone-mediated increase in enzyme activity was concentration and time dependent. Although the effect could be dissociated from cell growth and cell density, it was dependent on protein and RNA synthesis. A comparison of the enzyme isolated from control and dexamethasone-treated cells showed that the increased activity was not due to an alteration in the affinity of the enzyme for proline. These findings suggest that glucocorticoids induce the synthesis of proline oxidase in mammalian cells.     

Induction of Galactokinase in Saccharomyces cerevisiae: Kinetics of Induction and Glucose Effects

The induced synthesis of galactokinase and the repressing effects of glucose on this synthesis have been investigated in whole yeast cells rendered permeable by treatment with dimethyl sulfoxide. It was found that the induction response of uninduced cells to galactose is clearly dependent on the nature of the carbon source upon which the culture was grown prior to exposure to galactose. Glucose-grown cells exhibited a long lag before induction, whereas lactate-grown cells exhibited induced synthesis within 8 min. A concentration of 0.5% galactose was found to be optimal for induction. The addition of glucose to yeast cultures growing on galactose resulted in a severe transient repression of synthesis which was followed by a resumed rate of synthesis characteristic of a weaker permanent catabolite repression. Neither 2-deoxygalactose nor fucose acted as gratuitous inducers of the pathway, nor did they serve as a substrates for galactokinase.     

Oxygen dependence of promitochondrial and cytoplasmic protein synthesis in the formation of electron transfer complexes III and IV in adapting Bakers' yeast.

When anaerobically grown Saccharomyces cerevisiae cells are aerated in the presence of cycloheximide, they accumulate precursor components of electron transfer complexes III and IV. The formation of these precursors is dependent upon promitochondrial protein synthesis and can occur in the absence of concomitant cytoplasmic protein synthesis. The levels to which these precursor components can accumulate during the cycloheximide incubation (phase I) are three to fourfold greater when the cells are grown anaerobically in galactose rather than in glucose. When such galactose-grown cells are sequentially aerated first in cycloheximide and then in chloramphenicol, adaptation responses are elicited with respect to cyanide-sensitive oxygen consumption (QO₂), coenzyme QH₂-cytochrome c reductase (complex III) and cytochrome oxidase (complex IV), all of which are exhibited during the chloramphenicol incubation (phase II). These phase II adaptation responses for QO₂ and for both enzyme activities were observed to be dependent upon the continued presence of oxygen during both phase I (period of mitochondrial translation) and phase II (period of cytoplasmic translation). If one makes the assumption that mRNA's are neither imported into nor exported from promitochondria during adaptation, then one may conclude that oxygen independently and coordinately derepresses synthetic activity in both the mitochondrial and nucleo-cytoplasmic genetic systems. Other regulatory schemes are discussed.     

Polyamine Metabolism in Embryogenic Cells of Daucus carota: II. Changes in Arginine Decarboxylase Activity

Embryogenic cultured cells of Daucus carota have been shown to synthesize putrescine from exogenously supplied [¹⁴C]arginine at twice the rate of control nonembryogenic cells. In the present paper, the activity of arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19), an important enzyme in the synthesis of putrescine, was assayed and also found to be elevated by as much as 2-fold in embryogenic cells. This difference between embryogenic and nonembryogenic cells was observed as early as 6 hours after the induction of embryogenesis and appeared not to result from the presence of a diffusible inhibitor or activator. It seemed to be dependent upon concomitant RNA and protein synthesis, as judged using 6-methyl-purine and cycloheximide. After cycloheximide addition to the culture medium, arginine decarboxylase activity declined with a half-time of about 30 minutes in both embryogenic and nonembryogenic cells. It is suggested that elevated arginine decarboxylase activity is involved in the mechanism leading to elevated putrescine levels in these cells and hence may play a role in the embryogenic process.     

Tyramine oxidase and regulation of arylsulfatase synthesis in Klebsiella aerogenes.

The participation of tyramine oxidase in the regulation of arylsulfatase synthesis in Klebsiella aerogenes was studied. Arylsulfatase was synthesized when this organism was grown with methionine or taurine as the sulfur source (nonrepressing conditions) and was repressed by inorganic sulfate or cysteine; this repression was relieved by tyramine and related compounds (derepressing conditions). Under nonrepressing conditions, arylsulfatase synthesis was not regulated by tyramine oxidase synthesis. However, derepression of arylsulfatase and induction of tyramine oxidase synthesis by tyramine were both antagonized by glucose and other carbohydrate compounds. The derepressed synthesis of arylsulfatase, like that of tyramine oxidase, was released from catabolite repression by use of tyramine as the sole source of nitrogen. A mutant strain that exhibits constitutive synthesis of glutamine synthetase and high levels of histidase when grown in glucose-ammonium medium was subject to the catabolite repression of both tyramine oxidase and arylsulfatase syntheses. Mutants in which repression of arylsulfatase could not be relieved by tyramine could not utilize tyramine as the sole source of nitrogen and were defective in the gene for tyramine oxidase.