Production of l-Arginine by Arginine Hydroxamate-Resistant Mutants of Bacillus subtilis

l-Arginine hydroxamate inhibited the growth of various bacteria, and the inhibition was readily reversed by arginine. l-Arginine hydroxamate (10(-3)m) completely inhibited the growth of Bacillus subtilis. This inhibitory effect was prevented by 2.5 x 10(-4)ml-arginine, which was the most effective of all the natural amino acids in reversing the inhibition. l-Arginine hydroxamate-resistant mutants of Bacillus subtilis were isolated and found to excrete l-arginine in relatively high yields. One of the mutants, strain AHr-5, produced 4.5 mg of l-arginine per ml in shaken culture in 3 days.     

Effects of some antitumor agents on growth and glycolytic enzymes of the flagellate Crithidia

Some antitumor agents known to specifically inhibit certain tumor cell enzymes were examined for activity against glycolytic enzymes and growth of the insect trypanosomatid, Crithidia fasciculata. The cytoplasmic enzymes hexokinase, alpha-glycerophosphate dehydrogenase, malic dehydrogenase, and glucose-6-phosphate dehydrogenase were tested. Agaricic acid (2-hydroxy-1,2,3-nonadecane tricarboxylic acid) was highly inhibitory (50 to 100%) to malic and alpha-glycerophosphate dehydrogenases at approximately 3 x 10(-5)m; 2-(p-hydroxyphenyl)-2-phenylpropane (2 x 10(-4)m), and 5,6-dichloro-2-benzoxazolinone (5 x 10(-4)m) were less effective (50% inhibition) against them. The antiprotozoal agents primaquine (4 x 10(-4)m) and Melarsoprol (8 x 10(-4)m) were 30 to 40% inhibitory. Agaricic acid, 2-(p-hydroxyphenyl)-2-phenylpropane, and 5,6-dichloro-2-benzoxazolinone inhibited growth of Crithidia at less than 10(-4)m. Eight other test compounds from the Cancer Chemotherapy National Service Center (CCNSC) were not toxic to cell growth, although two (4-biphenylcarboxylic acid and 1-[p-chlorobenzyl]-2-ethyl-5-methyl-indole-3-acetic acid) inhibited Crithidia alpha-glycerophosphate dehydrogenase below 1 mm. All of the compounds used specifically inhibited cancer cell alpha-glycerophosphate dehydrogenase. The corresponding enzyme in pathogenic African trypanosomes is important in their terminal respiration. C. fasciculata may be useful in preliminary evaluation of chemotherapeutic agents as potential trypanocides.     

Amino acid transport in a polyaromatic amino acid auxotroph of Saccharomyces cerevisiae

The initiation of growth of a polyaromatic auxotrophic mutant of Saccharomyces cerevisiae was inhibited by several amino acids, whereas growth of the parent prototroph was unaffected. A comparative investigation of amino acid transport in the two strains employing (14)C-labeled amino acids revealed that the transport of amino acids in S. cerevisiae was mediated by a general transport system responsible for the uptake of all neutral as well as basic amino acids. Both auxotrophic and prototrophic strains exhibited stereospecificity for l-amino acids and a K(m) ranging from 1.5 x 10(-5) to 5.0 x 10(-5) M. Optimal transport activity occurred at pH 5.7. Cycloheximide had no effect on amino acid uptake, indicating that protein synthesis was not a direct requirement for amino acid transport. Regulation of amino acid transport was subject to the concentration of amino acids in the free amino acid pool. Amino acid inhibition of the uptake of the aromatic amino acids by the aromatic auxotroph did not correlate directly with the effect of amino acids on the initiation of growth of the auxotroph but provides a partial explanation of this effect.     

