The inhibition of β-glucosidase activity in Trichoderma reesei C30 cellulase by derivatives and isomers of glucose

The inhibition of β-glucosidase in Trichoderma reesei C30 cellulase by D -glucose, its isomers, and derivatives was studied using cellobiose and ρ-nitrophenyl-β-glucoside (PNPG) as substrates for determining enzyme activity. The enzymatic hydrolysis of both substrates was inhibited competitively by glucose with approximate K_i values of 0.5mM and 8.7mM for cellobiose and PNPG as substrate, respectively. This inhibition by glucose was maximal at pH 4.8, and no inhibition was observed at pH 6.5 and above. The α anomer of glucose inhibited β-glucosidase to a greater extent than did the β form. Compared with D -glucose, L -glucose, D -glucose-6-phosphate, and D -glucose-1-phosphate inhibited the enzyme to a much lesser extent, unlike D -glucose-L -cysteine which was almost as inhibitory as glucose itself when cellobiose was used as substrate. Fructose (2?100mM) was found to be a poor inhibitor of the enzyme. It is suggested that high rates of cellobiose hydrolysis catalyzed by β-glucosidase may be prolonged by converting the reaction product glucose to fructose using a suitable preparation of glucose isomerase.     

Immobilization of cellulase through its carbohydrate side chains — A rationale for its recovery and reuse

The major types of components of cellulase [see 1,4-(1, 3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] have been adsorbed onto concanavalin A immobilized on Sepharose 4B, suggesting that they are glycoproteins. These components were covalently coupled to cyanogen bromide-activated Sepharose after aminoalkylation of their periodate-oxidized carbohydrate side chains to provide additional points of attachment of the enzyme to the support. Although there was only a 9% recovery of starting avicelase activity, the immobilized enzyme catalysed the hydrolysis of insoluble cellulose to glucose with greater efficiency than did free cellulase.     

Some influences of CO2 enrichment, nitrogen nutrition and competition on grain yield and quality in spring wheat and barley

Spring wheat and spring barley were grown in elevated atmosphericCO₂ in controlled environments. Wheat was grown in monocultureand in competition with three weed species. In monoculture,wheat had 30% more grain yield and 28% less grain nitrogen inelevated compared to ambient atmospheric CO₂- In competition,wheat had no significant increase in yield with elevated atmosphericCO₂- In competition, grain nitrogen concentration was reducedin response to CO₂ with the largest reduction occurring withthe smallest competitor and the smallest reduction occurringwith the largest competitor. Spring barley was grown in monocultureat three nitrogen fertilizer supplies. In elevated atmosphericCO₂ there were significant increases in grain yield and reductionsin grain nitrogen concentration at all levels of nitrogen supply.In both species the reductions in grain nitrogen concentrationwere large enough to affect current bread making processes. Key words: Grain nitrogen, weeds, wheat, barley     

The influences of increased CO2 and water supply on growth,biomass allocation and water use efficiency of Sinapis alba L. grown under different wind speeds

We examined how independent and interactive effects of CO₂ concentrations, water supply and wind speed affect growth rates, biomass partitioning, water use efficiency, diffusive conductance and stomatal density of plants. To test the prediction that wind stress will be ameliorated by increased CO₂ and/or by unrestricted water supply we grew Sinapis alba L. plants in controlled chambers under combinations of two levels of CO₂ (350 ppmv, 700 ppmv), two water regimes and two wind speeds (0.3 ms^–1, 3.7 ms^–1). We harvested at ten different dates over a period of 60 days. A growth analysis was carried out to evaluate treatment effects on plant responses. Plants grown both in increased CO₂ and in low wind conditions had significantly greater stem length, leaf area and dry weights of plant parts. Water supply significantly affected stem diameter, root weight and leaf area. CO₂ enrichment significantly increased the rate of biomass accumulation and the relative ratio of biomass increase to leaf area expansion. High wind speed significantly reduced plant growth rates and the rate of leaf area expansion was reduced more than the rate of biomass accumulation. Regression analysis showed significant CO₂ effects on the proportion of leaf and stem dry weight to total dry weight. A marked plant-age effect was dependent on water supply, wind speed and CO₂ concentration. A reduced water supply significantly decreased the stomatal conductance, and water use efficiency significantly increased with a limited water supply, low wind and increased CO₂. We found significant CO₂ x wind effects for water diffusion resistance, adaxial number of stomata and water use efficiencies and significant wind x water effect for water use efficiency. In conclusion, wind stress was ameliorated by growing in unrestricted water but not by growing in increased CO₂.     

Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor beta subunits.

The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit-specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of alpha, precursor beta and processed beta subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of alpha and precursor beta but no processed beta subunits. These complexes possess short half-lives, are enzymatically inactive and their molecular weight is approximately 300 kDa. Radioactivity chases from these complexes into mature, long-lived approximately 700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing alpha subunits and the other, precursor beta subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor beta subunits PSMB2 (C7-I), PSMB3 (C10-II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the beta subunits and juxtaposition of the two beta rings) is accompanied by conformational changes in the (outer) alpha rings, and may be inefficient. Finally, interferon-gamma had no significant effect on the half-lives or total amounts of precursor or mature proteasomes.     

