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Nitronate monooxygenase (NMO; E.C. 1.13.12.16) oxidizes alkyl nitronates to aldehydes and nitrite. Although the biochemistry of the enzyme from fungal sources has been studied extensively, the physiological role is unknown. The ability of NMO to detoxify propionate-3-nitronate was tested by measuring growth of recombinant Escherichia coli containing the gene encoding for the enzyme in either the absence or presence of the nitronate and its conjugate acid 3-nitropropionate. The mixture propionate-3-nitronate/3-nitropropionate is toxic to E. coli cells lacking expression of NMO, but the toxicity is overcome through either induction of the gene for NMO or through addition of exogenous enzyme to the cultures. Both Williopsis saturnus and Neurospora crassa were able to grow in the presence of 0.4 mM propionate-3-nitronate and 19.6 mM 3-nitropropionate, while a knockout mutant of N. crassa lacking NMO was inhibited by concentrations of propionate-3-nitronate and 3-nitropropionate >0.3 and 600 μM, respectively. These results strongly support the conclusion that NMO functions to protect the fungi from the environmental occurrence of the metabolic toxin.

The purpose of this study was to determine whether mouse embryos at the two-cell to blastocyst stage have the capacity to reduce glutathione disulfide (GSSG) and to elucidate the mechanism that embryos utilize to recover from tertiary-butyl hydroperoxide (tBH)-induced oxidative stress. Experiments were conducted on embryos in vitro and tBH was used to induce oxidation of embryonic reduced glutathione (GSH). After derivatization of extracted embryo samples with dansyl chloride, GSH and GSSG were measured at picomole levels by fluorometric HPLC. Two-cell- and blastocyst-stage embryos were able to recover their GSH levels within 45 min after depletion of GSH by incubation in tBH for 15 min. Addition of 1,3-bis(2-chloroethyl)-1-nitrosourea to the culture medium blocked recovery of GSH and resulted in continued elevation of GSSG. Addition of buthionine sulfoximine (BSO) to the culture medium did not affect GSH levels in two-cell-stage embryos, but did reduce GSH levels in blastocysts by 1.5 h. Culture of two-cell embryos in the presence of BSO for 45 h decreased embryonic GSH content and percentage of embryos developing to the blastocyst stage. These results indicate that preimplantation mouse embryos have the capacity to reduce GSSG and suggest that under normal conditions, depletion and synthesis of GSH occur to a greater extent in the blastocyst than in the two-cell-stage embryo. A major protective role for glutathione reductase during specific stages of embryo development is indicated.

The presence of glucose in the cellular environment influences the response of cells to hydrogen peroxide. This study examines the effect of glucose on clonogenic cell survival of Chinese hamster ovary cells exposed to micromolar concentrations of exogenous hydrogen peroxide. Exposure to hydrogen peroxide (20 mumol/10(7) cells) resulted in considerable cytotoxicity that was unaffected by the presence or absence of glucose. However, glucose protected the cells from killing induced by milder exposure (1 mumol/10(7) cells) to the oxidant causing a shift in the dose-response curve. This effect was considered in terms of glucose metabolism via the pentose phosphate cycle. A low nontoxic concentration of hydrogen peroxide (0.1 mumol/10(7) cells) markedly increased pentose phosphate cycle activity in normal cells. Clonogenic survival and activity of the cycle for cells depleted of total glutathione to about 8.6% of its initial value and/or with catalase activity reduced to about 10% of control levels were also determined. Neither of these modifications alone completely abolished the protective effect of glucose. Efficacy of glucose protection against cytotoxicity of hydrogen peroxide diminished in cells depleted of glutathione, and this was not accompanied by any detectable increase in pentose phosphate cycle activity above the control level. Cells depleted of catalase alone had a profile of survival and pentose phosphate cycle activity similar to that of control cells when exposed to hydrogen peroxide. Cells depleted of both glutathione and catalase were almost as sensitive to hydrogen peroxide as the cells incubated without glucose. They also did not express any detectable increase in pentose phosphate cycle activity. Survival of those cells, when exposed to hydrogen peroxide, was almost the same regardless if glucose was present or not. These results demonstrate an important role for the glutathione redox cycle, catalase, and the pentose phosphate cycle in protection against hydrogen peroxide in Chinese hamster ovary cells. They confirm the essential role of glucose and pentose phosphate cycle activity for the detoxification of hydrogen peroxide via the glutathione redox cycle. The data suggest that the ability of catalase to metabolise peroxide may also depend on metabolism of glucose via the pentose phosphate cycle. A clear understanding of the protective mechanisms in cells against hydrogen peroxide has many applications since this common reactive oxygen species is implicated in several pathophysiologies and in the action of certain chemotherapeutic drugs.

Chinese hamster ovary cells cultured in vitro were used to assess the role of glutathione metabolism in the induction of the 32-kDa stress protein. Enhanced synthesis of the 32-kDa protein was observed after cells were incubated with CdCl 2 or diethylmaleate and protein was subjected to SDS-PAGE followed by fluorography. Concomitantly, in both cell preparations an increase in heme oxygenase activity was observed. Proteins from CdCl 2 -and diethylmaleate-treated cells were subjected to Western blotting and protein crossreacting with either rabbit antibody to rat liver heme oxygenase-1 (32,000 M r ) or rat testis heme oxygenase-2 (36,000 M r ) quantitated. The analysis indicated that the CdCl 2 treatment increased the intensity of the HO-1 band 5.5-fold while the diethylmaleate treatment increased it three-fold relative to control. Neither treatment affected the intensity of HO-2 antibody binding. Incubation of cells with buthionine sulfoximine, under conditions which resulted in ⩾90% of the intracellular glutathione being depleted, enhanced synthesis of a 32-kDa protein when assayed by SDS-PAGE. This protein exhibited a M r similar to the 32-kDa protein induced by either CdCl 2 or diethylmaleate treatment. Proteins from buthionine sulfoximine and diethylmaleate-treated cells were mixed together and subjected to 2D PAGE. The resulting fluorograph demonstrated that both treatments produced identical patterns. In contrast, incubation of cells in diamide, a thiol oxidizing compound, resulted in enhanced synthesis of the 110-, 90-, and 73-kDa heat shock proteins but not the 32-kDa protein. The data presented have shown that depletion of glutathione by two independent methods, conjugation and inhibition of synthesis, enhances the synthesis of a 32-kDa protein identified as heme oxygenase-1; oxidation of glutathione, on the other hand did not. We interpret this to indicate that glutathione depletion rather than conjugation or oxidation represents one pathway for induction of heme oxygenase-1.

