Rhus vernicifera (Rv) laccase was purified to electrophoretic homogeneity by hydrophobic interaction chromatography. A comprehensive study of the direct electrochemistry of Rv laccase covalently immobilized at a gold electrode using alkanethiol monolayers was undertaken. The observed midpoint potential was 410 mV versus the normal hydrogen electrode (NHE), consistent with reduction potentials obtained by potentiometric titration for the T1 copper site. Evidence is presented for a concerted 4-electron reversible process at slow scan rates (v) on the basis of peak current ratios (i(pa)/i(pc)). Catalytic currents were observed in the presence of the biological substrate oxygen, indicating that laccase activity is retained throughout the immobilization process. Electrochemical characteristics of the immobilized laccase were essentially invariant across the pH range 5.5-8.5 and the temperature range 5-35 degrees C. The purified enzyme displayed a pH optimum of 9.0, when assayed spectrophotometrically with syringaldazine as a substrate. Inhibition of the laccase activity with azide or fluoride showed an I(50)(NaN(3)) of 2.5 mM and an I(50)(NaF) of 18.5 mM. Electrochemistry in the presence of azide reduces the anodic current by ca. one-half, consistent with the 4-electron process decreasing to a 2-electron process. However, fluoride has no effect on anaerobic electrochemistry. These electrochemical results suggest that the pH dependence of laccase activity is related to the effects of pH on the structure or binding of the substrate.

The aim of this study was to investigate how mixed culture of wheat (Triticum aestivum L.) with white lupin (Lupinus albus L.) improves the growth and phosphorus (P) nutrition of the wheat. Wheat and white lupin plants were grown either in mixed culture or in monoculture in soil columns containing an adequate supply of nitrogen but a limited supply of P. Mixed-culture of the wheat with the lupins increased the shoot growth and shoot P uptake of the wheat by 33 and 45%, respectively, without significantly affecting the growth or P uptake of the lupins. After 6 weeks of plant growth, the soil columns were leached first with distilled water and then with 10 mM citric acid monohydrate (pH 4.0). In the initial fraction of the water leachate, the phosphate concentration for the lupin monocultures was very high, as high as for the uncultivated soil and approximately 10-fold higher than for the wheat monocultures. In contrast, in the initial fraction of the citric acid leachate, the phosphate concentration for the lupin monocultures was very low, approximately only 10% of the concentration for the wheat monocultures. For the mixed cultures, the phosphate concentrations for all fractions for both the water and the citric acid leachates were consistently low. Thus, the lupin monocultures preferentially depleted the citric acid-leachable soil P pool whereas the wheat monocultures preferentially depleted the water-leachable soil P pool and the mixed cultures depleted both pools. The lupin monocultures lowered the soil pH by 0.3 pH units whereas the wheat monocultures raised it by 0.8 pH units and the mixed cultures gave a soil pH intermediate between the two monocultures. Thus, the lupins in the mixed cultures partially offset the alkalinisation of the soil caused by the wheat and vice versa. This will be discussed in relation to the impact of soil pH on the plant availability of soil P.Abbreviations: P -phosphorus * FAX No: +61-8-8201 3015.

The rate of NH4+ assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH4+ assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H14CO-into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinations of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, [1990] Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C3 organism.The assimilation of NH4 and the net synthesis of amino acids requires the provision of carbon skeletons in the form of keto acids. Some of the most important keto acids used in amino acid synthesis are the TCA2 cycle intermediates 2-OG and OAA (5,8 by PEPC (4,9). This is also the case for many other C3 photosynthetic organisms (6, 7).Recently, we reported the partial purification ofPEPC from S. minutum and examined its kinetic and regulatory properties (9). Given the need for this enzyme to support the assimilation of N, there should be a tight coupling between the rate of N assimilation and its in vivo activity. The objectives of the present study were: (a) to determine the relationship between the rate of NW assimilation and the activity of PEPC in vivo and (b) to determine whether metabolite effectors of S. minutum PEPC in vitro, change in concentration in vivo in a manner consistent with metabolite regulation of this enzyme in intact cells. To fulfill these objectives we developed a system which allowed us to establish the rate of NfW+ assimilation as an independent variable. Using this system we show a linear relationship between the rate of NW assimilation and the activity of PEPC in vivo. We also show that changes in cellular levels of PEPC effectors in response to alterations in the rate of N assimilation are consistent with metabolite regulation of PEPC during N assimilation. MATERIALS AND METHODS Organism and Culture ConditionsThe green alga Selenastrum minutum (Naeg.) Collins (UTEX 2459) was grown in N-limited chemostats as previously described ( 12

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Phosphoenolpyruvate carboxylase (PEPC) was purified 40-fold from soybean (Glycine max L. Merr.) nodules to a specific activity of 5.2 units per milligram per protein and an estimated purity of 28%. Native and subunit molecular masses were determined to be 440 and 100 kilodaltons, respectively, indicating that the enzyme is a homotetramer. The response of enzyme activity to phosphoenolpyruvate (PEP) concentration and to various effectors was influenced by assay pH and glycerol addition to the assay. At pH 7 in the absence of glycerol, the Km (PEP) was about twofold greater than at pH 7 in the presence of glycerol or at pH 8. At pH 7 or pH 8 the Km (MgPEP) was found to be significantly lower than the respective Km (PEP) values. Glucose-6-phosphate, fructose-6-phosphate, glucose-I-phosphate, and dihydroxyacetone phosphate activated PEPC at pH 7 in the absence of glycerol, but had no effect under the other assay conditions. Malate, aspartate, glutamate, citrate, and 2-oxoglutarate were potent inhibitors of PEPC at pH 7 in the absence of glycerol, but their effectiveness was decreased by raising the pH to 8 and/or by adding glycerol. In contrast, 3-phosphoglycerate and 2-phosphoglycerate were less effective inhibitors at pH 7 in the absence of glycerol than under the other assay conditions. Inorganic phosphate (up to 20 millimolar) was an activator at pH 7 in the absence of glycerol but an inhibitor under the other assay conditions. The possible significance of metabolite regulation of PEPC is discussed in relation to the proposed functions of this enzyme in legume nodule metabolism.The anaplerotic fixation of CO2 by PEPC3 (EC 4.1.1.31) is believed to play a variety of roles in support of symbiotic N2 fixation by legume root nodules. These include: (a) the synthesis of dicarboxylic acids (malate, succinate) used as respiratory substrates by the bacteroids (10, 20), (b) provision of carbon skeletons for 20), and (c) synthesis of organic acids to maintain charge balance and neutral pH intracellularly and in the xylem (8). Most of the evidence for the first two functions comes from physiological studies ' Supported by the Natural Sciences and Engineering Research Council of Canada.2 Present address: