The NADP-linked glyceraldehyde-3-phosphate dehydrogenases of Anabaena variabilis and Synechocystis PCC 6803, which lack one of the cysteines found in the higher plant enzyme,are not reductively activated

Light activation of NADP-linked glyceraldehyde-3-P dehydrogenase involves reductive cleavage of a disulfide bond. We have proposed that the inactivating disulfide locks the two domains of the enzyme, preventing catalysis, and we have tentatively identified the two critical cysteine residues in the chloroplast enzyme (D. Li, F.J. Stevens, M. Schiffer and L.E. Anderson (1994) Biophys J. 67: 29–35). We reasoned that if activation of this enzyme involves these cysteines that enzymes lacking one or both should be active in the dark and insensitive to reductants. One of these cysteines is present in the enzymes from Anabaena variabilis and Synechocystis PCC 6803 but the other is not. Consistent with the proposed mechanism, glyceraldehyde-3-P dehydrogenase is not affected by DTT-treatment in extracts of either of these cyanobacteria. Fructosebisphosphatase is DTT-activated in extracts of both of these cyanobacteria and glucose-6-P dehydrogenase is inactivated in Synechocystis, as in higher plant chloroplasts. Apparently reductive modulation is possible in these cyanobacteria but glyceraldehyde-3-P dehydrogenase is not light activated.     

Influence of L-sorbose on the location of β-glucosidase in Trichoderma pseudokoningii

Abstract The effect of l -sorbose on growth, morphology, cell wall composition and β-glucosidase location has been examined with Trichoderma pseudokoningii . Sorbose-grown cultures exhibited a longer lag phase, a tendency to more frequent hyphal branching and showed a decreased cell wall content of β-1,3-glucan. In sorbose-containing cultures, a significant higher portion of total β-glucosidase was present in the culture fluid, whereas in sorbose-lacking control cultures the major part of activity was associated with the cell walls. The results support the previous hypothesis (Kubicek, C.P. (1982) Arch. Microbiol. 132, 349–354) that β-1.3-glucan is involved in cell wall binding of β-glucosidase in Trichoderma pseudokoningii .     

Cloning and characterization of β-galactoside and β-glucoside hydrolysing enzymes of Thermotoga maritima

Abstract A gene library of the hyperthermophilic bacterium Thermotoga maritima strain MSB8 was constructed in Escherichia coli . Two non-related T. maritima chromosomal DNA fragments were physically characterized. They conferred the synthesis of thermostable X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside)-hydrolysing activity upon the host organism. The biochemical properties of the recombinant enzymes indicated that genes for a β-galactosidase (BgaA) and a broad-specificity β-glucosidase (Bg1A) had been isolated. The genes were desiignted bgaA and bglA , respectively. According to analytical size exclusion chromatography data, BgaA and BglA had native molecular masses of approximately 240 kDa and 95 kDa, respectively. Both enzymes apparently have dimeric subunit structure. An additional β-glucosidase (designated BglB) activity, clearly distinct from BglA in terms of substrate specificity, could be detected in a crude extract of T. maritima .     

Purification and properties of a β-glucosidase from Penicillium oxalicum autolysates

A beta-glucosidase from the medium of an autolyzed culture of Penicillium oxalicum has been purified by tannic acid precipitation, sephacryl S-200, DEAE-Biogel, CM-Biogel and Mono Q successively. The purification process produced a homogeneous band in the SDS-PAGE that correspond to a Mr of 133,500. The enzyme had a pl of 4, and the active optima were found at pH 5.5 and 55 degrees C. The enzyme hydrolyzed different substrates showing maximum affinity against p-nitrophenyl-beta-D-glucoside with a Km value of 0.37 mM. The beta-glucosidase was inhibited by Glucono-D-lactone but not by glucose in the concentration range of 1 to 10 mM. The enzyme was adsorbed by Concanavalin-A-Sepharose.     

Selection and study of mutants of Dekkera intermedia and Candida wickerhamii derepressed for β-glucosidase production

Abstract Mutants of Candida wickerhamii and Dekkera intermedia , derepressed for β-glucosidase biosynthesis, were isolated. These mutants were also shown to hyperproduce this enzyme. In anaerobic culture, the C. wickerhamii mutant still hyperproduced β-glucosidase and was derepressed. Glucose-cellobiose anaerobic fermentation by this strain was thus improved. On the other hand, the D. intermedia mutant did not show any difference from the wild-type strain in anaerobic culture.     

Some antitumor derivatives of ellipticine deprived of mutagenic properties

Seven derivatives of the antitumor alkaloid ellipticine were assayed for activity against murine leukaemia L1210 and for mutagenicity in Ames' Salmonella-microsomes test. Not only did the results show a complete lack of correlation between these two properties, but it was possible to choose a highly efficient analog which was completely devoid of mutagenic and hence, probably, carcinogenic effect. The lack of interaction of this product (2-methyl-9-hydroxyellipticinium acetate) with Cytochrome P-450 of hepatic monooxygenases prevents the formation of reactive intermediates and their subsequent binding to DNA. These data are discussed in view of the currently admitted mode of action of ellipticines i.e., intercalation in DNA and their therapeutic use.     

