Metabolic products of microorganisms

The effect of the nucleoside-peptide antibiotics nikkomycin Z, nikkomycin X, and polyoxin A was tested on chitosomal chitin synthetase from yeast cells of the dimorphic fungus Mucor rouxii. The K _i was 0.6 M for polyoxin A and 0.5 M for nikkomycin X; nikkomycin Z was slightly less inhibitory (K _i=3.5M). Whereas the minimum inhibitory concentrations of the nikkomycins for growth and germination were quite low (about 1M, or lower), polyoxin A displayed no antifungal activity against yeast cells and sporangiospores of the test organism, even when present in high concentrations. These results are discussed with respect to structure/activity relationships.Abbreviations MIC minimum inhibitory concentration (i.e. concentration required to completely suppress growth: cf. Drews, 1979) - GlcNAc N-acetyl-d-glucosamine - UDP-GlcNAc uridine 5-diphospho-N-acetyl-d-glucosamine Metabolic products of microorganisms. 202. H. P. Kaiser and W. Keller-Schierlein: Strukturaufklärung von Elaiophylin: Spektroskopische Untersuchungen und Abbau. Helv. Chim. Acta 64: 407–424 (1981)     

Chitin synthetase mutants of Phycomyces blakesleeanus

Mutants resistant to nikkomycin, an inhibitor of chitin biosynthesis, were isolated after exposure of wild-type spores of the fungus Phycomyces blakesleeanus to N-methyl-N-nitro-N-nitrosoguanidine. Genetic analysis revealed that nikkomycin resistance was due to mutations in a single gene, chsA. Mutants and wild type grew equally well in the absence of nikkomycin. In contrast to the wild type, whose spore germination and mycelial growth were inhibited by 5 M nikkomycin, chsA mutants grew reasonably well in the presence of 50 M nikkomycin. Chitin synthesis in vivo was much less affected by the drug in the mutants than in the wild type. Resistance was not due to impaired uptake or detoxification of the drug. Analysis of the kinetics of chitin synthesis in vitro showed that the mutants had a decreased K_a for the allosteric activator, N-acetylglucosamine, and gross alterations in nikkomycin inhibition kinetics. These results indicate that chsA is the structural gene for chitin synthetase, or at least for the polypeptide that bears the catalytic and allosteric sites.     

Synthesis of chitin by particulate preparations from Aspergillus flavus

Cell-free extracts from Aspergillus flavus catalyzed the synthesis of chitin from UDP-GlcNAc. Most of the activity was associated with membrane-rich fractions whereas no activity was detected in the cell walls. Chitin synthetase was activated by fungal acid proteases; animal and plant proteases destroyed it. Upon incubation at 0 C and 28 C chitin synthetase was inactivated, probably by the action of proteases present in the particulate preparations. Maximal activity was obtained at pH 6.6–7.1 and 15 C. Arrhenius plot showed a biphasic curve with the transition at 7 C. E values were 3300 Kcal/mole above this temperature and 15500 Kcal/mole below it. The enzyme was activated by GlcNAc and required a divalent metal, the most active being Mg⁺⁺. By plotting v vs UDP-GlcNAc concentration a sigmoidal curve was obtained. Km calculated at high substrate concentrations was 20mm. Chitin synthetase was competitively inhibited by polyoxin D (Ki 6.5 m) and UDP (Ki 1.35mm), the latter giving complex kinetics.This work was supported by grants No. 020 and 847 of the Consejo Nacional de Ciencia y Tecnología, México.Part of this work was carried out while the authors were research visitors at the Department of Plant Pathology, University of California, Riverside.     