Growth of obligate autotrophic bacteria on glucose in a continuous flow-through apparatus

Nitrosomonas europaea, Nitrobacter agilis, Thiobacillus denitrificans, T. neapolitanus, and T. thioparus (all obligate autotrophic bacteria) have been grown in dialysis culture, on glucose salts media, in the absence of their specific inorganic energy source. Metabolic products for N. agilis grown on nitrite salts medium were identified as keto acids. Pyruvic acid inhibited this organism at 5 x 10(-5)m. Keto acids were not inhibitory for the thiobacilli grown on thiosulfate medium. However, when T. denitrificans was grown on glucose with dialysis, addition of 5 x 10(-4)m pyruvate inhibited growth. Thus, it appears pyruvate may be inhibitory for other autotrophs, as has been reported for T. thiooxidans.     

Mutagen Stability of Alkylation-Sensitive Mutants of Bacillus subtilis

The phosphotransacetylase of Veillonella alcalescens catalyzes a reversible reaction with Michaelis-Menten kinetics for all substrates. The rate of the reverse reaction (the synthesis of acetyl coenzyme A from acetyl phosphate) was 6.5 times greater than the rate of the forward reaction (the synthesis of acetyl phosphate from acetyl coenzyme A). The apparent K(m) values determined for the forward reaction were 8.6 x 10(-6)m for acetyl coenzyme A and 9.3 x 10(-3)m for phosphate. In the reverse reaction, the K(m) values were 3.3 x 10(-4)m for coenzyme A and 5.9 x 10(-4)m for acetyl phosphate. The results of an analysis of the inhibition by end products in the forward and reverse directions were compatible with a random bi- bi- mechanism. The enzyme was inhibited by adenosine triphosphate and adenosine diphosphate but was not affected by reduced nicotinamide adenine dinucleotide or pyruvate. The inhibition by adenosine triphosphate was noncompetitive with respect to acetyl phosphate and competitive with respect to coenzyme A. MgCl(2) reversed the inhibition by adenosine triphosphate or adenosine diphosphate. The role of Mg(2+) and adenylates in the regulation of phosphotranscetylase activity is discussed.     

Regulation of Starch Biosynthesis in Plant Leaves: Activation and Inhibition of ADPglucose Pyrophosphorylase

The ADPglucose pyrophosphorylases of 7 plant-leaf tissues were partially purified and characterized. In all cases the enzymes showed stability to heat treatment at 65 degrees for 5 minutes in the presence of 0.02 m phosphate buffer, pH 7.0. The leaf ADPglucose pyrophosphorylases were activated 5 to 15-fold by 3-phosphoglycerate. Fructose-6-phosphate and fructose 1, 6-diphosphate stimulated ADPglucose pyrophosphorylase to lesser extents. The A(0.5) (conc of activator required to give 50% of the observed maximal activation) of 3-phosphoglycerate for the barley enzyme was 7 x 10(-6)m while for the sorghum enzyme it was 3.7 x 10(-4)m. Inorganic phosphate proved to be an effective inhibitor of ADPglucose synthesis. The I(0.5) (conc of inhibitor that gave 50% inhibition of activity for the various leaf enzymes varied from 2 x 10(-5)m (barley) to 1.9 x 10(-4)m (sorghum). This inhibition was reversed or antagonized by the activator 3-phosphoglycerate. These results form the basis for an hypothesis of the regulation of leaf starch biosynthesis.     

Effect of spermine on the uptake of amino acids in Micrococcus lysodeikticus

Spermine inhibited the transport of neutral aliphatic amino acids (valine, leucine, isoleucine, alanine, and glycine) into cells of Micrococcus lysodeikticus. On the other hand, spermine did not affect the uptake of basic (arginine and histidine), acidic (glutamic acid), or aromatic (phenylalanine and tyrosine) amino acids. Inhibition of uptake of the neutral amino acids by spermine is apparently of a noncompetitive nature; the V(max) decreased, whereas the apparent K(m) remained unaltered. The inhibition is most likely due to a specific binding of spermine to the carrier(s) of these amino acids. Related polyamines, spermidine and cadaverine, also caused inhibition of valine uptake, though to a lesser extent; spermidine was less active than spermine, and cadaverine showed the weakest effect of all. Valine, leucine, and isoleucine were transported into M. lysodeikticus cells by a common carrier as evidenced from competition experiments. The uptake of these amino acids is an active process; it was temperature-dependent and inhibited by azide (10(-1)m to 2.5 x 10(-2)m) and dinitrophenol (10(-3)m). The intracellular concentration of valine was 100-fold higher than in the medium.     

