Purification and properties of erythro-β-hydroxyasparate dehydratase from Micrococcus denitrificans

1. The novel enzyme, erythro-beta-hydroxyaspartate dehydratase, a key enzyme of the beta-hydroxyaspartate pathway (Kornberg & Morris, 1963, 1965), has been purified 30-fold from extracts of glycollate-grown Micrococcus denitrificans. The purified preparation was devoid of erythro-beta-hydroxyaspartate-aldolase activity, and free from enzymes that act on oxaloacetate. 2. Properties of the purified dehydratase were studied by direct assay of the enzymic formation of oxaloacetate and ammonia from added erythro-beta-hydroxyaspartate. 3. The enzyme was highly substrate-specific, utilizing only the l-isomer of erythro-beta-hydroxyaspartate (K(m), 0.43mm, and V(max.), 99mumoles of oxaloacetate formed/min./mg. of protein at pH9.15 and 30 degrees ). Of many compounds tested, only maleate was a competitive inhibitor (K(i), 32mm at pH7.6). 4. The optimum pH for activity was about 9.5. The K(m) varied with pH, showing a marked optimum at pH7.8. The V(max.) also varied with pH in a manner suggesting the presence in the enzyme-substrate complex of a dissociable group of pK'(a) about 8.5. 5. Carbonyl reagents were inhibitory, but of three thiol reagents tested only p-chloromercuribenzoate was inhibitory. 6. A partially resolved preparation of the enzyme was activated four-fold by the addition of pyridoxal phosphate and thereby restored to half activity. 7. EDTA (0.1mm) was almost completely inhibitory, activity being restored by bivalent cations (Mg(2+), Ca(2+) and Mn(2+)); no activation by univalent cations was observed. 8. The findings are discussed in the light of reported properties of related hydroxyamino acid dehydratases.     

Influence of adenosine phosphates and magnesium on photosynthesis in chloroplasts from peas, sedum, and spinach

¹⁴CO₂ photoassimilation in the presence of MgATP, MgADP, and MgAMP was investigated using intact chloroplasts from Sedum praealtum, a Crassulacean acid metabolism plant, and two C₃ plants: spinach and peas. Inasmuch as free ATP, ADP, AMP, and uncomplexed Mg²⁺ were present in the assays, their influence upon CO₂ assimilation was also examined. Free Mg²⁺ was inhibitory with all chloroplasts, as were ADP and AMP in chloroplasts from Sedum and peas. With Sedum chloroplasts in the presence of ADP, the time course of assimilation was linear. However, with pea chloroplasts, ADP inhibition became progressively more severe, resulting in a curved time course. ATP stimulated assimilation only in pea chloroplasts. MgATP and MgADP stimulated assimilation in all chloroplasts. ADP inhibition of CO₂ assimilation was maximal at optimum orthophosphate concentrations in Sedum chloroplasts, while MgATP stimulation was maximal at optimum or below optimum concentrations of orthophosphate. MgATP stimulation in peas and Sedum and ADP inhibition in Sedum were not sensitive to the addition of glycerate 3-phosphate (PGA).

PGA-supported O₂ evolution by pea chloroplasts was not inhibited immediately by ADP; the rate of O₂ evolution slowed as time passed, corresponding to the effect of ADP on CO₂ assimilation, and indicating that glycerate 3-phosphate kinase was a site of inhibition. Likewise, upon the addition of AMP, inhibition of PGA-dependent O₂ evolution became more severe with time. This did not mirror CO₂ assimilation, which was inhibited immediately by AMP. In Sedum chloroplasts, PGA-dependent O₂ evolution was not inhibited by ADP and AMP. In chloroplasts from peas and Sedum, the magnitude of MgADP and MgATP stimulation of PGA-dependent O₂ evolution was not much larger than that given by ATP, and it was much smaller than MgATP stimulation of CO₂ assimilation. Analysis of stromal metabolite levels by anion exchange chromatography indicated that ribulose 1,5-bisphosphate carboxylase was inhibited by ADP and stimulated by MgADP in Sedum chloroplasts.

The appearance of label in the medium was measured when [U-¹⁴C] ADP-loaded Sedum chloroplasts were challenged with ATP, ADP, or AMP and their Mg²⁺ complexes. The rate of back exchange was stimulated by the presence of Mg²⁺. This suggests that ATP, ADP, and AMP penetrate the chloroplast slower than their Mg²⁺ complexes. A portion of the CO₂ assimilation and O₂ evolution data could be explained by differential penetration rates, and other proposals were made to explain the remainder of the observations.