Effects of polyoxin D on germination,morphological development and biosynthesis of the cell wall of Trichoderma viride

When polyoxin D is added to a spore suspension of Trichoderma viride at a concentration from 50–100 g/ml, it inhibits from 40–60% of germination. This percentage increases if dimethylsulfoxide (DMSO) is added.Mycelium growing in the presence of polyoxin D becomes irregular and loses its rigidity, showing several bulges along the hypha. Under the electron microscope the features of the cell wall and cytoplasmic content are apparently normal. Nevertheless, after incubation with different lytic systems or with (¹⁴C)glucose, it can be seen that polyoxin D partially inhibits the biosynthesis of -(1-3)glucan and the biosynthesis of chitin to a greater extent attaining inhibition of 83% at 100 g/ml of the antibiotic concentration.Regenerating protoplasts are less affected by polyoxin D. They do regenerate slower but the percentage of regeneration is more than 80%. Aberrant tubes synthesized by these protoplasts are not affected, they manifest their usual morphology and lack of chitin is confirmed in their composition.List of Abbreviations CECT Colecci Española de Cultivos Tipo - GAE Glucose, Asparagine and yeast Extract - DMSO Dimethylsulfoxide - PPO Diphenyloxazole - POPOP 1,4-bis-(4-methyl-5-phenyloxazol-2-yl)     

Polyamines and proline are affected by cadmium stress in nodules and roots of soybean plants

The effect of moderate (50 M) and high (200 M) doses of Cd were studied in relation to polyamine (Pas) metabolism, proline level and the glutamine synthetase/glutamate synthase system (GS/GOGAT) activity in nodules and roots of soybean plants during 6 days of treatment. The lower Cd concentration increased putrescine (Put) in both nodules and roots, while 200 M Cd increased Spm only in nodules and Put in roots. Spermidine (Spd) decreased in roots under both Cd concentrations. Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were both involved in Put biosynthesis in roots. In nodules, Put formation could mainly be attributed to ODC activity. Diamine oxidase (DAO) activity was severely reduced by 50 and 200 M Cd either in nodules or roots. The GS/GOGAT system activity was depressed either with 50 or 200 M Cd, but most significantly with the highest metal concentration. Under 200 M Cd, GS activity decayed to 25% or 60% of the control in nodules and roots, respectively, while GOGAT decreased 85% in nodules and 79% in roots by day 4 of treatment. Ammonium increased greatly in nodules (200% over the controls) and roots (100%) under 200 M Cd. Proline concentration increased significantly in nodules and roots under both Cd treatments, more markedly under 200 M Cd. The relationship between Pas and proline accumulation and nitrogen assimilation is discussed.     

Bioelectric and biosynthetic aspects of cell polarity inAllomyces macrogynus

Summary In contrast to all filamentous fungi examined to date, vegetative hyphae ofAllomyces macrogynus, whether extending or not, produced an outward flow of positive electrical current, at a maximum of 0.16 A cm^–2 around 40 m behind the apex, as measured with a vibrating probe. Inward currents of up to 0.55 A cm^–2 were recorded around the rhizoids. Increases in outward current were observed in hyphae pre-grown under oxygen deficiency and then allowed to widen backwards to the hyphal base in sufficient oxygen. When spores were germinated in an applied electrical field they produced rhizoids predominantly towards the anode. Hyphae were produced initially towards the cathode but later bent around towards the anode. Experiments with a range of chemicals provided no evidence for the involvement of calcium in vegetative growth and development inA. macrogynus. Polyoxin and nikkomycin, inhibitors of chitin synthesis, had no effect on swimming zoospores, but inhibited wall formation of cysts, rhizoids and forward and backward growing hyphae.     

Characterization of the NADH dehydrogenase and fumarate reductase of Fibrobacter succinogenes subsp. succinogenes S85

Conditions promoting maximal in vitro activity of the particulate NADH:fumarate reductase from Fibrobacter succinogenes were determined. This system showed a pH optimum of 6.0 in K⁺ MES buffer only when salt (NaCl or KCl) was present. Salt stimulated the activity eightfold at the optimal concentration of 150m M. This effect was due to stimulation of fumarate reductase activity as salt had little effect on NADH: decylubiquinone oxidoreductase (NADH dehydrogenase). The stimulation of fumarate reductase by salt at pH 6.0 was not due to removal of oxaloacetate from the enzyme. Kinetic parameters for several inhibitors were also measured. NADH dehydrogenase was inhibited by rotenone at a single site with a K _i of 1 M. 2-Heptyl-4-hydroxyquinonline-N-oxide (HOQNO) inhibited NADH: fumarate reductase with a K _i of 0.006 M, but NADH dehydrogenase exhibited two HOQNO inhibition constants of approximately 1 M and 24 M. Capsaicin and laurylgallate each inhibited NADH dehydrogenase by only 20% at 100 M. NADH dehydrogenase gave K _m values of 1 M for NADH and 4 M for reduced hypoxanthine adenine dinucleotide.Published with the approval of the Director of the Agricultural Experiment Station, North Dakota State University, as journal article no. 2201     