A number of early experiments suggested a relationship between methyl group metabolism and the exocrine secretion of the pancreas. These included nutritional studies showing that ethionine, the ethyl analog of methionine which inhibits cellular methylation reactions, is a specific pancreatic toxin. Other studies indicated that protein carboxymethylation might be involved. We now show that in vivo ethionine inhibits amylase secretion from freshly isolated rat pancreatic acini, while in vitro ethionine inhibits amylase secretion from the AR42J pancreatic cell line. S-Adenosylhomocysteine (SAH) is a product inhibitor of all methyltransferase reactions involving S-adenosylmethionine (SAM), and treatments that elevate cellular levels of SAH such as inhibition of S-adenosylhomocysteine hydrolase and the in vitro addition of adenosine and homocysteine result in the inhibition of amylase secretion in both isolated pancreatic acini and AR42J cells. Measurement of SAM and SAH levels in AR42J cells shows that inhibition of secretion is more closely related to elevation of SAH levels than to a decrease in the SAM/SAH ratio. Small G-proteins are carboxymethylated on the C-terminal prenylated cysteine and inhibitors of membrane-associated prenylcysteine methyltransferase, N-acetylfarnesylcysteine, N-acetylgeranylgeranylcysteine, and farnesylthioacetic acid (FTA), block secretion in AR42J cells. N-Acetylgeranylcysteine is not an inhibitor of the methyltransferase and does not inhibit amylase secretion. FTA inhibits membrane-associated prenylcysteine methyltransferase from AR42J cells with a Ki in the 45-69 microm range. These results suggest that a methylation event is needed for pancreatic exocrine secretion which may be the reversible methylation of a G-protein involved in signal transduction or membrane trafficking.

The solubilization and subsequent separation of the hepatic microsomal ethanol-oxidizing system from alcohol dehydrogenase and catalase activities by DEAE-cellulose column chromatography is described. Absence of alcohol dehydrogenase in the column eluates exhibiting microsomal ethanol-oxidizing system activity was demonstrated by the failure of NAD + to promote ethanol oxidation at pH 9.6. Differentiation of the microsomal ethanol-oxidizing system from alcohol dehydrogenase was further shown by the apparent K m for ethanol (7.2 m m , insensitivity of the microsomal ethanol-oxidizing system to the alcohol dehydrogenase inhibitor pyrazole (0.1 m m ) and by the failure of added alcohol dehydrogenase to increase the ethanol oxidation. Absence of catalatic activity in these fractions was demonstrated by spectrophotometric and polarographic assay. Differentiation of the microsomal ethanol-oxidizing system from the peroxidatic activity of catalase was shown by the apparent K m for oxygen (8.3 μ m ), insensitivity of the microsomal ethanol-oxidizing system to the catalase inhibitors azide and cyanide, and by the lack of a H 2 O 2 -generating system (glucose-glucose oxidase) to sustain ethanol oxidation in the eluates. The oxidation of ethanol to acetaldehyde by the alcohol dehydrogenase- and catalase-free fractions required NADPH and oxygen and was inhibited by CO. The column eluates showing microsomal ethanol-oxidizing system activity contained cytochrome P -450, NADPH-cytochrome c reductase, and phospholipids and also metabolized aminopyrine, benzphetamine, and aniline.

One hundred micromolar Ca 2+ added to rat liver mitochondria induces a transient uptake of Ca 2+ plus a rapid efflux of the mitochondrial Mg 2+ . Addition of a cytosolic molecule, cytosolic metabolic factor, to mitochondria prevents the efflux of the two divalent cations. ADP is required for this cytosolic metabolic factor action. This requirement for ADP is specific as it is shown by experiments with traps for nucleotides and inhibitors of the translocase. The implication of cytosolic metabolic factor in the mitochondrial regulation process is discussed.

A cell-free extract prepared from Fusarium solani pisi grown on cutin, catalyzed the hydration of 18-hydroxy-9,10-epoxyoctadecanoic acid to 9,10,18-trihydroxyoctadecanoic acid while extracts from glucose-grown cells contained g supernatant fraction. Rate of hydration of the epoxy acid was linear up to 15 min and up to a protein concentration of 30 μg/ml. This fungal epoxide hydrase has a molecular weight of 35,000, as determined by Sephadex G-100 gel filtration. It was partially purified by ammonium sulfate fractionation and gel filtration. The apparent K m and V of the enzyme was 2 × 10 −4 m and 222 nmoles/min/mg, respectively. Parachloromercuribenzoate strongly inhibited the enzyme, while N -ethylmaleimide was a less potent inhibitor. 1,1,1,-Trichloropropylene-2,3-oxide at 10 −3 m gave 50% inhibition of the hydration of 18-hydroxy-9,10-epoxyoctadecanoic acid. Kinetic analysis showed that trichloropropylene oxide was a competitive inhibitor. 18-Acetoxy-9,10-epox-yoctadecanoic acid, methyl 18-acetoxy-9,10-epoxyoctadecanoate, 9,10-epoxyoctadecanoic acid, and styrene oxide were not readily hydrated by this fungal epoxide hydrase showing that it has a stringent substrate specificity. Analysis of the enzymatic hydration product on boric acid-impregnated silica gel plates showed that the product obtained from the cis epoxide was exclusively erythro while acid hydrolysis of this epoxide gave rise to the expected threo product. This enzyme is novel in that it catalyzes cis hydration of epoxide while the other epoxide hydrases heretofore isolated catalyzed trans hydration of epoxides.

