Mechanism of microtubule assembly. Changes in polymer structure and organization during assembly of sea urchin egg tubulin

Assembly of tubulin, purified from eggs of the sea urchin Stronglyocentrotus purpuratus, was examined at physiological (18 degrees C) and nonphysiological (37 degrees C) temperatures. Critical concentrations for assembly were 0.71 mg/ml at 18 degrees C and 0.21 mg/ml at 37 degrees C. At tubulin concentrations above 1.2 mg/ml at 18 degrees C and 0.5 mg/ml at 37 degrees C, a concentration-dependent "overshoot" in turbidity and in small-angle light scattering was observed; turbidity and scattering increased rapidly to a peak, then decreased asymptotically toward a steady-state value. Quantitative sedimentation analysis revealed that the mass of assembled polymer reached and maintained a constant level during overshoot of turbidity. Changes in the wavelength dependence of turbidity were consistent with the initial formation of sheets of tubulin, followed by conversion of the sheets to microtubules, both at 18 and 37 degrees C. Examination by negative-stain electron microscopy showed that sheetlike structures predominated during the early stages of overshoot assembly, while complete microtubules were present at steady state. Furthermore, measurements of average polymer length revealed that the overshoots in turbidity and in light scattering are unlikely to be caused by polymer length redistribution. Qualitative observations of solution birefringence suggested that the polymer became progressively more aligned during assembly. These results suggest that the turbidity/light-scattering overshoots reflect changes in the form or in the organization of the assembling polymer, or both.     

Simultaneous and independent effects of abscisic acid on stomata and the photosynthetic apparatus in whole leaves

(±)-Abscisic acid (ABA) at 10^-5 M was added to the transpiration stream of leaves of 16 species (C₃ and C₄, monocotyledons and dicotyledons). Stomatal responses followed one of three patterns: i) stomata that were wide and insensitive to CO₂ initially, closed partially and became sensitive to CO₂; ii) for stomata that were sensitive to CO₂ before the application of ABA, the range of highest sensitivity to CO₂ shifted from high to low intercellular partial pressures of CO₂, for instance in leaves of Zea mays from 170–350 to 70–140 bar; iii) when stomata responded strongly to ABA, their conductance was reduced to a small fraction of the initial conductance, and sensitivity to CO₂ was lost. The photosynthetic apparatus was affected by applications of ABA to various degrees, from no response at all (in agreement with several previous reports on the absence of effects of ABA on photosynthesis) through a temporary decrease of its activity to a lasting reduction. Saturation curves of photosynthesis with respect to the partial pressure of CO₂ in the intercellular spaces indicated that application of ABA could produce three phenomena: i) a reduction of the initial slope of the saturation curve (which indicates a diminished carboxylation efficiency); ii) a reduction of the level of the CO₂-saturated rate of assimilation (which indicates a reduction of the ribulose-1,5-bisphosphate regeneration capacity); and iii) an increase of the CO₂ compensation point. Photosynthesis of isolated mesophyll cells was not affected by ABA treatments. Responses of the stomatal and photosynthetic apparatus were usually synchronous and often proportional to each other, with the result that the partial pressure of CO₂ in the intercellular spaces frequently remained constant in spite of large changes in conductance and assimilation rate. Guard cells and the photosynthetic apparatus were able to recover from effects of ABA applications while the ABA supply continued. Recovery was usually partial, in the case of the photosynthetic apparatus occasionally complete. Abscisic acid did not cause stomatal closure or decreases in the rate of photosynthesis when it was applied during a phase of stomatal opening and induction of photosynthesis that followed a transition from darkness to light.Abbreviations and symbols A rate of CO₂ assimilation - ABA (±)-abscisic acid - c _a partial pressure of CO₂ in the ambient air or in the gas supplied to the leaf chambers - c _i partial pressure of CO₂ in the intercellular spaces of a leaf - e _a partial pressure of H₂O in the air - g conductance for water vapor - J quantum flux - T ₁ leaf temperature     

Covalent immobilization of the multienzyme enniatin synthetase

Summary The multienzyme enniatin synthetase was covalently immobilized to N-hydroxysuccinimide activated agarose. The stability of the immobilized enzyme at 25°C was enhanced compared to the soluble enzyme. Immobilization experiments also indicated that the enniatins are synthesized by a single molecule and thus do not require interactions of several enzyme molecules.     

Isolation of mouse N-CAM-related cDNA: detection and cloning using monoclonal antibodies.

Clones coding for the mouse neural cell adhesion molecule (N-CAM) were isolated from a cDNA library prepared in the expression vector lambda gt 11 from mRNA extracted from a mouse neuroblastoma cell line. This library was screened with two anti-N-CAM monoclonal antibodies directed against different sites on the molecule and with rabbit anti-N-CAM serum. Two clones were identified with the first monoclonal antibody, three with the second one, none reacted with both. The relevance of these cDNA clones to N-CAM was confirmed by several observations. First, cDNA sequences detected with one monoclonal antibody cross-hybridized with those identified by the other antibody. Second, the different fusion proteins all bound the rabbit serum in addition to one monoclonal antibody. Finally, the probes hybridized to discrete mRNA species of sufficient lengths to code for the very large N-CAM polypeptides in RNA preparations from N-CAM-expressing, but not from N-CAM-negative cells. An additional mRNA species not seen in embryonic brain was expressed in adult mouse brain. Genomic blot experiments indicated that sequences corresponding to one of our probes are present only a few times in the mouse genome.     

