Substrate-dependent inhibition of yeast enolase by fluoride

A possibly physiologically significant inhibition of yeast enolase by fluoride occurs in the absence of inorganic phosphate. The inhibition increases with time, is strongly dependent on fluoride concentration and requires substrate and “catalytic” Mg²⁺. The inhibition increases more slowly in the presence of product (phosphoenolpyruvate) than substrate (2-phosphoglycerate). The dependence on fluoride concentration and the spans of substrate analogue displacement titrations suggest the inhibition is produced by two moles of fluoride per active site.     

Transgalactosylation and hydrolytic activities of commercial preparations of β-galactosidase for the synthesis of prebiotic carbohydrates

β-Galactosidases exhibit both hydrolytic and transgalactosylation activities; the former has been used traditionally for the production of delactosed milk and dairies, while the latter is being increasingly used for the synthesis of lactose-derived oligosaccharides: balance between both activities was highly dependent on the enzyme origin: β-galactosidases from Aspegillus oryzae and Bacillus circulans exhibited high transgalactosylation activity, while those from one from Kluyveromyces exhibited high hydrolytic activity but quite low transgalactosylation activity. Also the affinity for the donors (lactose or lactulose) and the acceptors (lactose, lactulose or fructose) of transgalactosylated galactose was dependent on the enzyme origin, as reflected by the Michaelis constants obtained in the synthesis of galacto-oligosaccharides, fructosyl-galacto-oligosaccharides and lactulose. Finally, the balance between transgalactosylation and hydrolytic activities of β-galactosidases could be tuned by changing the concentration of galactose donor.     

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot: III. Effects of Fluoride on ATPase Activity and Transport

The effects of fluoride on the tonoplast type ATPase and transport activities associated with sealed membrane vesicles isolated from sugarbeet (Beta vulgaris L.) storage tissue were examined. This anion had two distinct effects upon the proton-pumping vesicles. When ATP hydrolysis was measured in the presence of gramicidin D, significant inhibition (approximately 50%) only occurred when the fluoride concentration approached 50 millimolar. In contrast, the same degree of inhibition of proton transport occurred when the fluoride concentration was about 24 millimolar. Effects on proton pumping at this concentration of fluoride could be attributed to an inhibition of chloride movement which serves to dissipate the vesicle membrane potential. Valinomycin could partially restore ATPase activity in sealed vesicles which were inhibited by fluoride and this restoration occurred with a reduction in the membrane potential. Fluoride demonstrated a competitive interaction with chloride-stimulation of proton transport and inhibited the uptake of radioactive chloride into sealed vesicles. When the vesicles were allowed to develop a pH gradient in the absence of KCl, and KCl was subsequently added, fluoride reduced enhancement of the existing pH gradient by KCl. The results are consistent with a chloride carrier that is inhibited by fluoride.     

Catalytic versatility of trehalase: Synthesis of α-d-glucopyranosyl α-d-xylopyranoside from β-d-glucosyl fluoride and α-d-xylose

Trehalase was previously shown (see ref. 5) to hydrolyze α-d-glucosyl fluoride, forming β-d-glucose, and to synthesize α,α-trehalose from β-d-glucosyl fluoride plus α-d-glucose. Present observations further define the enzyme's separate cosubstrate requirements in utilizing these nonglycosidic substrates. α-d-Glucopyranose and α-d-xylopyranose were found to be uniquely effective in enabling Trichoderma reesei trehalase to catalyze reactions with β-d-glucosyl fluoride. As little as 0.2mm added α-d-glucose (0.4mm α-d-xylose) substantially increased the rate of enzymically catalyzed release of fluoride from 25mm β-d-glucosyl fluoride at 0°. Digest of β-d-glucosyl fluoride plus α-d-xylose yielded the α,α-trehalose analog, α-d-glucopyranosyl α-d-xylopyranoside, as a transient (i.e., subsequently hydrolyzed) transfer-product. The need for an aldopyranose acceptor having an axial 1-OH group when β-d-glucosyl fluoride is the donor, and for water when α-d-glucosyl fluoride is the substrate, indicates that the catalytic groups of trehalose have the flexibility to catalyze different stereochemical reactions.     