Reduced NADPH and a hemoprotein component are two specific chick kidney intramitochondrial cofactors essential for the conversion of 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol. It is concluded that the reversal of the respiratory electron flow is the major pathway for the production of NADPH during the succinate, malate, or isocitrate-supported 1-hydroxylation of 25-hydroxycholecalciferol. When mitochondria are swollen with 10 m m Ca 2+ and 5 m m Mg 2+ , extramitochondrial NADPH is capable of supporting the 1-hydroxylase reaction without Krebs cycle intermediates. In this case respiratory chain inhibitors and uncouples of oxidative phosphorylation are without effect. The succinate-supported 1-hydroxylation of 25-hydroxycholecalciferol is inhibited by carbon monoxide with a partition coefficient ( K ) of 0.5. The NADPH-supported hydroxylation is also inhibited by carbon monoxide ( K =2.5) but this inhibition can be partially reversed by exposure to white light.

Xanthine dehydrogenase, purine nucleoside phosphorylase, and tyrosine aminotransferase are all increased sharply in liver of chicks by dietary protein. Results in this paper show that most amino acids have this effect, with methionine and tryptophan being more effective than the remainder. This suggested that any source of amino nitrogen could cause a build-up of a nitrogenous intermedate(s) necessary for enzyme accumulation. The above hypothesis was tested by blocking the breakdown of amino acids using various antimetabolites. Results were uniformly successful in raising enzyme levels, and furthermore it was found that antimetabolites and amino acids are not additive, suggesting that they act by a similar mechanism.

Spinach leaf phosphoenolpyruvate carboxylase has been purified to homogeneity using salt fractionatjon, chromatography, and immunologie procedures to remove contaminating ribulose diphosphate carboxylase. From gel filtration and isoelectric focusing, the molecular weight (~560,000) and isoelectric point (pI = 4.9) are indistinguishable from those of ribulose diphosphate carboxylase. The subunit molecular weight of phosphoenolpyruvate carboxylase (130,000) suggests that the native enzyme is a tetramer. Kinetic studies using Mg2+ or Mn2+ as the activator indicate that the divalent cation lowers the Km of the substrate phosphoenolpyruvate by an order of magnitude and conversely, that the presence of the substrate similarly lowers the Km of the metal ion, suggesting an enzyme-metal-substrate bridge complex. Three analogs of phosphoenolpyruvate, l phospholactate, d -phospholactate, and phosphoglycolate are potent competitive inhibitors. The inhibitor constant (Ki) of l -phospholactate (2 μ m ) is 49-fold lower with Mn2+ as the activator than with Mg2+. An analysis of the competitive inhibition by portions of the l -phospholactate molecule (i.e., l -lactate, methyl phosphate, and phosphite) indicates this 49-fold lowering is due to increased interaction of the phosphoryl group and, to a lesser extent, of the carboxyl and C-O-P bridge oxygen of l -phospholactate with the enzyme metal complex. The results provide indirect evidence for phosphoryl coordination by the enzyme-bound divalent cation.

Synthesis of the glycoprotein hormone common α-subunit and alkaline phosphatase (placental isozyme) has been examined in HeLa S 3 cells. A variety of compounds that inhibit DNA synthesis lead to the increased production of both proteins. Experiments presented in this communication were undertaken to determine whether protein induction and DNA synthesis inhibition are coordinated. In general, nucleoside analogs and compounds that alter deoxynucleotide metabolism were good inducers of these ectopic products, whereas agents that altered DNA by intercalation, crosslinking, and covalent modification were poor inducers. The former class of effectors includes 5-bromo-2′-deoxyuridine, 5-fluoro-2′-deoxyuridine, 2′-deoxythymidine, 1-β- d -arabinofuranosylcytosine, methotrexate, hydroxyurea, N -phosphonoacetyl- l -aspartic acid, and sodium butyrate; and the latter class of compounds includes ethidium bromide, acridine, bleomycin, mitomycin C, cesalin, macromomycin, and cis -diamminedichloroplatinum(II). A direct correlation between protein induction and DNA synthesis inhibition is unlikely based on the following observations: (i) for some effectors, the concentrations required to induce α-subunit and PAP were significantly different from those necessary to inhibit DNA synthesis; (ii) several agents inhibit DNA replication but do not enhance hormone or enzyme production; (iii) the kinetics of ectopic protein induction were similar for a number of inducers whereas the kinetics of DNA synthesis inhibition elicited by the same compounds were quite different. It is difficult from the data obtained, however, to rule out the possibility that inhibition of DNA synthesis may be required but is not sufficient for protein induction.

The biosynthesis of cytidine nucleotides of liver rRNA was studied following single administration of seventeen different compounds. Only the substances recognized as inducers of the mixed-function oxidases of liver microsomes decrease the utilization of [2- 14 C]orotic acid for the synthesis of rRNA cytidine nucleotides.

Undeveloped encysted embryos of the brine shrimp, Artemia salina, contain a large quantity of metabolically repressed 80S ribosomes. These ribosomes appear to be inactive or nonfunctional due to the presence of an inhibitor protein on their 60S subunit. During development the inhibitor is released or inactivated and the 80S ribosomes and their constituent subunits become fully functional in a poly(U)-directed protein-synthesizing system. The inefficiency of most 80S ribosomes from undeveloped Artemia embryos appears to be due to their inability to form stable complexes with poly(U) and phe-tRNA in the presence of elongation factor, EF-1. A potent inhibitor of protein synthesis has also been found in the 105,000g supernatant fraction from undeveloped Artemia embryos. The exact nature of this inhibitor has not been ascertained but it appears to be a heat-labile protein devoid of RNase and protease activity. It is not known whether this inhibitor is the same as that associated with 60S ribosomal subunits of undeveloped cyst ribosomes.

Adult rat liver parenchymal cells in primary culture exhibit specific saturable binding of 125 I-labeled murine epidermal growth factor (EGF). The Scatchard plot of the binding data obtained at 36 °C was curvilinear yielding two apparent dissociation constants of 1.5 × 10 −10 m and 1.2 × 10 −9 m with 27,000 and 57,000 sites per cell, respectively. The binding data obtained at 2 °C yielded a linear Scatchard plot with an apparent dissociation constant of 4.4 × 10 −9 m and 78,000 sites per cell. Exposure of the hepatocytes to EGF at 36 °C resulted in a loss of EGF binding capacity due to down regulation of receptors. The cells recovered the capacity to bind EGF upon incubation in medium which did not contain EGF; this recovery was inhibited by cycloheximide. The cultures appeared to internalize and degrade bound EGF at 36 °C but not at 2 °C. The degradation of EGF was inhibited by chloroquine, an inhibitor of lysosomal enzymes. These data indicate that liver specifically binds and further processes EGF, and therefore, may be a physiological target tissue for this growth factor.