Regulation of erythrocyte membrane shape by Ca2+

In the presence of 1.0 mM ATP and MgCl₂, the specific viscosity of suspensions of human erythrocyte ghosts decreases 35% in 20 minutes at 22°C. The changes in viscosity are a sensitive index of Mg-ATP dependent shape changes in these membranes. Low concentrations of Ca²⁺ (1 to 5 μM) inhibit Mg-ATP dependent viscosity changes. If ghosts were preincubated with 1 mM Mg-ATP and 20 μM A23187 to produce a maximal decrease in viscosity, addition of 10 μM Ca²⁺ to the preincubated ghosts increased the viscosity to levels observed in ghosts preincubated without ATP. Ca²⁺ (1 to 5 μM) also inhibited Mg²⁺ dependent phosphorylation 30% and stimulated dephosphorylation 25% in ghost membranes. These effects of Ca²⁺ on viscosity and phosphorylation may be due to a membrane bound Ca²⁺ phosphatase activity which dephosphorylates membranes phosphorylated by a Mg²⁺ dependent kinase activity.     

Respiratory energy costs for the maintenance of biomass, for growth and for ion uptake in roots of Carex diandra and Carex acudformis

van der Werf, A., Kooijman, A., Welschen, R. and Lambers, H. 1988. Respiratory energy costs for the maintenance of biomass, for growth and for ion uptake in roots of Carex diandra and Carex acutiformis. - Physiol. Plant. 72: 483–491. The respiratory characteristics of the roots of Carex diandra Schrank and Carex acutiformis Ehrh. were investigated. The aims were, firstly to determine the respiratory energy costs for the maintenance of root biomass, for root growth and for ion uptake, and secondly to explain the higher rate of root respiration and ATP production in C. diandra. The three respiratory energy components were derived from a multiple regression analysis, using the relative growth rate and the net rate of nitrate uptake as independent variables and the rate of ATP production as a dependent variable. Although the rate of root respiration and ATP production was significantly higher in C. diandra than in C. acutiformis, the two species showed no significant difference in their rate of ATP production for the maintenance of biomass, in the respiratory energy coefficient for growth (the amount of ATP production per unit of biomass produced) and the respiratory energy coefficient for ion uptake (amount of ATP production per unit of ions absorbed). It is concluded that the higher rate of root respiration of C. diandra is caused by a higher rate of nitrate uptake. At relatively high rates of growth and nitrate uptake, the contribution of the rate of ATP production for ion uptake to the total rate of ATP production amounted to 38 and 25% for C. diandra and C. acutiformis, respectively. At this growth rate, the respiratory energy production for growth contributed 37 and 50%, respectively, to the total rate of ATP production. The relative contribution of the rate of ATP production for the maintenance of biomass increased from 25 to 70% with increasing plant age for both species. The results suggest that ion uptake is one of the major sinks for respiratory energy in roots. These experimentally derived values for the rate of ATP production for the maintenance of biomass, the respiratory energy coefficient for growth and the respiratory energy coefficient for ion uptake are discussed in relation to other experimentally and theoretically derived values.     

Possible involvement of NADPH requirement in regulation of Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase levels in rat liver

Summary Previous studies examining regulation of synthesis of Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase in rat liver have focussed on the induction of these enzymes by different diets and some hormones. However, the precise mechanism regulating increases in the activities of these enzymes is unknown and the factors involved remain unidentified. Considering that many of these metabolic conditions occur simultaneously with the increase of some NADPH consuming pathway, in particular fatty acid synthesis, we suggest that the activities of Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase could be regulated through a mechanism involving changes in the NADPH requirement. Here, we have studied the effect of changes in the flux through different NADPH consuming pathways on the NADPH/NADP ratio and on Glucose-6-Phosphate and 6-Phosphogluconate levels. The results show that: i) an increase in consumption of NADPH, caused by activation of fatty acid synthesis or the detoxification system which consumes NADPH, is paralleled by an increase in levels of these enzymes; ii) when increase in consumption of NADPH is prevented, Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase levels do not change.Abbreviations G6PDH Glucose-6-Phosphate Dehydrogenase - 6PGDH 6-Phosphogluconate Dehydrogenase - ME Malic Enzyme - NF Nitrofurantoin - CumOOH Cumene Hydroperoxide - t-BHP t-Butyl hydroperoxide - BCNU 1,3,-Bis (2-chloroethyl)-1-nitrosourea - GR Glutathione Dehydrogenase - 2-ME 2-Mercaptoethanol - DTT Dithiothreitol - NADP B-Nicotinamide-Adenine Dinucleotide Phosphate - NADPH B-Nicotinamide-Adenine Dinucleotide Phosphate Reduced - EDTA Ethylenediaminetetraacetic Acid - GSH Glutathione Reduced Form - GSSG Glutathione Oxidized Form     