Immobilization of β-glucosidase from different sources on nylon tube

Cellulase from four different fungi and β-glucosidase from almonds were immobilized on the inner surface of nylon tubing. The highest values of β-glucosidase activity retention on the support were obtained when P. funiculosum and N. crassa were used as the enzyme source. A comparative study of the thermal stability referring to β-glucosidase activity was developed using free and immobilized enzymes. The most stable β-glucosidases (from P. funiculosum and A. niger) did not show an appreciable change in its thermal stability after immobilization. An important increase in thermal stability was observed when less stable β-glucosidases (from T. reesei, N. crassa and almonds) were immobilized.     

Effects of windspeed on the growth and biomass allocation of white mustard Sinapis alba L.

Summary We examined how different wind speeds and interactions between plant age and wind affect growth and biomass allocation of Sinapis alba L. (white mustard). Physiological and growth measurements were made on individuals of white mustard grown in controlled-environment wind tunnels at windspeeds of 0.3, 2.2 and 6.0 ms^–1 for 42 days. Plants were harvested at four different dates. Increasing wind speed slightly increased transpiration and stomatal conductance. We did not observe a significant decline in the photosynthetic rate per unit of leaf area. Number of leaves, stem length, leaf area and dry weights of total biomass and plant parts were significantly lower in plants exposed at high wind speed conditions. There were no significant differences in the unit leaf rate nor relative growth rates, although these were always lower in plants grown at high wind speed. Allocation and architectural parameters were also examined. After 42 days of exposure to wind, plants showed higher leaf area ratio, root and leaf weight ratios and root/shoot ratio than those grown at control treatment. Only specific leaf area declined significantly with wind speed, but stem and reproductive parts also decreased. The responses of plants to each wind speed treatment depended on the age of the plant for most of the variables. It is suggested that wind operates in logarithmic manner, with relatively small or no effect at lower wind speeds and a much greater effect at higher speeds. Since there is no evidence of a significant reduction in photosynthetic rate of Sinapis with increasing wind speed it is suggested that the effect of wind on plant growth was due to mechanical effects leading to changes in allocation and developmental patterns.     

Angiotensin II receptors in Xenopus oocytes.

Electrical recordings were used to study the sensitivity of native Xenopus oocytes to the octapeptide angiotensin II (AII). AII elicited oscillatory currents associated with an increase in membrane conductance to Cl-. Responsiveness to AII varied greatly between oocytes taken from different frogs, and to a lesser extent between oocytes from the same ovary. Oocytes from frogs showing high sensitivity had response thresholds between 0.5-1.0 nM AII, and at a holding potential of -60 mV, responded to 1 microM AII with currents greater than 3 microA. In contrast, oocytes from some frogs gave no response, even to 10 microM AII. A total of 618 oocytes from 79 frogs were tested for sensitivity to AII, and oocytes from 85% of frogs gave detectable electrical responses. Oscillatory Cl- currents elicited by AII were largely independent of extracellular Ca2+, were abolished by chelation of intracellular Ca2+ using EGTA and were mimicked by intraoocyte injection of inositol 1,4,5-trisphosphate (IP3). In addition to oscillatory Cl- currents, AII also evoked an influx of extracellular Ca2+, giving rise to a transient inward Cl- current on membrane hyperpolarizing steps. These experiments all suggested that AII responses were elicited through activation of an intracellular messenger pathway triggered by hydrolysis of inositolphospholipids, mobilization of intracellular Ca2+ by inositol polyphosphates, and activation of Ca(2+)-gated Cl- channels. The effect of manual or enzymic defolliculation on AII responses was studied in nine separate experiments recording from 70 defolliculated oocytes. Efficacy of defolliculation procedures was assayed using scanning electron microscopy, which confirmed removal of 90 to greater than 98% of follicular cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethanol Inhibition of N-Methyl-D-Aspartate-Stimulated Endogenous Dopamine Release from Rat Striatal Slices: Reversal by Glycine

N-Methyl-D-aspartate stimulated a concentration-dependent release of endogenous dopamine from rat striatal slices. The threshold for activation was between 10 and 25 microM and reached a maximum at 1 mM. Release was completely blocked by magnesium or tetrodotoxin. Ethanol (10-200 mM) significantly inhibited the N-methyl-D-aspartate-stimulated release of dopamine by 20-45%, with half-maximal inhibition occurring at approximately 21 mM. Addition of ethanol plus increasing concentrations of magnesium resulted in a greater inhibition of N-methyl-D-aspartate-stimulated dopamine release than that observed with magnesium alone. However, this effect appeared to be due to a noninteractive additive effect of the two antagonists, as the IC50 value for magnesium inhibition was not significantly altered by ethanol. Glycine, which had no effect on dopamine release by itself, completely reversed the inhibitory effects of ethanol (25 mM) at low micromolar concentrations. These results suggest that ethanol may produce its effects in striatal slices by interfering with a glycine modulatory site of the N-methyl-D-aspartate receptor-ionophore complex.