Growth Inhibition in Thiobacillus neapolitanus by Histidine, Methionine, Phenylalanine, and Threonine

Thiobacillus neapolitanus, a strict chemoautotroph, is sensitive to the addition of 10(-4)m methionine, histidine, threonine, or phenylalanine to the thiosulfate medium on which it grows. When histidine, threonine, or phenylalanine are added at the time of inoculation, spontaneous mutants tolerant to the three amino acids are selected. These mutants appear to result from a single genetic change; of 18 independently isolated histidine-tolerant mutants, all are also tolerant to phenylalanine and threonine. The uptake of (14)C-phenylalanine into exponentially growing cells of one such mutant is negligible in contrast with the uptake observed in the phenylalanine-sensitive parent. The addition of methionine to the medium slows growth, but spontaneous mutants are not selected. Inhibition of growth by these amino acids is observed only under conditions of amino acid imbalance; the addition of an equimolar mixture of 16 amino acids, in which each component is present at a concentration of 10(-3)m, causes no inhibition. Histidine and threonine inhibition may be released by equimolar amounts of any one of seven amino acids: serine, alanine, glycine, leucine, valine, tryptophan, or tyrosine; histidine inhibition is also released by isoleucine, and threonine inhibition by methionine. None of the inhibiting amino acids inhibits oxidation of thiosulfate in cell suspensions. A group of hexoses, pentoses, and Krebs cycle intermediates were tested for inhibition of growth or release of inhibition by histidine, phenylalanine, or threonine, but no effects, either inhibition or relief of inhibition, were found.     

Comparative physiologial studies of the yeast and mycelial forms of Histoplasma capsulaum: uptake and incorporation of L-leucine

l-Leucine entered the cells of both morphological forms of Histoplasma capsulatum by a permease-like system at low external concentrations of substrate. However, at levels greater than 5 x 10(-5)m l-leucine, the amino acid entered the cells both through a simple diffusion-like process and the permease-like system. The rate of the amino acid diffusion into yeast and mycelial forms appeared to be the same, whereas the initial rate of accumulation through the permease-like system was 5 to 10 times faster in the mycelial phase than it was in the yeast phase. The Michaelis constants were 2.2 x 10(-5)m in yeast phase and 2 x 10(-5)m in mycelial phase cells. Transport of l-leucine at an external concentration of 10(-5)m showed all of the characteristics of a system of active transport, which was dependent on temperature and pH. Displacement or removal of the alpha-amino group, or modification of the alpha-carboxyl group abolished amino acid uptake. The process was competitively inhibited by neutral aliphatic side-chain amino acids (inhibition constants ranged from 1.5 x 10(-5) to 6.2 x 10(-5)m). Neutral aromatic side-chain amino acids and the d-isomers of leucine and valine did not inhibit l-leucine uptake. These data were interpreted to mean that the l-leucine transport system is stereospecific and is highly specific for neutral aliphatic side-chain amino acids. Incorporation of l-leucine into macromolecules occurred at almost the same rate in both morphological forms of the fungus. The mycelial phase but not the yeast phase showed a slight initial lag in incorporation. In both morphological forms the intracellular pool of l-leucine was of limited capacity, and the total uptake of the amino acid was a function of intracellular pool size. The initial rate of l-leucine uptake was independent of the level of intracellular pool. Both morphological forms deaminated and degraded only a minor fraction of the accumulated leucine.     