Beta-glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose

An inducible mycelial beta-glucosidase from Scytalidum thermophilum was characterized. The enzyme exhibited a pI of 6.5, a carbohydrate content of 15%, and an apparent molecular mass of about 40 kDa. Optima of temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable up to 1 h at 50 degrees C and exhibited a half-life of 20 min at 55 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-d-glucopyranoside, p-nitrophenyl-beta-d-xylopyranoside, o-nitrophenyl-beta-d-galactopyranoside, p-nitrophenyl-alpha-arabinopyranoside, cellobiose, laminaribiose and lactose. Kinetic studies indicated that the same enzyme hydrolyzed these substrates. Beta-Glucosidase was activated by glucose or xylose at concentration varying from 50 to 200 mM. The apparent affinity constants (K0.5) for glucose and xylose were 36.69 and 43.24 mM, respectively. The stimulatory effect of glucose and xylose on the S. thermophilum beta-glucosidase is a novel characteristic which distinguish this enzyme from all other beta-glucosidases so far described.     

Effect of β-glucanases on Penicillium oxalicum cell wall fractions

The polysaccharidic effect of a purified 1,3- β -glucanase, a purified β -glucosidase, and of partially purified endo-1,3- β -glucanase from autolysed Penicillium oxalicum cultures on cell wall isolate fractions from the same fungus were studied.
Fractionation of 5-day-old cell wall gave rise to a series of fractions that were identified using infrared spectrophotometry. The fractions used were: F₁, an α -glucan; F₃, a β -glucan; F₄, a chitin-glucan; and F_4b, a β -glucan. The fractions were incubated with each of the enzymes and with a mixture of equal parts of the three enzymes and the products of the enzymatic hydrolysis were analyzed after 96 h incubation.
The enzymes were found to degrade fraction F_4b ( β -glucan); the greatest degree of hydrolysis was reached when the three enzymes were used together, suggesting the need for synergic action by these enzymes in the cell wall degradation process.     

Induction and preliminary characterization of intracellular β-glucosidases from a cellulolytic Streptomyces strain

Abstract The cellulolytic actinomycete Streptomyces sp. QM-B814 posasses an intracellular β-glucosidase system which is induced by cellobiose and carboxymethylcellulose. Maximal β-glucosidase activity was attained 8–10 h after inducer addition to exponential phase growing cultures. The induction is depressed in the presence of glucose. The system is composed of two electrophoretically different β-glucosidase forms showing relative molecular masses of about 60 and 35 kDa, and p I values in the range 4.2–4.5. Both β-glucosidases are synthesized de novo. The enzymes share substrate preference and are both inhibited by δ-gluconolactone and p -chloromercuribenzoate. The induction pattern and glucose inhibition are similar for both enzymes.     

β-Glucosidase and β-Galactosidase in Primary Cultures of Rat Astrocytes: Comparison to the Brain Enzymes

In primary astrocyte cultures beta-glucosidase (EC 3.2.1.21) and beta-galactosidase (EC 3.2.1.23) showed pH optima and Km values identical to rat brain enzymes, using methylumbelliferyl glycosides and labeled gluco- and galactocerebrosides as substrates. The activities of both glycosidases increased in culture up to 3-4 weeks. In rat brain only galactosidase increased; glucosidase activity declined between 12-20 days after birth. The specific activities were two- to sixfold higher in astrocyte cultures than in rat brain. These activities were not due to uptake of enzymes from the growth medium. Secretion of beta-galactosidase, but not beta-glucosidase nor acid phosphatase could be demonstrated. These results support the suggestion of a degradative function for astrocytes in the brain.     

Nicotinamide Adenine Dinucleotide Phosphate Phosphatase Facilitates Dark Reduction of Nitrate: Regulation by Nitrate and Ammonia

Leaves of 15 - 30-d-old plants of sunflower and jute were harvested at 10.00 or 23.00 (local time) and measured immediately, or those harvested at 10.00 were incubated for one hour in sunlight either in water or 5 mM methionine sulfoximine (MSX) solution and then for three hours in dark either in water or 15 mM KNO3 solution. Nitrate feeding during dark incubation, in general, increased nitrate reductase (NR) and nitrite reductase (NiR) activities, and NADH and soluble sugar contents. Increase in tissue nitrate concentration in MSX fed but not in control samples suggested reduction of nitrate in dark. NADPH-dependent NR activity increased considerably upon feeding with nitrate in dark. Concomitantly, NADPH phosphatase activity was also increased in nitrate treated, dark incubated leaves. It is proposed that nitrate regulates dark nitrate reduction by facilitating generation of NADH from NADPH by NADPH phosphatase. High amounts of ammonia accumulated in MSX treated, but not in control leaves, upon dark incubation. Relative activities of NR and NADPH phosphatase, and amounts of soluble sugar and NADH were low in MSX fed samples compared to that of control. So, high amount of ammonia might partially repress NADPH phosphatase and consequently deprive NR of reducing equivalents. This revised version was published online in July 2006 with corrections to the Cover Date.