The toxicity of legume seed diffusates toward rhizobia and other bacteria

Summary Seed coat diffusates from the legumesCentrosema pubescens and subterranean clover were found to contain a water-soluble, thermostable, antibiotic inhibiting a wide range of rhizobia and other gram-negative organisms as well as gram-positive organisms. Lucerne seed diffusate showed little activity. The extent of inhibition varied with the micro-organism. Diffusate from subterranean clover was generally more active than fromC. pubescens.It was shown that seed diffusates can have a depressive or a stimulative effect on multiplication of organisms around seeds germinating in sand and soil.The significance of the results, particularly with respect to inoculation with Rhizobium is discussed.     

Mass spectrometry imaging–based assays for aminotransferase activity reveal a broad substrate spectrum for a previously uncharacterized enzyme

Aminotransferases (ATs) catalyze pyridoxal 5′-phosphate–dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in the biosynthesis and degradation of both proteinogenic and nonproteinogenic amino acids and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data. Even for characterized ATs, the full spectrum of substrate specificity, among many potential substrates, has not been explored in most cases. This is largely due to the lack of suitable high-throughput assays that can screen for AT activity and specificity at scale. Here we present a new high-throughput platform for screening AT activity using bioconjugate chemistry and mass spectrometry imaging–based analysis. Detection of AT reaction products is achieved by forming an oxime linkage between the ketone groups of transaminated amino donors and a probe molecule that facilitates mass spectrometry-based analysis using nanostructure-initiator mass spectrometry or MALDI–mass spectrometry. As a proof-of-principle, we applied the newly established method and found that a previously uncharacterized Arabidopsis thaliana tryptophan AT-related protein 1 is a highly promiscuous enzyme that can utilize 13 amino acid donors and three keto acid acceptors. These results demonstrate that this oxime–mass spectrometry imaging AT assay enables high-throughput discovery and comprehensive characterization of AT enzymes, leading to an accurate understanding of the nitrogen metabolic network.     

Effect of Src kinase phosphorylation on disordered C-terminal domain of N-methyl-D-aspartic acid (NMDA) receptor subunit GluN2B protein

NMDA receptors are ligand-gated ion channels with a regulatory intracellular C-terminal domain (CTD). In GluN2B, the CTD is the largest domain in the protein but is intrinsically disordered. The GluN2B subunit is the major tyrosine-phosphorylated protein in synapses. Src kinase phosphorylates the GluN2B CTD, but it is unknown how this affects channel activity. In disordered proteins, phosphorylation can tip the balance between order and disorder. Transitions can occur in both directions, so it is not currently possible to predict the effects of phosphorylation. We used single molecule fluorescence to characterize the effects of Src phosphorylation on GluN2B. Scanning fluorescent labeling sites throughout the domain showed no positional dependence of the energy transfer. Instead, efficiency only scaled with the separation between labeling sites suggestive of a relatively featureless conformational energy landscape. Src phosphorylation led to a general expansion of the polypeptide, which would result in greater exposure of known protein-binding sites and increase the physical separation between contiguous sites. Phosphorylation makes the CTD more like a random coil leaving open the question of how Src exerts its effects on the NMDA receptor.     

Laws of the Postcolonial

Laws of the Postcolonial. Eve Darian-Smith and Peter Fitzpatrick. eds. Ann Arbor: University of Michigan Press, 1999. 309 pp.     

Inability to detect organo-silicon compounds in Equisetum and Thuja

Silicon in Equisetum arvense sap has low molar mass and is probably monomeric silicic acid. Tris(β-thujaplicine)silicon X complexes (X  Cl, I₃) were synthesized but could not be found in extracts of Thuja plicata.