Activity of dihydrofolate reductase in the mollicutesAcholeplasma laidlawii andMycoplasma gallisepticum and their susceptibility to antifolates

Mycoplasma gallisepticum andAcholeplasma laidlawii were found to possess dihydrofolate reductases exhibiting similar specific activities and kinetics, with values in the range of those reported for other microorganisms. The apparent Km values for dihydrofolate in enzymes ofM. gallisepticum andA. laidlawii are 7.95±0.13 and 7.50±0.11 M, and for NADPNH 8.46±0.25 and 9.32±0.18 M, respectively.M. gallisepticum is 3300-fold more resistant to methotrexate than isA. laidlawii; concentrations causing 50% inhibition were 200.00 and 0.06 M, respectively. This is in contrast to almost the same sensitivity to that drug exhibited by the dihydrofolate reductases of both microorganisms.M. gallisepticum is also 3600-fold more resistant to trimethoprim than isA. laidlawii, and the concentrations for 50% inhibition of growth were 1800.0 and 0.5 M, respectively. The high resistance was found to be due partially to a 130-fold lower affinity of the target enzyme for this antifolate, but another mechanism, presumably impaired transport, must also be involved. This is the first report of dihydrofolate reductase activity in Mollicutes.     

Endogenous ethylene requirement for adventitious root induction and growth in tomato cotyledons and lavandin microcuttings in vitro

The role of ethylene in the formation of adventitious roots in vitro was studied in tomato (Lycopersicon esculentum Mill. cv. UC 105) cotyledons and lavandin (Lavandula officinalis Chaix × Lavandula latifolia microshoots. Both systems were able to form roots on hormone-free medium evolving low amounts of ethylene. The addition of 20–50 M indole-3-acetic acid (IAA) inhibited root formation in tomato cotyledons while increasing ethylene production. Naphthaleneacetic acid (NAA, 3 M) stimulated root number in lavandin explants and induced a transient rise in ethylene evolution. Enhanced ethylene levels via the endogenous precursors 1-aminocyclopropane-1-carboxylic acid (ACC, 25–50 M) drastically impaired root regeneration and growth in tomato. In lavandin, 10 M ACC stimulated ethylene production and significantly inhibited the rooting percentage and root growth. Conversely, ACC enhanced the root number in the presence of NAA only. Severe inhibition of rooting was also caused by ethylene reduction via biosynthetic inhibitors, aminoethoxyvinylglycine (AVG, 5–10 M) in tomato, and salicylic acid (SA, 100 M) in lavandin. A strict requirement of endogenous ethylene for adventitious root induction and growth is thus suggested.Abbreviations LS Linsmaier and Skoog medium - BA N⁶-benzyladenine - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - AVG Aminoethoxyvinylglycine - SA Salicylic acid - ACC 1-aminocyclopropane-1-carboxylic acid     

Binding of [3H]muscimol to calf cerebrocortical synaptic membranes and the effects of sulphur-containing convulsant and non-convulsant compounds