A Mycobacterium species that synthesizes carotenoids only after light stimulation has been used to study the inhibition of carotenoid synthesis by a variety of compounds. In addition to diphenylamine, which has been shown to inhibit carotenogenesis in many systems, benzophenone, acridine orange, methylene blue, neutral red, toluidine blue, proflavine, acridine, and 9,10-dihydroacridine were found to inhibit the formation of the more highly unsaturated carotenoids. In all instances phytofluene accumulated when the formation of the more highly unsaturated carotenoids was inhibited. Under conditions of high oxygen tension all of these compounds except diphenylamine and benzophenone no longer inhibit carotenogenesis. Molecular models show that the dihydro derivatives of the heterocyclic inhibitors, benzophenone, and diphenylamine have a close structural similarity to carotenoids in the region of subsequent dehydrogenation. It is postulated that these compounds bind with the carotenoid dehydrogenases, thereby inhibiting carotenogenesis.

The effect of 2-deoxy- d -glucose on the conversion of palmitate-1-C14, pyruvate-1-C14 and -2-C14, and acetate-1-C14 to CO2 by ascites tumor cells has been investigated. The oxidation of the labeled substrates was affected by concentrations of deoxyglucose which depressed the oxygen uptake. Similar to glucose, deoxyglucose strongly depressed the oxidation of palmitate, while it had only a small effect on the oxidation of acetate. Since deoxyglucose does not provide respiratory metabolites, the depression of fatty acid oxidation cannot be explained by replacement. At high concentrations of pyruvate (above 10−2M), deoxyglucose, in contrast to glucose, depressed the oxidation of pyruvate carbon-2 to a greater degree than the oxidation of pyruvate carbon-1. The results suggest that oxidative decarboxylation is less affected by the depressed respiratory rate than is the utilization of the carbon-2 units in the citric acid degradation cycle. From the experiments in the presence of monoiodoacetic acid and from the experimental conditions employed in general, it appears unlikely that the effects of deoxyglucose on substrate oxidation are correlated with known effects of deoxyglucose on the enzymes of glucolysis or on the intracellular transport of carbohydrates.

The metabolism of the new compound l - O -ethylthreonine by both mammalian and bacterial cells has been investigated and the results obtained showed that l - O -ethylthreonine was a metabolic antagonist of l -isoleucine. With a partially purified enzyme system from sarcoma 180 cells, the analogue was found to suppress the synthesis of l -isoleucyl-tRNA and the inhibition was competitively reversed by the natural amino acid. The mechanism of the inhibition appeared to be caused by the incorporation of the analogue into tRNA. In Escherichia coli B cells, l - O -ethylthreonine was found to be growth-inhibitory as well as to inhibit the incorporation of a number of amino acids into proteins. The analogue was incorporated into protein in bacterial cells although at a slower rate than that of l -isoleucine.

The 503 mμ band, which is detectable in the absorption spectrum of aerobically growing cells of baker's yeast, was further intensified in cells collected after prolonged cultivation. The cells showed another new band at 475 mμ, besides the 503 and 550 mμ bands in the visible region. Upon addition of various carbonyl reagents, the 475 and 503 mμ bands disappeared. These two bands, as well as the 550 mμ band, were flattened upon oxygenation and reappeared upon deoxygenation of cell suspensions. The reduction of the 503 mμ pigment and the c -type cytochrome (550 mμ pigment) started at the same oxygen tension of the suspension. In the presence of respiratory substrates or inhibitors other than cyanide and azide, onset of the reduction of both pigments was accelerated or delayed. In almost all of these cases, reduction of both pigments also started simultaneously. With the inhibitors, the reduction rate of the 503 mμ pigment was lowered while the oxidation rate was not appreciably affected. With cyanide or azide, oxidation of both pigments by aeration was completely inhibited.

Conditions are described for the in vitro synthesis of the T-even bacteriophageinduced deoxycytidylate deaminase using either T2, T4, or T6 phage DNA or mRNA as template. The deaminase appears after dihydrofolate reductase, but before thymidylate synthetase in this system. The T-odd phage DNA were not active in the synthesis of deoxycytidylate deaminase, a fact consistent with the inability of these phages to induce deoxycytidylate deaminase on infection of Escherichia coli . Proof that the activity being measured was from the deaminase was obtained with antibody to this enzyme and through the use of a specific inhibitor, tetrahydrodeoxyuridylate. Attempts to demonstrate the feedback regulation of the deoxycytidylate deaminase synthesized in vitro were unsuccessful initially due to the presence of a phosphotransferase capable of converting deoxycytidylate to deoxycytidine triphosphate with thymidine triphosphate and other nucleoside triphosphates as donors. Removal of this activity, however, enabled a definitive pH dependence of activation by deoxycytidine triphosphate to be shown. In addition, other properties associated with the pure deaminase, such as the inhibition by thymidine triphosphate and its reversal by deoxycytidine triphosphate, became evident.

