The interaction of nucleotides and yeast hexokinase

The kinetics of ethenoadenosine triphosphate (?ATP) as the phosphate donor in the phosphoryl transfer reaction of hexokinase were examined to obtain the K_m′s, V's, and K_α's for the nucleotide and sugar. Dissociation constants for eATP and ?ADP with hexokinase were obtained from fluorometric measurements and compared with similar constants obtained kinetically. Other selected nucleoside triphosphates were used as phosphate donors in the hexokinase reaction and their kinetic constants were obtained. Reactions were also performed using two nucleotides simultaneously as phosphorylating substrates for the hexokinase reaction in an attempt to find the individual dissociation constants, K_m′s and K_i′s. These were compared with the K_m′s obtained from using the nucleotides separately in the hexokinase reaction. From these kinetic and fluorescence binding studies, evidence is presented supporting the postulate that the K_m′s are primarily dissociation constants in a random bi-bi mechanism. Analysis of the K_m values provides additional evidence to support the importance of the amino group in position 6 on the purine ring as a hydrogen-bond acceptor during binding. It was found that ?CTP was a much better hexokinase substrate than CTP. These observations suggest that the V for this reaction is highly dependent upon the size of the nucleotide.     

Heterospecific pollen transfer between sympatric species in a midsuccessional old-field community

The cumulative (season-long) incidence of heterospecific pollen transfer (HPT) was examined using nine sympatric species in a midsuccessional old field. Inflorescences were collected weekly during the flowering season, and the proportion of foreign pollen/stigma was recorded. Flowering phenologies of sympatric species and ovule and seed counts of study species were also recorded. Heterospecific pollen was detected on some stigmas of each species. Medicago sativa (Fabaceae) received the most foreign pollen; in some cases, all of the grains on a stigma were heterospecific. Lotus corniculatus (Fabaceae) received the least amount of foreign pollen; the incidence of heterospecific pollen was near zero in most cases. The mean and range of foreign pollen received varied by as much as an order of magnitude between species. The six species with zygomorphic flowers, all Fabaceae, received more heterospecific pollen than the three species with actinomorphic flowers, Potentilla recta and P. simplex (Rosaceae) and Ranunculus acris (Ranunculaceae). This probably reflects a bias because our data were analyzed on a cumulative basis and the Fabaceae had longer flowering phenologies. HPT was not correlated with the species' relative abundance within the community. Proportion of foreign pollen received varied temporally within species, and this variation generally was not related to phenology of any sympatric taxa or the species' own phenology. Pollen grain diameter was positively related to levels of foreign pollen received by species. This might be caused by poor adhesion of large pollen grains to small stigmatic papillae or if generalist pollinators carrying large amounts of heterospecific pollen visit the large-grained species and specialists with little foreign pollen visit the small-grained species. The large proportions of heterospecific pollen on stigmas of many species indicate that HPT occurs frequently in the community we studied and the implications may include reduced seed set because of occlusion by foreign grains. As yet, however, it is unclear how important a factor HPT is in mediating pollen limitation of reproductive success.     

24-methylenepollinastanol and 24-dehydropollinastanol—two sterols of pollen

The esterified and unesterified sterol fractions of bee-gathered mixed pollens were examined, and total sterol composition was determined. Two new sterols of pollens, 14α-methyl-9β,19-cyclo-5α-cholest-24-en-3β-ol (24-dehydropollinastanol) and 14α-methyl-5α-ergost-24(28)-en-3β-ol (24-methylenepollinastanol) were isolated and identified. Both sterols were found primarily in the esterified sterol fraction, and 24-methylenepollinastanol accounted for 43% of the sterols of this fraction. 24-Dehydropollinastanol and four other sterols which also contain a 9β,19-cyclopropane ring were found only in the esterified sterol fraction. 24-Methylenecholesterol was the major sterol of the unesterified sterol fraction.     