Endogenous and xenobiotic sulphur-containing convulsant and non-convulsant compounds containing structural moieties of, or bearing a structural resemblance to, GABA and homocysteine were tested in binding studies for their potency in displacing the GABA-mimetic [³H]muscimol from specific, high-affinity sites (K _d=3.6 nM;B _max=3.94 pmol/mg protein) on freeze-thawed, Triton-treated calf-brain synaptic membranes. The xenobiotic convulsants, 4-mercaptobutyric acid (MBA), 3-mercaptopropionic acid (3-MPA) and 2-mercaptopropionic acid (2-MPA) were found to be two-site competitive inhibitors exhibiting apparent inhibition affinity constants (K _i ^app ) of 5000 M, 3750 M, and 4800 M, respectively; while homocysteic acid (K _i ^app =4800 M) was shown to be a one-site partial competitive inhibitor. Intermediary metabolites of methionine: S-adenosyl-l-homocysteine,l-cysteine, the convulsantl-homocysteine, and its non-convulsant disulphide oxidation product, homocystine, were found to be one-site partial competitive inhibitors exhibitingK _i ^app values of 5750 M, 8350 M, 5000 M, and 510 M, respectively. The endogenous anticonvulsant neuroeffector, taurine, and the tripeptide, reduced glutathione (GSH) were shown to be, respectively, one-site (K _i=20 M) and two-site (K _i ^app =4300 M) competitive inhibitors of [³H]muscimol binding. These findings are discussed with regard to a previously proposed mechanism for the convulsant action of homocysteine.     

Responses of nitrogen-fixing and nitrate-supplied alfalfa (Medicago sativa L.) to iron chelates in an alkaline hydroponic medium

Ferric ethylenediamine di-(o-hydroxyphenylacetate) (FeEDDHA) and ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) were evaluated as Fe sources for hydroponic growth of alfalfa (Medicago sativa L., cv. Mesilla), either dependent on N₂ fixation or supplied with NO₃. The hydroponic medium was maintained at pH 7.5 by addition of CaCO₃. Nitrogen-fixing cultures were inoculated with Rhizobium meliloti 102 F51 and grown in medium without added nitrogen. After five to seven weeks of growth under greenhouse conditions, plants were harvested. Nitrogen fixation was measured by the acetylene reduction method.When FeEDDHA was supplied, growth of alfalfa, whether dependent on N₂ fixation or supplied with NO₃, was severely limited at concentrations typically used in hydroponic medium (10 or 20 M). Maximum yield of NO₃-supplied alfalfa was obtained at 100 M while maximum yield of N₂-fixing alfalfa was obtained in the range of 33 to 200 M FeEDDHA. Nodule fresh weights and N₂ fixation rates increased with FeEDDHA concentration up to 33 M and remained essentially constant up to 200 M. With FeHEDTA, maximum yields of both NO₃-grown and N₂-fixing alfalfa were obtained at 10 M. Growth of NO₃-supplied plants was inhibited at 200 M FeHEDTA while growth of N₂-fixing plants was inhibited at 100 M FeHEDTA. The numbers of nodules per plant increased between 3.3 and 10 M FeHEDTA; however, inhibition of nodule formation occurred at a concentration of 33 M or higher. Nodule weights per plant and N₂ fixation rates were depressed at 3.3 M as well as at 100 M FeHEDTA. The results suggest that alfalfa dependent on N₂ fixation is more sensitive to limited Fe availability than alfalfa supplied with NO₃.     

AT-1 Receptor and Phospholipase C Are Involved in Angiotensin III Modulation of Hypothalamic Noradrenergic Transmission

SUMMARY 1. We previously reported that angiotensin III modulates noradrenergic neurotransmission in the hypothalamus of the rat. In the present work we studied the effects of angiotensin III on norepinephrine release and tyrosine hydroxylase activity. We also investigated the receptors and intracellular pathways involved in angiotensin III modulation of noradrenergic transmission.2. In rat hypothalamic tissue labeled with [³H]norepinephrine 1, 10, and 100 nM and 1 M losartan (AT1 receptor antagonist) had no effect on basal neuronal norepinephrine release, whereas 10 and 100 nM and 1 M losartan partially diminished norepinephrine secretion evoked by 25 mM KCl. The AT2 receptor antagonist PD 123319 showed no effect either on basal or evoked norepinephrine release. The increase in both basal and evoked norepinephrine output induced by 1 M angiotensin III was blocked by 1 M losartan, but not by 1 M PD 123319.3. The phospholipase C inhibitor 5 M neomicin inhibited the increase in basal and evoked norepinephrine release produced by 1 M angiotensin III.4. Tyrosine hydroxylase activity was increased by 1 M angiotensin III and this effect was blocked by 1 M LST and 5 M neomicin, but not by PD 123319. On the other hand, 1 M angiotensin III enhanced phosphatidyl inositol hydrolysis that was blocked by 1 M losartan and 5 M neomicin. PD 123319 (1 M) did not affect ANG III-induced phosphatidyl inositol hydrolysis enhancement.5. Our results confirm that angiotensin III acts as a modulator of noradrenergic transmission at the hypothalamic level through the AT1-phospholipase C pathway. This enhancement of hypothalamic noradrenergic activity suggests that angiotensin III may act as a central modulator of several biological processes regulated at this level by catecholamines, such as cardiovascular, endocrine, and autonomic functions as well as water and saline homeostasis.     