The malate-aspartate, fatty acid, and α-glycerophosphate shuttles for the transport of reducing equivalents into mitochondria were reconstituted, using isolated hepatic mitochondria and the extramitochondrial components of the shuttles. Clofibrate and thyroxin increased, while propylthiouracil treatment decreased, the activity of mitochondrial α-glycerophosphate dehydrogenase. Despite these changes, the activity of the reconstituted α-glycerophosphate shuttle was similar in mitochondria from control rats and those from rats treated with clofibrate and propylthiouracil. There was an increase in the activity of the shuttle using mitochondria from thyroxin-treated rats. Rotenone caused 60–90% inhibition of this shuttle, suggesting that rotenone-sensitive NADH dehydrogenase participates in the pathway of oxidation of extramitochondrial hydrogen. Palmitate, oleate, and octanoate were equally effective in reconstituting a cyclic fatty acid shuttle. The shuttle was inhibited by various compounds affecting mitochondrial metabolism, including oligomycin, dinitrophenol, cyanide, rotenone, atractyloside, and α-bromopalmitate. Carnitine and several dicarboxylic and tricarboxylic acids which stimulate fatty acid elongation, augmented fatty acid shuttle activity. The malate-aspartate shuttle was inhibited by cycloserine, amino-oxyacetic acid, and hydrazine, and stimulated by pyridoxal phosphate, at the same concentrations which affected the activities of cytoplasmic and mitochondrial glutamic oxalacetic transaminase. This shuttle was inhibited by uncouplers, antimycin, azide, cyanide, rotenone, amobarbital, oligomycin, and several inhibitors of anion transport including iodobenzylmalonate and avenaciolide. The reconstituted shuttle is sufficiently active to provide about 70–80% of the oxalacetate required for maximal rates of gluconeogenesis. Extrapolations based on the rates of mitochondrial oxidation of acetaldehyde and the activity of the microsomal ethanol oxidizing system suggest that any one of the shuttles could account for the rate of ethanol metabolism in vitro by the alcohol dehydrogenase pathway.

The action of certain derivatives of benzal malononitrile and of β-nitrostyrene on respiration and phosphorylation processes was examined in mitochondria, Escherichia coli , and chloroplasts. The 4-hydroxy derivatives were found to be particularly effective uncouplers of oxidative phosphorylation in mitochondria and inhibitors of cyclic photosynthetic phosphorylation in chloroplasts. These findings may provide a basis for the oftennoted physiologic action of benzal malonitriles and β-nitrostyrenes. In mitochondria, benzal malononitriles and β-nitrostyrenes appear to interact with the energy rather than the electron transfer pathway. Thus the substances interfere with various energy-dependent processes such as the succinate-linked pyridine nucleotide reduction and mitochondrial swelling-shrinkage phenomena.

β-Aspartylhydroxamic acid (BAH) has been found to mimic asparagine in cells of Lactobacillus arabinosus 17-5 by inhibiting asparagine synthetase activity and repressing the formation of the same enzyme. The action of BAH is apparently specific, since the compound does not inhibit the formation of the repressible glutamine synthetase of L. arabinosus . Further, extracts of the organism catalyze a BAH-dependent and an asparagine-dependent ATP-PP i exchange, and evidence is given which indicates that only one enzyme activity is involved with both the BAH and asparagine activations. This latter finding is in agreement with the hypothesis that certain amino acids which repress biosynthetic enzyme formation must be capable of being activated and transferred to an appropriate tRNA molecule. However, enzymatic transfer of BAH to tRNA has not been demonstrated in this study.

Cycloheximide (actidione) was shown to be a potent inhibitor of protein synthesis and the estrogen response in the immature rat uterus. Increases in uterine wet weight and cytidine-H 3 incorporation into RNA associated with the four hour response to estrogen were blocked by actidione administration. Increases in orthophosphate-P 32 and acetate-C 14 incorporation into lipid and cytidine-H 3 incorporation into RNA at 2 hours after estrogen were also blocked by actidione. Protein synthesis in the uterus was not significantly increased by estrogen at 2 hours and was blocked to 10% of controls by a single injection of 200 μg. of actidione into immature female rats.

dl-3,4-Dehydroproline inhibits the growth of Lactobacillus arabinosus, Streptococcus lactis, Pediococcus cerevisiae, Leuconostoc dextranicum, and Escherichia coli, and the toxicities are competitively reversed by l-proline with inhibition indices of 3, 3, 3, 10, and 10, respectively. A noncompetitive reversal of dehydroproline toxicity by a peptide, glycylproline, is observed with E. coli, but for S. lactis, P. cerevisiae, and L. arabinosus a competitive reversal of the inhibitory effect of the analog is observed with glycylproline. Proline peptides are either more or less active than the free amino acid depending upon the assay organism. A synergistic rather than an additive effect of glycylproline in combination with proline in reversing the toxicity of dehydroproline for L. arabinosus is observed.

By the use of any one of three halogen-containing analogs of riboflavin, it has been possible to discover and isolate a mutant strain of Lactobacillus casei . This mutant strain continues to require an exogenous source of flavin, but the limits within which the physicochemical properties of the flavin must fall for utilization by the organism are not as narrowly defined as those of the stock strain. It appears that the mutant strain exists in all cultures of this organism, but would have remained undetected except for the use of one of these three flavins. The existence of the mutant in the cultures of the organism does not interfere with the use of L. casei for the microbiological assay of riboflavin.

Cell-free preparations of Nitrosomonas europaea are capable of effecting a number of organic transformations. These include the fixation of CO 2 with adenosinediphosphate and ribose-5-phosphate or inosinediphosphate and phosphopyruvic acid. The formation of organic phosphate compounds concomitant with the oxidation of hydroxylamine is demonstrated and the reduction of diphosphopyridine nucleotide is shown to occur both endogenously and upon the oxidation of hydroxylamine. Absorption and difference spectra of cytochromes are presented.

Samples of extractable collagen from normal rats purified by different means were compared with similar samples from rats treated with β-aminopropionitrile (BAPN) with respect to gelling at 37 °C. and with respect to the extent to which the gels redissolve on cooling to 4 °C. The in vitro experiments confirm the in vivo findings of a defect in the intermolecular bonding of lathyritic collagen fibrils. The results are consistent with either an intrinsic defect in lathyritic collagen or the presence in normal connective tissue of a factor extrinsic to collagen which is absent in lathyritic connective tissue. The results are inconsistent with the presence of an inhibitor in lathyritic connective tissue preventing intermolecular bonding of collagen.

The function of the aorta in aortic calcification in vitro has been investigated. In this system calcification does not depend on metabolic processes in the aorta but on the nucleating activity of elastin fibers. Homogenates of aorta calcify, but fractions isolated by centrifugation from the homogenate do not. However, the 1000 g sediments from aortas incubated in serum for a relatively short time prior to homogenization do calcify. These results suggest that more than one component in the aorta is necessary for mineral induction. Treatment of aortas and nuchal ligaments, a tissue consisting chiefly of elastin, with group-blocking reagents indicates that both sulfhydryl and amino groups are involved in the calcification process. Amino groups appear to participate at a later stage in the process than sulfhydryl groups.

l -Ethionine (tested at 0.26, 0.52 and 1.04 m M ) was found to be a potent inhibitor of tropolone biosynthesis in Penicillium stipitatum . The effect of d -ethionine was identical to that of the l -enantiomorph. Although cell synthesis was always diminished in the presence of ethionine, the effect on tropolone formation was much greater than could be accounted for by this fact alone. Neither the simultaneous addition of equimolar amounts of methionine nor pretreatment with methionine prevented the ethionine inhibition. Sulfanilamide (0.50 and 1.00 m M ), succinylsulfathiazole (0.23 m M ), and dl -methionine sulfoxide (0.13, 0.26, and 0.52 m M ) had no significant effect on tropolone biosynthesis. From ethioninetreated P. stipitatum cultures, a sample of pure, crystalline orsellinic acid was isolated by partition and paper chromatographic techniques. Methods for the detection of orsellinic acid were supplemented by the use of gas liquid chromatography. Orsellinic acid had a retention time of 10.5 minutes on 3% nitrile silicone columns at 194 °C. Orsellinic acid was also identified in extracts of the culture fluids of the following molds by paper chromatographic methods: Chaetomium globosum, Cladosporium fulvum, Curvularia lunata, Gibberetta fujikuroi , and Penicillium cyclopium . No evidence for the presence of orsellinic acid was obtained in uninhibited cultures of Penicillium stipitatum, Gliocladium roseum, Aspergillus quadrilineatus , and a Cephalosporium sp.

A method is presented for the isolation of a respiratory inhibitor(s) from an alfalfa forage which caused bloat in grazing cattle. Solvents were used to extract the crude respiratory inhibitor, and ion-exchange chromatography separated it from an anticholinesterase constituent of the forage. Carbon-column chromatography afforded purification of the respiratory inhibitor, which was eluted with methanol and then further purified by precipitation with cholesterol. The respiratory inhibitor was thus purified 350–400-fold from the original dried alfalfa. The behavior of the respiratory inhibitor during isolation would suggest it to be a saponin.

Several β-substituted aspartic acid analogs were tested as growth inhibitors of Escherichia coli and Leuconostoc mesenteroides. Of the compounds tested, the two racemic pairs of β-hydroxyaspartic acid were shown to be the most active inhibitors of growth. In most instances, this antagonism was readily reversed by l -aspartic acid. In L. mesenteroides, however, d -aspartic acid appears to be a more effective reversal agent than the l isomer in alleviating the growth inhibition caused by dl -erythro-β-hydroxyaspartic acid. None of the β-substituted aspartic acid analogs tested supported growth of L. mesenteroides, a culture which requires preformed aspartic acid, in the absence of aspartic acid.

Vancomycin inhibits growth and mucopeptide synthesis in Bacillus subtilis strain W23. These effects can be minimized by added magnesium. The metal was most effective in alleviating growth inhibition when added simultaneously with the antibiotic. Addition of magnesium at later time intervals showed less response. High concentrations of vancomycin (8.5 μg. per milligram dry wt.) caused a rapid lysis of this organism when incubated in a complete medium. Leakage of cell metabolites was observed with sublytic vancomycin concentrations (4.6 μg per milligram dry wt.). Magnesium was unable to prevent either lysis or leaching of cell metabolites. A mechanism of inhibition based on these findings is suggested.

C14-Labeled ethanol, acetate, pyruvate, or butyrate added to pigeon liver slices in the presence of sulfanilamide contributed about 70, 50, 45, and 20%, respectively of the acetyl groups found in the acetyl sulfanilamide. The simultaneous addition of unlabeled alcohol or acetate reduced the incorporation of C14 from pyruvate or butyrate, while the simultaneous addition of unlabeled pyruvate, glycerol, or butyrate did not seriously reduce the incorporation of C14 from ethanol or acetate. Some 15 times as much C14 from butyrate was found in the CO2 as in the acetyl sulfanilamide; the corresponding ratios for pyruvate, acetate and alcohol were 3, 1 2 , and 1 3 , respectively. The oxidation of labeled butyrate and pyruvate was not seriously decreased by the simultaneous presence of unlabeled ethanol or acetate, but the oxidation of acetate and ethanol was inhibited by pyruvate and especially butyrate. The results are interpreted to show a compartmentation of the acetyl CoA formed from the different substrates with a preferential utilization of alcohol and acetate for the acetylation reaction, and a preferential oxidation of butyrate and pyruvate.

Antagonism at the oxidative deamination stage may well be one of the factors responsible for the poorer growth observed when a d -amino acid requiring inversion is fed in a diet which contains a large proportion of other d -amino acids. The rate of oxygen uptake when two d -amino acids were incubated together with crude and purified extracts of hog kidney and with slices of rat kidney was less than the sum of the rates when each was oxidized separately. Data based upon the Chromatographic separation and estimation of the α-keto acids produced, or upon the spectrophotometric measurement of one keto acid, with calculation of the second from extra oxygen uptake, showed that the ratios of their degrees of involvement when they were oxidized simultaneously were not the same as their ratios when they were oxidized separately. Lineweaver-Burk treatment of data obtained with purified d -amino acid oxidase indicated that the inhibition was competitive. The maximum velocity of the oxidation of two amino acids jointly approximated that of the amino acid with the lower Michaelis constant.

Atractyloside (20 μ m ) has been found to decrease the incorporation of dATP- 3 H into DNA by 90% in intact mitochondria (at a level of 3 mg of protein) but to have no such effect on sonic extracts of mitochondria nor on the DNA polymerase from Escherichia coli . Inhibition of dATP- 3 H incorporation by atractyloside can be overcome by increasing the level of ATP present. The incorporation of TTP- 3 H is not similarly inhibited by atractyloside. TTP- 3 H incorporation, which is stimulated by added ATP, is inhibited by atractyloside to the extent of 40% in the absence of and 13% in the presence of ATP (2 m m ). Atractyloside can be used with labeled dATP to ascertain that DNA synthesis is occurring intramitochondrially in intact organelles, in vitro , rather than externally as a result of contaminating nuclear DNA polymerase.

A series of analogs of acetate, α- p -bipbenylylbutyric acid, α-phenylbutyric acid, γ-phenylbutyric acid, cyclopropanecarboxylic acid, α-phenylacetic acid, and β,β-dimethylacrylic acid, was tested for inhibition of the growth of Tetrahymena pyriformis . The inhibitory effects of all but the last were released by acetate or lower fatty acids, although an exponential relationship to the acetate concentration was observed in some cases. The inhibitory effects of none of the analogs were completely reversed by sterols, but for some, sterols partially restored growth. No reversal was obtained with mevalonic acid. Soybean lecithin and an enzymic hydrolyzate of casein were effective.

The glycolytic and respiratory capacities of five cultured mammalian cell lines and a mouse ascites cell were compared. Mouse lymphoma cells (L5178Y) were studied in more detail. Inhibition of lymphoma cell respiration is linear and complete by rotenone, antimycin and Amytal, complete but nonlinear by cyanide, and linear but incomplete by oligomycin and aurovertin. The cells are substantially more sensitive to many of these inhibitors than are isolated rat liver mitochondria and fail to show the threshold phenomenon characteristic of mitochondria in vitro. These data show the feasibility of studying mitochondrial properties in vivo through the use of selective inhibitors. Of the agents tested, only atractyloside failed to inhibit respiration in intact cells. Studies of phosphate transport by Chinese hamster ovary cells and of valine incorporation by the lymphoma cells indicate that the energy requirements for these two processes are met in very different ways. Energy for the first process is supplied nearly equally by glycolytic and respiratory chain ATP; nonphosphorylated high-energy intermediates of the respiratory chain appear to play no part. Valine incorporation, in contrast, appears to be driven exclusively by respiratory chain energy with both ATP and nonphosphorylated high-energy intermediates contributing.

Submitochondrial particles obtained by treatment with digitonin and by sonication were compared in order to examine the specificity of the phosphate acceptor in oxidative phosphorylation. The digitonin particles behave like intact mitochondria. They have an absolute specificity for ADP in oxidative phosphorylation and for ATP in the P i -ATP exchange reaction. These reactions, and the 2,4-dinitrophenol-stimulated ATPase, are sensitive to atractyloside. The inhibition of oxidative phosphorylation by atractyloside is competitively reversed by ADP. The finding of Low, Vallin, and Alm that sonic particles are not absolutely specific for ADP as phosphate acceptor and are insensitive to atractyloside has been confirmed. The atractyloside sensitivity and nucleotide specificity of digitonin particles is not altered by sonication and the properties of sonic particles are not affected by treatment with digitonin. The broad phosphate acceptor specificity of sonic particles is markedly altered by the concentration of divalent cation present in the assay medium. It is concluded that it is unnecessary to invoke the existence of a nucleotide translocase enzyme to account for the differences in phosphate acceptor specificity between the two preparations. The reasons for this conclusion are discussed.

The reaction of 1,4-dichloro-2-butyne with potassium phthalimide yielded N -(4-chloro-2-butynyl)phthalimide, which was subsequently condensed with ethyl acetamidomalonate. Acid hydrolysis of the condensation product gave 2,6-diamino-4-hexynoic acid. Growth inhibition of Leuconostoc dextranicum 8086 by 2,6-diamino-4-hexynoic acid is reversed in a competitive manner by lysine over a hundredfold range of increasing concentrations with an inhibition index of about 10.

The sensitivity of acetylcholine receptor to eleven cholinergic drugs, phospholipase A, heat and pH provided evidence that the so-called high-affinity binding ( K d for acetylcholine 11 n m in 1% Triton) and low-affinity binding ( K d 562 n m ) were related to two distinct binding sites. The low-affinity binding site was less sensitive to heat and several of the cholinergic drugs, but was a little more sensitive to bungarotoxin than the high-affinity site. Zinc (0.4 m m ) and EDTA (10 m m ) abolished acetylcholine binding to both sites; the EDTA inhibition was time-dependent.

The pyrrolopyrimidine nucleosides, toyocamycin, sangivamycin, and tubercidin are isolated from the culture filtrates of 14 species of the Streptomyces. Although earlier experiments showed that the biosynthesis of the pyrrolopyrimidine nucleosides require GTP as the common precursor, there was no experimental evidence to demonstrate the interconversion of these naturally occurring nucleoside analogs. The data presented here describe two types of experiments to prove that toyocamycin is the precursor for sangivamycin. First, in vivo experiments show that radioactive toyocamycin is converted to sangivamycin. Second, the enzyme, toyocamycin nitrile hydrolase, that catalyzes the conversion of toyocamycin to sangivamycin has been isolated and partially purified from the soluble fraction of S. rimosus. The nitrile hydrolase is not present in cell-free extracts of the Streptomyces that synthesize tubercidin or toyocamycin. Activity can be assayed by measuring the formation of radioactive sangivamycin from toyocamycin. The enzyme has been purified 24-fold with an over-all yield of 5%. The pH optimum is 6.5 and the K m is 0.5 m m . Most nitriles tested are competitive inhibitors but they are not substrates. The activity of the hydrolase is limited to the conversion of the nitrile group to the carboxamide group. Hydrolase activity is observed in cell-frre estracts of S. rimosus before toyocamycin production begins. The in vivo and in vitro studies demonstrate that toyocamycin is not a precursor for tubercidin. The experimental evidence strongly suggests that there must be a branch point in the biosynthesis of the pyrrolopyrimidine nucleoside antibiotics.