     

Within‐group relatedness is correlated with colony‐level social structure and reproductive sharing in a social fish

In group‐living species, the degree of relatedness among group members often governs the extent of reproductive sharing, cooperation and conflict within a group. Kinship among group members can be shaped by the presence and location of neighbouring groups, as these provide dispersal or mating opportunities that can dilute kinship among current group members. Here, we assessed how within‐group relatedness varies with the density and position of neighbouring social groups in Neolamprologus pulcher, a colonial and group‐living cichlid fish. We used restriction site‐associated DNA sequencing (RADseq) methods to generate thousands of polymorphic SNPs. Relative to microsatellite data, RADseq data provided much tighter confidence intervals around our relatedness estimates. These data allowed us to document novel patterns of relatedness in relation to colony‐level social structure. First, the density of neighbouring groups was negatively correlated with relatedness between subordinates and dominant females within a group, but no such patterns were observed between subordinates and dominant males. Second, subordinates at the colony edge were less related to dominant males in their group than subordinates in the colony centre, suggesting a shorter breeding tenure for dominant males at the colony edge. Finally, subordinates who were closely related to their same‐sex dominant were more likely to reproduce, supporting some restraint models of reproductive skew. Collectively, these results demonstrate that within‐group relatedness is influenced by the broader social context, and variation between groups in the degree of relatedness between dominants and subordinates can be explained by both patterns of reproductive sharing and the nature of the social landscape.     

Pollination and Breeding System of two Co-Occurring Hirtella Species (Chrysobalanaceae) in Central Brazil

The phenology, major floral characteristics, breeding systems and fruiting success of two co-occurring species of Hirtella: H. glandulosa and H.gracilipes (Chrysobalanaceae) were studied in Central Brazil. The two species occur as trees in mesophyllous forests but H. glandulosa is frequent also in dense savanna areas. Both species flower at the end of the dry season (September) and have flowers with one-day longevity. In both species flowers produce nectar and are pollinated exclusively by butterflies. The number of visits recorded and pollination rate in each species were significantly different and indicate that pollinators prefer H. glandulosa flowers. Natural and controlled fruit sets were low in both species. The index of self-incompatibility (ISI) was 0.17 in H. glandulosa and 0.86 in H. gracilipes. Although ISI in H. glandulosa could denote an incompatibility system, the presence of fruits with aborted embryos at different degrees of development point to an inbreeding depression situation. Low pollen viability and fruit set in H. gracilipes suggested reproductive problems which may be linked to hybridization events.     

The synthesis and biological evaluation of a series of potent dual inhibitors of farnesyl and geranyl-Geranyl protein transferases

We have prepared a series of potent, dual inhibitors of the prenyl transferases farnesyl protein transferase (FPTase) and geranyl-geranyl protein transferase I (GGPTase). The compounds were shown to possess potent activity against both enzymes in cell culture. Mechanistic analysis has shown that the compounds are CAAX competitive for FPTase inhibition but geranyl-geranyl pyrophosphate (GGPP) competitive for GGPTase inhibiton.     

A stable isotope study of linkages between stream and terrestrial food webs through spider predation

SUMMARY 1. Transfer of carbon from freshwater to terrestrial ecosystems can occur through predation on adult aquatic insects, but the significance of this trophic pathway to the energetics of riparian communities is poorly understood. We used stable isotopes of carbon and nitrogen to explore linkages between aquatic insect production and the nutrition of web‐building and free‐living spiders alongside two streams in the North Island of New Zealand. 2. δ¹³C values for riparian tree leaves (means for each site = ?32.2 and ?30.3‰) were distinct from those of lichens collected from stream channel rocks and instream algae, both of which were similar (?23.4 to ?22.4‰). δ¹⁵N values for leaves were similar at both sites (?3.4 and ?2.7‰), but algae were considerably more depleted in δ¹⁵N atonesite suggesting significant differences in instream nitrogen sources between the twostreams. 3. Isotope values for potential aquatic prey of spiders indicated that aquatic algal production was their primary carbon source at both sites. Terrestrial invertebrates collected and assumed to be potential prey reflected a range of carbon sources and represented several trophic levels. 4. At one site, δ¹³C values indicated a primarily algae‐aquatic insect pathway of carbon transfer to both web‐building and free‐living spider guilds. The other site appeared to have a primarily terrestrial carbon pathway for the free‐living spider guild, and a mixed aquatic‐terrestrial pathway for the web‐building guild. 5. Overall, web‐building spiders were estimated to obtain around 61% of their body carbon from aquatic production compared with 55% for free‐living spiders. Our findings suggest that consumption of prey derived from aquatic sources can provide significant nutrition for spiders living along some stream channels. This pathway may represent an important feedback mechanism contributing to the energetics of riparian communities at sites where aquatic insect production is high.     