Studies on the Mode of Action of Polyoxins

(1) Chitin-UDP acetylglucosaminyltransferase (E.C. 2.4.1.16., chitin synthetase) in the cell-free system from phytopathogenic fungus Piricularia oryzae, and effects of various polyoxins and related compounds on the enzyme activity were studied. Polyoxins A~M, polyoxin A derivatives, polyoxin C derivatives, 5′-amino-5′-deoxyuridine, uridine and thymidine inhibited equally the incorporation of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc) into chitin.

(2) Competition between the above inhibitors and UDP-GlcNAc was observed by kinetic studies. The Km for UDP-GlcNAc was determined to be 3.3 × 10^?3 m and the Ki values for polyoxins A~M, except polyoxin C, were found to be in the range of 3.3 × 10^?5 m to 3.4 × 10^?6 m. For polyoxin C, 5′-amino-5′-deoxyuridine and uridine, the Ki values of 2.7 × 10^?3 m, 8.0 × 10^?3 m and 3.0 × 10^?3 m were given, respectively. The inhibitor constants for other related compounds were also calculated.

(3) The values of binding affinity, ?ΔG, for formation of substrate- or inhibitor-enzyme complexes were calculated from the Km or Ki values. In addition, partial binding affinities, ?Δg, for certain moieties or groups of polyoxins were estimated from the ?ΔG. For instance, the ?ΔG values for UDP-GlcNAc and polyoxin L were 5.7 kcal/mole and 9.2 kcal/mole, respectively. And the ?Δg values for the nucleoside moiety (part I), the carbamylpolyoxamic acid moiety (part II) and the carboxyl group at C5′ position of polyoxin L were 5.2, 3.5 and 0.7 kcal/mole, respectively.

(4) From the results obtained, the mechanism of action and relation between chemical structure and competitive inhibition of chitin synthetase were discussed.

     

Clomipramine and Related Structures as Inhibitors of the Skeletal Sarcoplasmic Reticulum Ca2+ Pump

The Ca²⁺-pumping activity of skeletal sarcoplasmic reticulum vesicles is half-maximallyinhibited by 120 M clomipramine, 250 M desipramine, and 500 M imipramine or trimipramine.The inhibition is attributed to the dihydrodibenzazepine moiety, since3-(dimethylamino)propionitrile, reproducing the aliphatic amine chain, has no inhibitory action. The inhibitionis shown as a marked decrease of Ca²⁺ binding at equilibrium in theabsence of ATP and asa reduction of phosphorylation of the Ca²⁺-free conformation byinorganic phosphate. Therefore,the drug effect is consistent with preferential interaction of tricyclic antidepressants withthe Ca²⁺-free conformation of the nonphosphorylated enzyme. An additional decrease in theapparent rate constant of enzyme dephosphorylation, i.e., in the release of phosphate fromATP during enzyme cycling was also noticed.     

An alternative propagation method ofAnanas through nodule culture

A micropropagation scheme forAnanas comosus Merr. was developed using nodule culture. Nodules were induced from leaf-base or chopped shoot-base explants on modified half-strength MS medium supplemented with 2.69-5.37 M NAA and 4.44 M BA and could be maintained long-term as nodules. The nodules proliferated into more nodules when chopped into pieces of 1–3 mm and placed onto the same medium. They regenerated shoots when transferred to medium supplemented with 0.54–10.74 M NAA and 0.44–8.88 M BA. The regeneration capacity of nodules is higher than that of direct regeneration or callus. Maximum regeneration was obtained from culture medium containing 0.54 M NAA and 0.44 M BA, where shoots could be observed as early as within 2 weeks. Many shoots formed roots in the same medium in which they were regenerated after 10 subcultures, but the best rooting occurred in medium containing 0.54 M NAA and 0.44 M BA. Rooted plantlets ofA. comosus Merr. could be routinely produced at 6-week intervals.Abbreviations NAA naphthaleneacetic acid - BA 6-benzylaminopurine     

