The effects of dark-induced stress on the evolution of the soluble metabolites present in senescent soybean (Glycine max L.) nodules were analysed in vitro using (13)C- and (31)P-NMR spectroscopy. Sucrose and trehalose were the predominant soluble storage carbons. During dark-induced stress, a decline in sugars and some key glycolytic metabolites was observed. Whereas 84% of the sucrose disappeared, only one-half of the trehalose was utilised. This decline coincides with the depletion of Gln, Asn, Ala and with an accumulation of ureides, which reflect a huge reduction of the N(2) fixation. Concomitantly, phosphodiesters and compounds like P-choline, a good marker of membrane phospholipids hydrolysis and cell autophagy, accumulated in the nodules. An autophagic process was confirmed by the decrease in cell fatty acid content. In addition, a slight increase in unsaturated fatty acids (oleic and linoleic acids) was observed, probably as a response to peroxidation reactions. Electron microscopy analysis revealed that, despite membranes dismantling, most of the bacteroids seem to be structurally intact. Taken together, our results show that the carbohydrate starvation induced in soybean by dark stress triggers a profound metabolic and structural rearrangement in the infected cells of soybean nodule which is representative of symbiotic cessation.

Bradyrhizobium japonicum is a symbiotic nitrogen-fixing soil bacteria that induce root nodules formation in legume soybean (Glycine max.). Using (13)C- and (31)P-nuclear magnetic resonance (NMR) spectroscopy, we have analysed the metabolite profiles of cultivated B. japonicum cells and bacteroids isolated from soybean nodules. Our results revealed some quantitative and qualitative differences between the metabolite profiles of bacteroids and their vegetative state. This includes in bacteroids a huge accumulation of soluble carbohydrates such as trehalose, glutamate, myo-inositol and homospermidine as well as Pi, nucleotide pools and intermediates of the primary carbon metabolism. Using this novel approach, these data show that most of the compounds detected in bacteroids reflect the metabolic adaptation of rhizobia to the surrounding microenvironment with its host plant cells.

The enzyme P-galactosidase (EC 3.2.1.23) from Aspergillus niger was purified and resolved into three multiple forms, using molecular sieving, ion-exchange, and hydrophobic chromatography. The isolated enzyme forms accounted for 83 %, 8 % and 9 % of the total P-galactosidase activity, respectively. They were glycoproteins with estimated molecular weights of 124000, 150000 and 173 000, isoelectric points of about 4.6, and pH optima between 2.5 and 4.0. Amino acid and carbohydrate analyses showed that multiplicity was mainly due to dissimilar carbohydrate contents (about 12.5 %, 20.5 % and 29 % neutral carbohydrates, respectively). The multiple form pattern might depend on the culture conditions.The P-galactosidase forms were heat-stable up to about 60 "C. The K, values for lactose ranged from 85 mM to 125 mM, whereas those for the synthetic substrate o-nitrophenyl-P-D-galactopyranoside were equal to about 2.4 mM. The V values obtained at 30°C for lactose and o-nitrophenyl-P-D-galactopyranoside were 104 units/mg enzyme protein and 121 units/mg enzyme protein, respectively (weighted averages for the three enzyme forms). The slight reactional dissimilarities between the three enzyme forms are unlikely to be physiologically relevant. The biological significance of A. niger p-galactosidase multiplicity might be related to the observed differences in carbohydrate content, as suggested by recent reports on other microbial glycoprotein enzymes.

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Mechanisms involved in the action of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa (EC 1.6.1.1) have been investigated by means of kinetic studies and fluorescence titration. Our results, as well as those from previous investigations, suggest that the allosteric MWC model (Monod, J., Wyman, J., and Changeux, J. P. (1965), J. Mol. Biol. 12, 88-118) may be used as a first step for the explanation of the properties of the transhydrogenase. The basic reaction of the enzyme is the oxidation of reduced triphosphopyridine nucleotide (TPNH) by diphosphopyridine nucleotide (DPN+). In terms of the model, the functional R state is favored by TPNH, whereas the product triphosphopyridine nucleotide (TPN+) behaves as an allosteric inhibitor, and is therefore assumed to favor the nonfunctional T state. To a slight extent, the T state is also favored by inorganic phosphate. On the other hand, adenosine 2'-monophosphate and several other 2'-phosphate nucleotides function as activators, and hence are presumed to shift the allosteric equilibrium toward the R state. The studies in this paper suggest a specific regulatory site for the transhydrogenase.