Microsomal membranes isolated from sugar beet (Beta vulgaris L. var. GWD-2) storage tissue were found to contain a Na3VO4-dependent system for the oxidation of NADH. The system was demonstrated to be enzymatic in nature and specific for Na3VO4. Maximal Na3VO4-dependent NADH oxidation was observed at pH 6.5, when Na3VO4 was present at 200 microM and when NADH was present at 100 microM. The oxidation activity was insensitive to rotenone and antimycin A but was inhibited by NaN3, NaCN, and quinacrine. Sodium vanadate-dependent NADH oxidation occurred with a concomitant uptake of O2 from the assay solution. Both NADH oxidation and O2 consumption were dependent upon the presence of Na3VO4, inhibited by manganese, and preferred NADH to NADPH. Catalase prevented Na3VO4-dependent O2 consumption but accelerated NADH oxidation. The effects of manganese and catalase suggest that superoxide anion and hydrogen peroxide may be involved in this process. While it is unclear as to the physiological significance of Na3VO4-dependent NADH oxidation in plant cells, the presence of this system indicates that caution must be exercised when coupled ATPase assays depending upon NADH oxidation are used with plant membranes in the presence of Na3VO4.

Electrons can be transferred to the respiratory chain in whole cells and in membrane vesicles of Bacillus subtilis W 23 by the membrane impermeable electron donor reduced 5-N-methyl-phenazonium-3-sulfonate as efficiently as by the membrane permeable electron donor reduced 5-N-methyl-phenazonium methyl-sulfate, indicating that the respiratory chain is accessible from the outside of the membrane. Succinate is oxidized by whole cells and membrane vesicles at a low rate and does not energize transport of l -glutamate. In the presence of 5-N-methyl-phenazonium-3-sulfonate or 5-N-methyl-phenazonium methyl-sulfate, the oxidation rate and the rate of l -glutamate transport are increased considerably. The electrons are transferred directly from succinic dehydrogenase to these acceptors. Succinic dehydrogenase must therefore be exposed to the outside surface of the membrane in both membrane vesicles and whole cells. The exposure of succinic dehydrogenase to the outside is also indicated by the observations that only a 5% increase in the oxidation rates of succinate-5-N-methyl-phenazonium methylsulfate and succinate-5-N-methyl-phenazonium-3-sulfonate is observed upon solubilization of the membrane with the nonionic detergent Brij-58. Furthermore, treatment of membrane vesicles with trypsin decreases by more than 95% these oxidation rates. NADH is oxidized at a high rate and energizes transport of l -glutamate in whole cells and membrane vesicles effectively. The NADH-oxidation is not effected by trypsin treatment of the vesicles indicating that the oxidation occurs at the inside-surface of the membrane. Trypsin treatment of the vesicles, however, significantly decreases the rate of l -glutamate transport driven by NADH. Therefore component(s) of the transport system for l -glutamate must be effected by trypsin treatment. No apparent differences could be observed in the localization of membrane-bound functions between membrane vesicles and whole cells. This strongly supports the contention that the vesicle membrane of B. subtilis has the same orientation as the cytoplasmic membrane of whole cells.

Isolated rat adrenal cells were used to study the possible pathways of intramitochondrial NADPH generation for 11β-hydroxylation of 11-deoxycorticosterone. Pyruvate was efficiently utilized by the mitochondria as shown by evolution of 14 CO 2 from [1- 14 C]- and [2- 14 C]pyruvate. Citrate, isocitrate, succinate, and malate were not utilized by intact cells due to their inability to permeate the plasma membrane. For every mole of corticosterone formed, 1.9 and 0.8 moles of 14 CO 2 were formed from [1- 14 C]- and [2- 14 C]pyruvate, respectively, indicating that pyruvate dehydrogenase was quite active and supplied acetyl CoA to the Krebs cycle. Fluorocitrate and 2,4-dinitrophenol inhibited 11β-hydroxylation of 11-deoxycorticosterone as well as the production of 14 CO 2 from [2- 14 C]pyruvate. Comparison of data with the two inhibitors showed that for the same percentage of inhibition of 14 CO 2 production, the inhibition of 11β-hydroxylation was greater with 2,4-dinitrophenol than with fluorocitrate. It is concluded that operation of the Krebs cycle may be essential for 11β-hydroxylation to occur primarily because NADH generated by the cycle provides ATP, via the respiratory chain, as well as the substrate for the energy-linked transhydrogenase that forms NADPH. The NADPH required for 11β-hydroxylation seems to be derived to a large extent via the energy-linked transhydrogenase.

The glucocerebrosidase of human placenta was studied with various potential inhibitors. Several compounds that resemble the lipoidal product of enzyme action, ceramide, proved to be excellent inhibitors, acting by mixed modes (primarily noncompetitively). These were N-decyl-oL-erythro-3-phenyl-2-amino-l.3-propanediol and several p-substituted derivatives. These compounds were also highly effective in rat spleen toward glucocerebroside and p-nitrophenyl fi-glucoside as substrates. The compounds were inactive toward the analogous enzyme, galactocerebrosidase of rat brain, and were slightly stimulatory toward the rat brain enzyme which makes galactocerebroside. Longer and shorter N-alkyl groups proved to be less effective. Decanoic acid amides of phenylaminopropanediol and related compounds proved to be relatively inert, although some were stimulatory. Deoxycorticosterone P-glucoside was a moderately effective noncompetitive inhibitor and is apparently hydrolyzed by a different glucosidase. p-Nitrophenyl P-glucoside was also a moderately effective inhibitor, acting by mixed modes. p-Chloromercuribenzenesulfonate was a good inhibitor, presumably acting on a sensitive cysteine residue. It is concluded that cerebrosidase contains two sensitive sites, one catalytic and the other allosteric. each containing an important anionic group and able to bind glucosides and ceramide-like compounds.

Ribonuclease resistance of RNA from mammalian cells was studied as a possible measure of the extent of base pairing. Ribonuclease-sensitive and resistant components of the RNA were discerned from the biphasic kinetics of the ribonuclease reaction. The amount of resistant component increased progressively with increasing salt concentration until it comprised 20–40% of the total RNA. Comparisons between different RNA fractions from L1210 cells revealed differences in the content of resistant component, in the order: nucleolar ribosomal-precursor RNA > mature ribosomal RNA > nucleoplasmic RNA. In each RNA fraction, the amount of resistant component was greater when measured by a cytidine label than by a uridine label. Comparison with known double-stranded RNA indicated that the bulk of the resistant component consists of lessstable structures, such as short or imperfect helices.