Smaller species experience mild adversity under shading in an old‐field plant community

Plant competition experiments commonly suggest that larger species have an advantage, primarily in terms of light acquisition. However, within crowded natural vegetation, where competition evidently impacts fitness, most resident species are relatively small. It remains unclear, therefore, whether the size advantage observed in controlled experiments is normally realized in habitats where competition is most intense. We characterized the light environment and tested for evidence of a size advantage in competition for light in an old‐field plant community composed of perennial herbaceous species. We investigated whether larger species contributed to reduced light penetration (i.e., greater shading), and examined the impact of shade on smaller species by testing whether their abundance and richness were lower in plots with less light penetration. Light penetration in plots ranged from 0.3% to 72.4%. Significant effects were more common when analyses focused on small plants that reached reproduction (i.e., flowering rooted units); focusing on only flowering plants (i.e., excluding nonflowering rooted units) can clarify community patterns. Plots with a greater mean species height had significantly lower light penetration, and plots with lower light penetration had significantly lower flowering abundance and richness of small species. However, the impact of shade on the flowering abundance and richness of small species was relatively small (R ² values between 8% and 15%) and depended on how we defined “small species.” Synthesis: Our results confirm that light penetration in herbaceous vegetation can be comparable to levels seen in forests, that plots with taller species cast more shade, and that flowering smaller species are less abundant and diverse in plots where light penetration is low. However, variation in mean plot height explained less than 10% of variation in light penetration, and light penetration explained between 5 and 15% of variation in the flowering abundance and richness of small species. Coupled with the fact that flowering small species were present even within the most heavily shaded plots, our results suggest that any advantage in light competition by large species is limited. One explanation is that at least some small species in these communities are shade‐tolerant. Shade tolerance in predominantly herbaceous communities, particularly among small plant species, requires further research.     

Pyruvate decarboxylase: a key enzyme for the oxidative metabolism of lactic acid by Acetobacter pasteurianus

Acetobacter pasteurianus, an obligately oxidative bacterium, is the first organism shown to utilize pyruvate decarboxylase (PDC) as a central enzyme for oxidative metabolism. In plants, yeast, and other bacteria, PDC functions solely as part of the fermentative ethanol pathway. During the growth of A. pasteurianus on lactic acid, the central intermediate pyruvate is cleaved to acetaldehyde and CO(2) by PDC. Acetaldehyde is subsequently oxidized to its final product, acetic acid. The presence of the PDC enzyme in A. pasteurianus was confirmed by zymograms stained for acetaldehyde production, enzyme assays using alcohol dehydrogenase as the coupling enzyme, and by cloning and characterization of the pdc operon. A. pasteurianus pdc was also expressed in recombinant Escherichia coli. The level of PDC activity was regulated in response to growth substrate, highest with lactic acid and absent with mannitol. The translated PDC sequence (548 amino acids) was most similar to that of Zymomonas mobilis, an obligately fermentative bacterium. A second operon ( aldA) was also found which is transcribed divergently from pdc. This operon encodes a putative aldehyde dehydrogenase (ALD2; 357 amino acids) related to class III alcohol dehydrogenases and most similar to glutathione-dependent formaldehyde dehydrogenases from alpha-Proteobacteria and Anabeana azollae.     

Modulating the Intrinsic Disorder in the Cytoplasmic Domain Alters the Biological Activity of the N-Methyl-d-aspartate-sensitive Glutamate Receptor

The NMDA-sensitive glutamate receptor is a ligand-gated ion channel that mediates excitatory synaptic transmission in the nervous system. Extracellular zinc allosterically regulates the NMDA receptor by binding to the extracellular N-terminal domain, which inhibits channel gating. Phosphorylation of the intrinsically disordered intracellular C-terminal domain alleviates inhibition by extracellular zinc. The mechanism for this functional effect is largely unknown. Proline is a hallmark of intrinsic disorder, so we used proline mutagenesis to modulate disorder in the cytoplasmic domain. Proline depletion selectively uncoupled zinc inhibition with little effect on receptor biogenesis, surface trafficking, or ligand-activated gating. Proline depletion also reduced the affinity for a PDZ domain involved in synaptic trafficking and affected small molecule binding. To understand the origin of these phenomena, we used single molecule fluorescence and ensemble biophysical methods to characterize the structural effects of proline mutagenesis. Proline depletion did not eliminate intrinsic disorder, but the underlying conformational dynamics were changed. Thus, we altered the form of intrinsic disorder, which appears sufficient to affect the biological activity. These findings suggest that conformational dynamics within the intrinsically disordered cytoplasmic domain are important for the allosteric regulation of NMDA receptor gating.