Methionine analogs inhibit production of β-subunit of soybean 7S protein

We have previously reported that exogenous methionine inhibits production of the β-subunit of the 7S storage protein in cultured soybean cotyledons, and that this inhibition involves lack of functional mRNA for the β-subunit. Analogs of methionine were used to study this inhibition. Cycloleucine, norleucine, norvaline and S-ethylcysteine treatments prevented accumulation of the β-subunit. The effects of cycloleucine and norleucine on β-subunit synthesis might have been indirect, since these compounds inhibited growth and caused a 2- to 3-fold increase in free methionine concentration. Norvaline did not affect free methionine concentration, but it did inhibit growth. Treatment with a combination of S-ethylcysteine and aminoethoxyvinylglycine prevented appearance of the β-subunit without inhibiting growth or raising the S-adenosylmethionine concentration. Thus, accumulation of S-adenosylmethionine does not appear to mediate the effect of exogenous methionine on β-subunit production. Treatment with S-ethylcysteine raised free methionine concentration only 34%, so S-ethylcysteine was probably acting directly to inhibit β-subunit production. Measurements of free methionine concentrations in seeds of different sizes, taken from intact plants, suggested that the relatively late appearance of the β-subunit in normal soybean seed development may be due to the presence of high levels of free methionine in very young seeds.     

Inhibition by heparin and dextran sulfate of stimulated rat pancreatic adenylate cyclase

Heparin inhibited the adenylate cyclase activity of semipurified rat pancreatic plasma membranes stimulated by hormones and by Gpp(NH)p but not by fluoride or when in the persistently active state. When observed, the inhibition was rapid and sustained. It was of a noncompetitive type and never exceeded 20% for secretin. The inhibition of Gpp(NH)p-stimulated activity was more pronounced (48% inhibition at a heparin concentration of 50 μg/ml). For the C-terminal octapeptide of pancreozymin (CCK-8)-stimulated adenylate cyclase, the inhibition amounted to 93% at 50 μg/ml. This inhibition was competitive at low heparin concentration and of a mixed type above 10 μg/ml. Besides, heparin inhibited (I₅₀ = 6 μg/ml) the binding of peptides of the CCK family to their specific receptors without affecting the apparent K_d value of binding. Taken together, these relatively specific effects of heparin gave evidence in favor of the existence of CCK spare receptors. Dextran sulfate was more potent than heparin as an inhibitor of adenylate cyclase activation while chondroitin-4-sulfate and chondroitin-6-sulfate were ineffective. Dansylated pancreatic plasma membranes exhibited characteristics of adenylate cyclase activation by CCK-8 which were similar to those found for untreated membranes exposed to heparin.     

Rheological and microstructural investigation of oat β-glucan isolates varying in molecular weight

The rheological properties and microstructure of aqueous oat β-glucan solutions varying in molecular weight were investigated. The structural features and molecular weights (MW) were characterized by ¹³C NMR spectroscopy and high performance size-exclusion chromatography (HPSEC), respectively. The microstructure of the β-glucans dispersions was also examined by atomic force microscopy (AFM). The samples with β-glucan content between 78 and 86% on a dry weight basis had MW, intrinsic viscosity ([η]) and critical concentration (c*) in the range of 142-2800 × 10³ g/mol, 1.7-7.2 dl/g and 0.25-1.10 g/dl, respectively. The flow and viscoelastic behaviour was highly dependent on MW and on the concentration of the β-glucans dispersions. Pseudoplastic behaviour was exhibited at high concentrations and Newtonian behaviour was evident at low concentrations. At the same concentration, the viscosity was higher for higher MW samples. The Cox-Merz rule was applicable for the lower molecular weight samples at higher concentrations whereas the high molecular weight sample deviated at concentrations greater than 1.0%, w/v. The mechanical spectra with variation of both MW and concentration were typical of entangled biopolymer solutions. AFM images revealed the formation of clusters or aggregates linked via individual polymer chains scattered heterogeneously throughout the system. The aggregate size increased with the molecular weight of the samples investigated and has been linked to the rheological behaviour of the samples.     

Changes in the level of lectins in the mantle of the mussel Mytilus trossulus in response to anthropogenic contaminants

The lectin activity in an extract from the mantle of the mussel Mytilus trossulus was tested for the first time. Using the method of the hemagglutination inhibition assay, it was shown that lectins were Gal/GalNAc-specific and best agglutinated with rabbit erythrocytes. The influence of foreign compounds on the lectin level in the M. trossulus mantle was examined. Upon cadmium acetate exposure, the level of lectin activity exhibited phasic alterations and depended on the contaminant concentration or the time of exposure. During exposure of mussels to a synthetic detergent or diesel fuel, changes in lectin contents were dependent on the time of exposure.     

Author index

The effect of the accumulation of β-galactosides on the uptake of P_i into cells and cell nucleotides was examined in ML strains of Escherichia coli. Nonmetabolizable sulfur analogs of lactose, which are accumulated only in the presence of the product of y gene of the Lac operon, inhibited the uptake of P₁ into whole cells and into cell nucleotides. This inhibition was most pronounced in starved cells, those with a low rate of ATP production. When the cell membrane was disrupted by sonication or detergents, the inhibition was lost. No significant inhibition was seen in y^? strains or in inducible y⁺ strains which were not induced. Hence. inhibition of the uptake of phosphate into nucleotides is dependent on the presence of the product of the y gene and a β-galactoside.A technique using ³²P_i and ³³P₁ was developed for simultaneously measuring the turnover and level of nucleotides. β-Galactosides inhibited ATP synthesis in aerobic cells, but stimulated ATP synthesis in anaerobic cells, indicating that an intermediate of oxidative phosphorylation was the source of energy for β-galactoside accumulation.     