The use of cultured Acer cells as a screening tool for mitotic inhibitors. I. Reference herbicides

Several commercially available mitosis inhibitors have been studied for their effects on an Acer pseudoplatanus cell suspension culture; these were colchicine, N-(1,1-dimethylpropynyl)3-chlorobenzamide, propyzamide and trifluralin. The last two compounds are known as preemergence herbicides. In the studied cultures, the concentrations necessary to arrest mitosis were between 50 and 100 M for colchicine, and between 10 and 100 M for propyzamide, N-(1,1-dimethylpropynyl)3-chlorobenzamide and trifluralin. However, protein synthesis, plastid and mitochondria division and dry weight increase occurred in the treated cells, leading to the formation of giant cells. These results suggest that mitosis inhibition is the fundamental effect of these compounds at the concentrations studied. At these concentrations, metabolic activities seem unaffected, in contrast with what was found previously in the case of chlorpropham. This phenylcarbamate inhibited mitosis in the same cells at 100 M, but also prevented protein synthesis and weight increase, probably as a result of its uncoupling properties.The effect of another compound, glyphosate, on Acer cell suspension cultures, was studied concurrently. At concentrations between 10 and 100 M, it only slightly decreased protein formation in the culture, and was unable to prevent mitosis from occurring.The formation of giant cells in Acer cell suspension cultures seems to be a good criterion to demonstrate that selective mitosis inhibition has taken place. This criterion might be used in the case of mitosis inhibitors acting on the G ₂-phase.

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Résumé Les effets de divers inhibiteurs de la division cellulaire ont été étudiés sur des suspensions cellulaires d' Acer pseudoplatanus. Ces produits sont la colchicine, le N-(1,1-diméthylpropynyl)3-chlorobenzamide, le propyzamide et la trifluraline. Les 2 derniers composés sont des herbicides de pré-levée. Les concentrations nécessaires pour bloquer la mitose dans les suspensions cellulaires se situent de 50 à 100 M pour la colchicine et de 10 à 100 M pour les 3 autres composés.Dans les cellules traitées la synthèse protéique, la multiplication des plastes et des mitochondries et l'augmentation de masse sèche ont lieu, entraînant la formation de cellules géantes. Ces résultats suggèrent que, aux concentrations étudiées, le blocage de la mitose est l'effet primaire de ces produits; les activités métaboliques des cellules, à ces concentrations, ne semble pas affecté contrairement à ce que l'on a observé précédemment avec le chlorprophame. Ce phénylcarbamate, en effet, inhibe la mitose des cellules d'Acer á 100 M mais aussi, et probablement du fait de ses propriétés découplantes, empêche la synthèse protéique et l'augmentation de masse sèche. Le glyphosate étudié parallèlement est incapable de bloquer la mitose entre 10 et 100 M; il freine légèrement la synthèse protéique.L'obtention de cellules géantes dans les suspensions cellulaires d'Acer pseudoplatanus semble un bon test pour mettre en évidence une inhibition sélective de la mitose. Ce critère pourrait être utilisé pour les composés antimitotiques agissant sur la phase G ₂.

     

Distribution of magnesium in wheat (Triticum aestivum L.) in relation to supply

The aims of this study were to describe the distribution of magnesium (Mg) and its retranslocation within wheat, in order to develop diagnostic procedures for Mg deficiency. Plants were grown in solution culture with both constant supply (0, 5, 10, 20, 40, 80, 160 MMg) and discontinued supply (40 M and 160 M decreased to nil).Magnesium was depleted from old leaves when Mg supply to the roots was halted. However, initial deficiency symptoms occurred on young leaves under constant but inadequate supply, contrasting with previous reports. Magnesium concentrations were also lower in young leaves compared to old leaves. Symptoms of yellowing and necrosis occurred if the leaf tissue contained <1194 gg^–1, irrespective of leaf age. The minimum Mg concentration in whole shoots associated with maximum shoot weight was 932 gg^–1; for the youngest emerged blade (YEB) it was 861 gg^–1. Symptoms were apparent on the young leaf before a reduction in shoot weight was measurable. The concentration of Mg in the YEB and whole shoot were better related to solution Mg concentration than was the Mg concentration in the old leaf.     