Antibacterial nitroacridine, Nitroakridin 3582: effects on bacterial growth and macromolecular biosynthesis in vivo

The antibacterial drug Nitroakridin 3582 inhibited the growth of selected grampositive bacteria more strongly than it inhibited the growth of gram-negative bacilli. Nitroakridin at concentrations of the order of 5 x 10(-5)m induced lysis of Bacillus licheniformis and Micrococcus lysodeikticus. At concentrations less than 10(-4)m, Nitroakridin 3582 reduced the exponential growth rate of Escherichia coli C-2; at 10(-4)m the drug was bacteriostatic, and, at concentrations greater than 10(-4)m, it was bactericidal. Prolonged bacteriostasis resulted in the formation of long filaments by E. coli, Serratia marcescens, Shigella sonnei, and Proteus mirabilis. The reversible effects of Nitroakridin 3582 on the growth of E. coli correlated with partial inhibitions of deoxyribonucleic acid biosynthesis; ribonucleic acid and protein syntheses were inhibited less strongly. Nitroakridin 3582 at concentrations greater than 2 x 10(-4)m, which block deoxyribonucleic acid biosynthesis, produced an accelerated bactericidal action.     

Mechanism of action of nalidixic acid on Escherichia coli. Vi. Cell-free studies

The effects of nalidixic acid in vitro on deoxyribonucleic acid (DNA)- polymerase (deoxyribonucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7), deoxyribonucleotide kinases (ATP: deoxymono- and diphosphate phosphotransferases), and deoxyribosyl transferase (nucleoside: purine deoxyribosyltransferase, EC 2.4.2.6) were examined employing partially purified and crude extracts of Escherichia coli ATCC 11229 and E. coli 15TAU. Nalidixic acid had no inhibitory effect on the DNA-polymerase of the wild-type strain E. coli ATCC 11229 at concentrations of 1.4 x 10(-3) to 2.8 x 10(-3)m. No inhibition of deoxyribonucleotide kinase activity was observed at concentrations of nalidixic acid ranging from 2 x 10(-3) to 8.6 x 10(-3)m. Nalidixic acid (0.43 x 10(-4) to 0.43 x 10(-3)m) had no inhibitory effect on the deoxyribosyl transferase activity of crude extracts obtained from E. coli ATCC 11229 or E. coli 15TAU. Analytical CsCl density gradient centrifugation demonstrated that the DNA obtained after treatment of E. coli 15TAU with nalidixic acid was not cross-linked. These results suggest that the prevention of DNA synthesis in vivo by nalidixic acid is not attributable to inhibition of DNA polymerase, deoxyribonucleotide kinase, deoxyribosyl transferase, or to cross-linking of the DNA of treated cells.     

Dimerization of ferulic and caffeic acids by purified peroxidase isolated from Bupleurum salicifolium callus culture

A novel peroxidase that catalyses the transformation of caffeic acid and ferulic acid via oxidative coupling was purified from callus cultures of Bupleurum salicifolium petioles. The enzyme, which was purified over 2,900-fold, is a glycoprotein with a molecular weight of 38,000, determined by SDS/PAGE and gel filtration. The K(m) values obtained were 2.4x10(-4) M for caffeic and 2.6x10(-4) M for ferulic acid, while the K(m) values for H2O2 with caffeic acid was 4x10(-5) M and for H2O2 with ferulic acid was 4.8x10(-4) M. The purified peroxidase exhibits lower activity with typical peroxidase substrates (guaiacol and pyrogallol) than it does with caffeic and ferulic acids, but does not exhibit any activity with other phenylpropanoids tested (cinnamic acid, coumaric acid, and 3,4-dimethoxycinnamic acid).     