Glyconamides as inhibitors of human beta-glucosidases and beta-galactosidases.

d-Gluconamide, d-gluconyl hydrazide, and N-(6-aminohexyl)-d-gluconamide were prepared from d-glucono-1,5-lactone by treatment with ammonia, hydrazine, and 1,6-diaminohexane, respectively. These d-gluconamide derivatives were tested for their inhibitory action on human liver lysosomal glucocerebrosidase and human spleen neutral aryl β-glucosidase. Analogous d-galactonamide derivatives were evaluated for their inhibition of human spleen galactocerebrosidase and G_M1-ganglioside β-galactosidase. d-Gluconyl hydrazide and d-gluconamide were effective inhibitors of the lysosomal glucocerebrosidase, attaining 50% inhibition at 5 and 12 mm, respectively. In contrast, N-(6-aminohexyl)-d-gluconamide did not inhibit the glucocerebrosidase. d-Gluconyl hydrazide was also the most effective inhibitor of human liver and spleen aryl β-glucosidase, 50% inhibition being achieved at 4 mm concentration (competitive inhibition, K_i = 0.4–0.9 mM). d-Galactonamide was the most effective inhibitor of spleen galactocerebrosidase; 4 mm d-galactonamide caused 50% inhibition of the enzyme activity (noncompetitive inhibition). N-(6-Aminohexyl)-d-galactonamide is a potent inhibitor (90% inhibition, 5 mm) of G_M1-ganglioside β-galactosidase but is without effect on galactocerebrosidase. It has, therefore, the potential usefulness in distinguishing between two of the galactosphingolipid β-galactosidases.     

Pyrodoxal 5′-phosphate inhibition of lactate production in rat liver supernatant fluid

The inhibitory effect of pyridoxal 5′-phosphate was examined on lactate production in rat liver 100000 × g supernatant fluid. Inhibition by pyridoxal 5′-phosphate was dependent on the duration of preincubation with supernatant fluid; lactate production exhibited a half-life of 5.5 min in the presence of 25 μM pyridoxal 5′-phosphate. The concentration of pyridoxal 5′-phosphate for 50% inhibition of lactate production from glucose as substrate was 10 μM and from glucose 6-phosphate as substrate, 185 μM.     

Effect of cations on the tyrosine kinase activity of the insulin receptor: Inhibition by fluoride is magnesium dependent

We have recently reported that fluoride interacts directly with the insulin receptor, which causes inhibition of its phosphotransferase activity. The inhibitory effect of fluoride on phosphotransferase activity is not due to the formation of complexes with aluminium and occurs in the absence of alterations to the binding of ATP or insulin. In this report we substantiate that the tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle shows a strict requirement of Mg²⁺ ions (Ka near 11 mM). This effect of Mg²⁺ was inhibited in a competitive manner by Mn²⁺, which is compatible with competition of both divalent ions for binding sites. The inhibition of tyrosine kinase activity caused by fluoride was dependent on the concentration of Mg²⁺ in the medium and no inhibitory effect was detected at low concentrations of Mg²⁺. Moreover, the addition of increasing concentrations of Mn²⁺ in the presence of a constant high concentr rease in the inhibitory effect of fluoride. These results indicate that the Mg-insulin receptor complex is the major fluoride-susceptible form. Based on the characteristics of the inhibition of tyrosine kinase shown by fluoride it might be proposed that its action is exerted by the formation of multi-ionic MgF complexes analogous to Pi, which bind to the insulin receptor kinase.     

Fed-batch cultures of recombinant Escherichia coli with inhibitory substance concentration monitoring

Fed-batch cultures of recombinant Escherichia coli HB101 were investigated to obtain high cell density and large amounts of β-galactosidase (β-gal). E. coli HB1010 was transformed with a hybrid plasmid pTREZ1, which contained a β-gal gene controlled by the trp promoter. In fed-batch cultures of recombinant E. coli, when the cell concentration reached around 13 g/l, the cell growth stopped and large amounts of inhibitory substances have accumulated in the broth. These inhibitory substances were isolated and identified. Acetate produced by the cells was evidently the main inhibitor of cell growth and β-gal production. Since the cells proved to assimilate acetate, the feed rate was controlled with acetate concentration monitoring in the fed-batch culture. As a result, the acetate concentration was maintained at a low level and cells grew smoothly without acetate-induced inhibition. Cell concentration and β-gal quantity reached high values of 28 g/l and 64 U/ml, respectively.     