Purification and characterization of fatty acid synthetase from Cryptococcus neoformans

Fatty acid synthetase has been purified from Cryptococcus neoformans 450 fold to a specific activity of 3.6 units per mg protein with an overall yield of 23%. The purified enzyme contained two non-identical subunits, Mr approximately 2.1×10⁵ and 1.8×10⁵. Under optimum conditions, 100 mM KCl and pH 7.5, apparent Km values for the substrates were: Acetyl CoA, 19 M; Malonyl CoA, 5 M; and NADPH, 6 M. Product inhibition patterns were determined to be: CoA, competitive versus acetyl CoA and malonyl CoA, uncompetitive versus NADPH; NADP, competitive versus NADPH, uncompetitive versus acetyl CoA and malonyl CoA; Palmitoyl CoA, competitive versus malonyl CoA, noncompetitive versus acetyl CoA and NADPH; Bicarbonate, uncompetitive versus malonyl CoA. These product inhibition patterns are consistent with the multisite ping-pong mechanism previously proposed for the avian fatty acid synthetase complex. The cryptococcal fatty acid synthetase was inhibited by the polyanionic polymers, heparin and dextran sulfate, an effect never before demonstrated for a fatty acid synthetase. This inhibition exhibited a marked dependence on the length of the polymer chain, with dextran sulfate fractions with Mr of 6×10⁵ and above having K _i values below 100 nanomolar. A model is presented that involves initial binding of the anionic polymer to the enzyme complex at a region of high positive charge density, followed by interaction of the end of the tethered polymer with the catalytic site. This study represents the first purification of fatty acid synthetase from a basidiomycete.     

Antagonistic interaction between auxins and the growth regulator NC 9634

The synthetic growth regulator NC 9634, [(3-phenyl-1,2,4-thiadiazol-5-yl)thio] acetic acid (NC 9634) was shown to have anti-auxin properties in bioassay tests. The inhibition of wheat coleoptile extension growth by concentrations of NC9634 up to 260 M was completely overcome by 60 M IAA, 50 M NAA or 50 M 2,4-D. In cress root tests 2.6 × 10^–7 M NC 9634 stimulated root elongation and relieved the growth inhibiting effect of 2,4-D. Extension growth of intact apple shoots was inhibited by NAA, which proved lethal at concentrations above 6.25 × 10^–4 M. NC 9634 applied in combination with the NAA, reduced this growth inhibiting effect and prevented death of shoots sprayed with high auxin concentrations.     

The action of divalen Zn,Cd, Hg,Cu and Pb ions on the ATPase activity of a plasma membrane fraction isolated from roots ofZea mays

The effects of the divalent metal ions Zn, Cd, Hg, Cu and Pb on the ATPase activity of a plasma membrane fraction isolated from roots ofZea mays have been investigated. When Mg-ions (3 mM), with or without K-ions (50mM) are included in the reaction medium, inhibition of ATPase activity was found in all cases, the relative order of the inhibitors over the concentration range 10 to 100M, being Hg>>CuCd>ZnPb. Below 1.0M only Hg caused substantial inhibition. In the absence of Mg ions, Zn and to a lesser extent Cd, activated the enzyme up to a concentration of 1 mM, activity being further stimulated in the presence of K-ions (50mM). No activation of ATPase activity was found for Hg, Cu or Pb. It was concluded that Zn-ATP and Cd-ATP are both alternative substrates for the enzyme. Further experiments showed that both K_m and V_max for the substrates Zn-ATP and Cd-ATP are very much lower than for the usual substrate Mg-ATP.These present results are discussed in relation to the known actions of these divalent cations on the trans-root potential and H-ion efflux in excised maize roots (Kennedy and Gonsalves, 1987).