Tetracycline Inhibition of a Lipase from Corynebacterium acnes

A lipase which hydrolyzes triglycerides (tricaprylin and trilaurin) and naphthyl laurate was obtained from the broth of Corynebacterium acnes cultures by ammonium sulfate fractionation. Ca(2+) and sodium taurocholate stimulated activity of the enzyme. Ethylenediaminetetraacetic acid (EDTA) did not inhibit activity of the Ca(2+)-activated enzyme, but lipolytic activity was inhibited by EDTA in the absence of Ca(2+). Tetracycline (10(-4)m) produced a slight inhibition of the lipase activity with 5 x 10(-5)m or less showing no effect on the lipase activity. However, complete inhibition by tetracycline at 10(-4)m was observed for Ca(2+)-activated enzyme. Tetracycline inhibition of the C. acnes lipase could be demonstrated at concentrations as low as 10(-6)m.     

Regulation of synthesis of glutamine synthase in Bacillus subtilis

A study of the regulation of the synthesis of the enzyme glutamine synthase in Bacillus subtilis was initiated. An assay, based on the measurement of glutamo-hydroxamate, was used to characterize the enzyme in crude preparations and in toluene-treated cells. Determinations were made of the Michaelis constants for adenosine triphosphate, hydroxylamine, and glutamate (9 x 10(-3), 4 x 10(-3), and 2.2 x 10(-2)m, respectively), the pH optimum (7.6 to 7.7), and the stability. The differential rate of synthesis was determined under various growth conditions. The enzyme was found to be relatively insensitive to regulation. Partial repression was caused by glutamine, arginine, asparagine, and glutamate, or by carbon limitation in a chemostat. Derepression was caused by exhaustion of externally added amino acids or by nitrogen limitation in a chemostat.     

Amino Acid Transport into Cultured Tobacco Cells: I. LYSINE TRANSPORT

Lysine transport into suspension-cultured Wisconsin-38 tobacco cells was observed. Uptake was linear (up to 90 minutes) with respect to time and amount of tissue only after 4 to 6 hours preincubation in calcium-containing medium. The observed cellular accumulation of lysine was against a concentration gradient and not due to exchange diffusion. Transport was stimulated by low pH and characterized by a biphasic uptake isotherm with two K(m) values for lysine. System I (K(m) approximately 5 x 10(-6) molar; V(max) approximately 180 nanomoles per gram fresh weight per hour) and system II (K(m) approximately 10(-4) molar; V(max) approximately 1900 nanomoles per gram fresh weight per hour) were inhibited by N-ethylmaleimide and a variety of respiratory inhibitors. This inhibition was not due to increased efflux. In antagonism experiments, system I was inhibited most effectively by basic amino acids, followed by the sulfur amino acids. System I was only slightly inhibited by the neutral and aromatic amino acids and was not inhibited by the acidic amino acids aspartic and glutamic acids. Transport by system II was inhibited by all of the tested amino acids (including aspartic and glutamic acids) and analogs; however, this system was not inhibited by d-arginine. Neither system was strongly inhibited by d-lysine or the lysine analog S-2-aminoethyl-l-cysteine. Arginine was shown to be a competitive inhibitor of both systems with values for K(i) similar to the respective K(m) values.These studies suggest the presence of at least two amino acid permeases in W-38 tobacco cells.     

大麻素抑制大鼠三叉神经节神经元ATP激活电流

本文在培养的大鼠三叉神经节(trigeminal ganglion,TG)神经元上采用全细胞膜片钳技术,探讨大麻素对大鼠TG神经元ATP激活电流(ATP-activated currents,IATP)的影响.结果显示(1)胞外给予ATP,大部分受检细胞(67/75,89.33%)可记录到一个内向电流,且具有剂量依赖性.该电流可被P2X嘌呤受体特异性拮抗剂PPADS所阻断.(2)预加WIN55212-2[大麻素受体1(cannabinoid receptor 1,CB1受体)激动剂]可对IATP产生抑制作用,此作用呈剂量依赖性,并可被CB1受体特异性拮抗剂AM281阻断.预加不同浓度的WIN55212-2(1x10-13、1x10-12、1x10-11、1x10-10、1x10-9和1x10-8mol/L)对IATP(1x10-4mol/L ATP)的抑制作用分别为(8.14±3.14)%、(20.11±2.72)%、(46.62±3.51)%、(72.16±5.64)%、(80.21±2.80)%和(80.59±3.55)%.(3)预加WIN55212-2后IATP的浓度-反应曲线明显下移;最大反应浓度时的IATP幅值减小了(58.02±4.21)%,而阈值基本不变;预加WIN55212.2前后曲线的EC50值非常接近(1.15x10-4mol/L vs 1.27x10-4 mol/L).(4)预加forskolin[腺苷酸环化酶(adenylyl cyclase,AC)激动剂]或8-Br-cAMP可以逆转WIN55212-2对IATP的抑制作用.以上结果表明,大麻素可能作用于CB1受体,通过抑制AC-cAMP-PKA途径发挥对IATP的抑制作用.     