Effect of dissolved inorganic carbon on the expression of carboxysomes,localization of Rubisco and the mode of inorganic carbon transport in cells of the cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon (CO₂+HCO _inf3 ^sup- ) in the growth medium. In cells grown under 5% CO₂ and in those bubbled with air, carboxysomes were present in low numbers (<2 · longitudinal section^-1) and were distributed in an apparently random manner throughout the centroplasm. In contrast, cells grown in standing culture and those bubbled with 30 l CO₂ · 1^-1 possessed many carboxysomes (>8 · longitudinal section^-1). Moreover, carboxysomes in these cells were usually positioned near the cell periphery, aligned along the interface between the centroplasm and the photosynthetic thylakoids. This arrangement of carboxysomes coincided with the full induction of the HCO _inf3 ^sup- transport system that is involved in concentrating inorganic carbon within the cells for subsequent use in photosynthesis. Immunolocalization studies indicate that the Calvin cycle enzyme ribulose bisphosphate carboxylase was predominantly carboxysome-localized, regardless of the inorganic carbon concentration of the growth medium, while phosphoribulokinase was confined to the thylakoid region. It is postulated that the peripheral arrangement of carboxysomes may provide for more efficient photosynthetic utilization of the internal inorganic carbon pool in cells from cultures where carbon resources are limiting.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon (CO₂+HCO _inf3 ^sup- +CO _inf3 ^sup2- ) - PRK phosphoribulokinase - RuBP ribulose 1,5-bisphosphate - Rubisco LS large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase     

IMMUNOCYTOCHEMICAL CHARACTERIZATION OF THE INTRAPYRENOID THYLAKOIDS OF CRYPTOMONADS1

Thylakoid lamellae extend into the pyrenoids of only two genera of cryptomonad algae, Chroomonas and Hemiselmis, We used immunoelectron microscopy to assess the photosynthetic competency of cryptomonad intrapyrenoid thylakoids. Intrapyrenoid thylakoids possess phycobiliproteins and the chlorophyll a/c₂ light-harvesting complex, both of which are associated with photosystem (PS) II in a light-harvesting capacity. In addition, thylakoids that extend into the pyrenoid of Hemiselmis brunnescens were immunolabelled by anti-PSI. These results indicate that cryptomonad intrapyrenoid thylakoids likely function in a manner analogous to thylakoids of the chloroplast stroma. Moreover, our observation that the Calvin cycle enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is pyrenoid-localized in these two cryptophytes indicates that the processes of photosynthetic O₂-evolution and ribulose 1,5-bisphosphate (RuBP) carboxylation/oxygenation are not spatially separated in these algae.     

Interactions of porphyrins with purified DNA and more highly organized structures

Studies of the solution properties of gold(III)tetrakis(4-N-methylpyridyl) porphine and its DNA binding characteristics have been conducted utilizing uv/vis absorption spectroscopy, circular dichroism (CD), Mossbauer spectroscopy, and temperature-jump relaxation techniques. These studies indicate that over the concentration range considered this water soluble gold(III) porphyrin does not aggregate, binds axial ligands only weakly with a preference for soft Lewis bases, and is capable of intercalation into nucleic acids of appropriate base pair content. The interaction of this and several other porphyrins with the synthetic polynucleotide poly(dA-dC).poly(dT-dG) has been studied. Spectroscopic signatures for intercalation were found for those derivatives not having axial ligands. Intercalation into chromatin in vitro can also occur with those porphyrins and metalloporphyrins which do not have axial ligands. Finally, studies utilizing microinjection techniques indicate that once within the cell, tetrakis(4-N-methylpyridyl)porphine tends to localize in the nucleus.