Effects of cotton condensed tannin on feeding and digestion in the cotton pest, Heliothis zea

Condensed tannin of cotton incorporated into an artificial diet caused growth inhibition in H. zea larvae whose susceptibility was dependent upon the concentration of the dietary tannin, and also upon the size of the feeding larvae.Larvae feeding on tannin-treated diet exhibited both decreased protease and invertase activities in the midgut caecal wall, as well as lowered total protein and sugar levels in the haemolymph as compared to controls. These differences, however, had no effect on assilimation and efficiency of conversion of digested matter into animal biomass and therefore may be secondary effects. Ultimately, inhibition of growth is attributed to a reduction in food consumption.     

The effect of CPTA analogs and other nitrogenous compounds on the biosynthesis of carotenoids in Phycomyces blakesleeanus mutants

The inhibitory effect of a series of analogs of CPTA, 2-(4-chlorophenylthio)-triethylamine-HCl, and ammonia derivatives on carotenoid biosynthesis in Phycomyces blakesleeanus mutants was studied. The types of inhibition exhibited allowed no firm conclusions about the biosynthetic route to β-carotene from either β-zeacarotene or lycopene. However, the evidence suggests at present that both pathways are operative. It was found that a slight change in structure of inhibitor resulted in a different type of action. Conclusions based on a single inhibitor could be cited as “evidence” for a certain pathway.     

Lipopeptides from Bacillus strain AR2 inhibits biofilm formation by Candida albicans

The ability of the human fungal pathogen Candida albicans to reversibly switch between different morphological forms and establish biofilms is crucial for establishing infection. Targeting phenotypic plasticity and biofilm formation in C. albicans represents a new concept for antifungal drug discovery. The present study evaluated the influence of cyclic lipopeptide biosurfactant produced by Bacillus amyloliquefaciens strain AR2 on C. albicans biofilms. The biosurfactant was characterized as a mixture of iturin and fengycin by MALDI-TOF and amino acid analysis. The biosurfactant exhibited concentration dependent growth inhibition and fungicidal activity. The biosurfactant at sub-minimum growth inhibition concentration decreased cell surface hydrophobicity, hindered germ tube formation and reduced the mRNA expression of hyphae-specific gene HWP1 and ALS3 without exhibiting significant growth inhibition. The biosurfactants inhibited biofilm formation in the range of 46–100 % depending upon the concentration and Candida strains. The biosurfactant treatment dislodged 25–100 % of preformed biofilm from polystyrene plates. The biosurfactant retained its antifungal and antibiofilm activity even after exposure to extreme temperature. By virtue of the ability to inhibit germ tube and biofilm formation, two important traits of C. albicans involved in establishing infection, lipopeptides from strain AR2 may represent a potential candidate for developing heat stable anti-Candida drugs.     

Tyrosinase Inhibitory Effects and Antioxidative Activities of Saponins from Xanthoceras Sorbifolia Nutshell

Certain saponins are bioactive compounds with anticancer, antivirus and antioxidant activities. This paper discussed inhibitory effects of saponins from Xanthoceras Sorbifolia on tyrosinase, through the research of the rate of tyrosinase catalyzed L-DOPA oxidation. The inhibition rate of tyrosinase activity presented non-linear changes with the saponins concentration. The rate reached 52.0% when the saponins concentration was 0.96 mg/ml. Antioxidant activities of saponins from Xanthoceras Sorbifolia were evaluated by using hydroxyl and superoxide radical scavenging assays. The hydroxyl radical scavenging effects of the saponins were 15.5–68.7%, respectively at the concentration of 0.18–2.52 mg/ml. The superoxide radical scavenging activity reduced from 96.6% to 7.05% with the time increasing at the concentration of 1.44 mg/ml. All the above antioxidant evaluation indicated that saponins from Xanthoceras Sorbifolia exhibited good antioxidant activity in a concentration- dependent manner.     

Formation of β-cyanoalanine and pyruvate by Acacia georginae

Pyruvate is formed on incubation of l-cysteine with acetone powder preparations of Acacia georginae but in the presence of cyanide, β-cyanoalanine is produced and pyruvate production is highly depressed. The pH optimum for pyruvate production is 8·5. In the presence of fluoride (1·5 mM), the pH profile is unchanged and in the presence of cyanide (1·5 mM), minimal pyruvate production occurs at pH 8·5. Although addition of pyridoxal phosphate had no influence on pyruvate or β-Cyanoalanine production, these processes were prevented by sodium borohydride, an inhibitor of pyridoxal enzymes. Neither l-serine nor O-acetyl-l-serine serve as alternative substrates for pyruvate production. β-Fluoroalanine was not detected on incubating fluoride with an enzyme preparation from A. georginae acetone powders.