Responsiveness of RNA degradation to amino acids in cultured rat hepatocytes: comparison with isolated rat hepatocytes.

The role of amino acids in the regulation of RNA degradation was investigated in cultured hepatocytes from fed rats previously labeled in vivo with [6-14C]orotic acid. Rates of RNA degradation were determined between 42 and 48 h of culture from the release of radioactive cytidine in the presence of 0.5 mM unlabeled cytidine. The fractional rate was about 4.4 +/- 0.4%/h in the absence of amino acids (0x). The catabolism of RNA was decreased to basal level (1.5 +/- 0.3%/h) by the addition of amino acids at 10 times normal plasma concentration (10x). The inhibition of RNA degradation, expressed as percentage of maximal deprivation-induced response (0x minus 10x), averaged 60% at normal plasma levels of amino acids. The degree of responsiveness was greatly improved as compared to freshly isolated hepatocytes (20%) and was similar to the sensitivity previously observed with perfused livers. In cultured hepatocytes, the sensitivity of RNA degradation to amino acids was not affected by varying the volume of medium from 1 to 4 ml per dish. In freshly isolated hepatocytes, the inhibitory effect of amino acids was not modified by changing the cell density from 0.5 to 5 x 10(6) cells per ml. In the range of normal plasma concentration of amino acids, the low sensitivity of RNA degradation in isolated hepatocytes persisted with inhibition ranging from 10 to 20%. These findings suggest that the control of RNA degradation in both cultured and isolated hepatocytes is not affected by the total quantity of amino acids available in the medium, but their concentration is crucial. Electron microscopy observations and the inhibitory effect of 3-methyl-adenine in cultured rat hepatocytes partially confirmed the role of the lysosomal system in the increase of RNA degradation and its regulation by amino acids.     

Comparative study of oestrogenic properties of eight phytoestrogens in MCF7 human breast cancer cells

Previous studies have compared the oestrogenic properties of phytoestrogens in a wide variety of disparate assays. Since not all phytoestrogens have been tested in each assay, this makes inter-study comparisons and ranking oestrogenic potency difficult. In this report, we have compared the oestrogen agonist and antagonist activity of eight phytoestrogens (genistein, daidzein, equol, miroestrol, deoxymiroestrol, 8-prenylnaringenin, coumestrol and resveratrol) in a range of assays all based within the same receptor and cellular context of the MCF7 human breast cancer cell line. The relative binding of each phytoestrogen to oestrogen receptor (ER) of MCF7 cytosol was calculated from the molar excess needed for 50% inhibition of 3H]oestradiol binding (IC50), and was in the order coumestrol (35x)/8-prenylnaringenin (45x)/deoxymiroestrol (50x)>miroestrol (260x)>genistein (1000x)>equol (4000x)>daidzein (not achieved: 40% inhibition at 10(4)-fold molar excess)>resveratrol (not achieved: 10% inhibition at 10(5)-fold molar excess). For cell-based assays, the rank order of potency (estimated in terms of the concentration needed to achieve a response equivalent to 50% of that found with 17beta-oestradiol (IC50)) remained very similar for all the assays whether measuring ligand ability to induce a stably transfected oestrogen-responsive ERE-CAT reporter gene, cell growth in terms of proliferation rate after 7 days or cell growth in terms of saturation density after 14 days. The IC50 values for these three assays in order were for 17beta-oestradiol (1 x 10(-11)M, 1 x 10(-11)M, 2 x 10(-11)M), and in rank order of potency for the phytoestrogens, deoxymiroestrol (1 x 10(-10)M, 3 x 10(-11)M, 2 x 10(-11)M)>miroestrol (3 x 10(-10)M, 2 x 10(-10)M, 8 x 10(-11)M)>8-prenylnaringenin (1 x 10(-9)M, 3 x 10(-10)M, 3 x 10(-10)M)>coumestrol (3 x 10(-8)M, 2 x 10(-8)M, 3 x 10(-8)M)>genistein (4 x 10(-8)M, 2 x 10(-8)M, 1 x 10(-8)M)/equol (1 x 10(-7)M, 3 x 10(-8)M, 2 x 10(-8)M)>daidzein (3 x 10(-7)M, 2 x 10(-7)M, 4 x 10(-8)M)>resveratrol (4 x 10(-6)M, not achieved, not achieved). Despite using the same receptor context of the MCF7 cells, this rank order differed from that determined from receptor binding. The most marked difference was for coumestrol and 8-prenylnaringenin which both displayed a relatively potent ability to displace [3H]oestradiol from cytosolic ER compared with their much lower activity in the cell-based assays. Albeit at varying concentrations, seven of the eight phytoestrogens (all except resveratrol) gave similar maximal responses to that given by 17beta-oestradiol in cell-based assays which makes them full oestrogen agonists. We found no evidence for any oestrogen antagonist action of any of these phytoestrogens at concentrations of up to 10(-6)M on either reporter gene induction or on stimulation of cell growth.     

The kinetics of the reduction of isocyanides, acetylenes and the cyanide ion by nitrogenase preparation from Azotobacter chroococcum and the effects of inhibitors

1. Nitrogen-fixing preparations from Azotobacter chroococcum reduced substrates with the following K(m) values: methyl isocyanide, 1.8x10(-4)m; ethyl isocyanide, 2.5x10(-2)m; cyanide ion, 1.4x10(-3)m; acetylene, 1.2x10(-4)m. 2. Nitrogen, carbon monoxide or hydrogen competitively inhibited isocyanide reduction with the following K(i) values: hydrogen, 1.3x10(-3)m; carbon monoxide, 6.8x10(-6)m; nitrogen, 4.3x10(-4)m. 3. Living nitrogen-fixing bacteria, and isolated clover nodules, formed methane from methyl isocyanide. 4. These results are discussed in relation to other work and possible mechanisms of nitrogen fixation.     

Mechanism of D-cycloserine action: alanine racemase from Escherichia coli W

The antibiotic d-cycloserine is an effective inhibitor of alanine racemase. The lack of inhibition by l-cycloserine of alanine racemase from Staphylococcus aureus led Roze and Strominger to formulate the cycloserine hypothesis. This hypothesis states that d-cycloserine has the conformation required of the substrates on the enzyme surface and that l-cycloserine cannot have this conformation. Alanine racemase from Escherichia coli W has been examined to establish whether these observations are a general feature of all alanine racemases. The enzyme (molecular weight = 95,000) has Michaelis-Menten constants of 4.6 x 10(-4)m and 9.7 x 10(-4)m for d- and l-alanine, respectively. The ratio of V(max) in the d- to l-direction is 2.3. The equilibrium constant calculated from the Haldane relationship is 1.11 +/- 0.15. Both d- and l-cycloserine are competitive inhibitors with constants (K(i)) of 6.5 x 10(-4)m and 2.1 x 10(-3)m, respectively. The ratio of K(m)d-alanine to K(i)d-cycloserine is 0.71, and the ratio of K(m)l-alanine to K(i)l-cycloserine is 0.46. Since l-cycloserine is an effective inhibitor, it is concluded that the cycloserine hypothesis does not apply to the enzyme from E. coli W.