Anaerobic degradation of 3,4,5-trimethoxybenzoate by a defined mixed culture of Acetobacterium woodii, Pelobacter acidigallici, and Desulfobacter postgatei

Abstract Acetobacterium woodii was continuously grown on 3,4,5-trimethoxybenzoate as pure culture or in commensalistic combination with Pelobacter acidigallici and Desulfobacter postgatei . Under pure culture conditions the following growth parameters were determined: μ _max= 0.112 h⁻¹, K _s= 1.07 mM, Y _max= 35 g/mol, and m = 0.22 mmol·g⁻¹·h⁻¹. In coculture with P. acidigallici the affinity for the substrate increased and the K _s value was found to be 135 μM. Under batch culture conditions mixed populations of A. woodii, P. acidigallici , and D. postgatei completely mineralized 3,4,5-trimethoxybenzoate to CO₂, whereas under continuous culture conditions more than 3 mM acetate remained unused.     

Vergleichende Mikromorphologie der Narbenoberflächen bei den Angiospermen; Zusammenhänge mit Pollenoberflächen bei heterostylen Sippen

Stigmatic surfaces of about 250 species from more than 100 families were examined by scanning electron microscopy. There are five main groups which differ by the amount of secretion, the morphology of the surface and the distribution of receptive cells. The types of stigmatic surfaces are often remarkably constant on the family level.Ericaceae andLiliaceae are examples for very heterogenous families. Particular interest was paid to heteromorphic (heterostylous and cleistogamous) species. In most heterostylous species there is a close morphological correlation between the stigmatic surface and the sculpture of compatible pollen. Heterostyly was established for the genusGoniolimon. The dimorphism of plants with chasmogamous and cleistogamous flowers is a phenomenon which is not comparable to the polymorphism of heterostylous forms.

     

Contrasting roles of tau and microtubule-associated protein 2 in the vinblastine-induced aggregation of brain tubulin

Two different proteins, tau and microtubule-associated protein 2 (MAP 2), are able to stimulate tubulin polymerization into microtubules in vitro, but it is not certain if both proteins act by the same mechanism. We have examined the effects of tau and MAP 2 on the vinblastine-induced polymerization of tubulin into spiral filaments. In the presence of tau, vinblastine induced extensive aggregation of tubulin as shown by a large increase in turbidity. The increase in turbidity was accompanied by the formation of large numbers of spirals composed of a filament 40-60 A in diameter. The rate and extent of this aggregation into spirals were dependent on the concentrations of tubulin, tau, and vinblastine. Unlike normal microtubule assembly, this type of aggregation was not inhibited by colchicine or podophyllotoxin. In contrast, MAP 2, even at high concentrations, was less effective than tau at promoting the vinblastine-induced increase in turbidity of tubulin. In fact, MAP 2 strongly inhibited the effect of tau. These results indicate that tau and MAP 2 interact differently with the tubulin molecule in the presence of vinblastine and suggest that the two proteins may play different roles in regulating or promoting microtubule assembly. Vinblastine may thus be a useful probe in analyzing the modes of interactions of tau and MAP 2 with tubulin.     

Heme regulation of HeLa cell transferrin receptor number

The number of diferic transferrin receptors on HeLa cells decreases when cells are grown in iron-supplemented media. The experiments reported here suggest that heme is the iron-containing compound which serves as the signal for receptor number regulation. When HeLa cells were grown in the presence of hemin, transferrin receptor number decreased to a greater degree than when cells were grown in equivalent amounts of iron supplied as ferric ammonium citrate. Incubation of cells in conditions which increased cellular heme content resulted in a decrease in cellular transferrin receptors. Incubating cells with 5-aminolevulinic acid (thus bypassing the rate-limiting step in heme biosynthesis, 5-aminolevulinic acid synthase) led to a decrease in transferrin receptor number. Incubation of cells with an inhibitor of heme oxygenase, Sn-protoporphyrin IX, also led to a decrease in transferrin receptor number. When cellular heme content was decreased by inhibiting heme synthesis with succinylacetone (an inhibitor of 5-aminolevulinic acid dehydratase), or by depriving cells of iron with deferoxamine, an increase in HeLa cell transferrin receptor number was seen. When HeLa cells were incubated with inducers of heme oxygenase (CoCl2, SnCl2, Co-protoporphyrin IX), transferrin receptor number also increased. The effects of all compounds which alter transferrin receptor number were dependent on the concentration of the supplement, as well as the duration of the supplementation. These experiments suggest that intracellular heme content may be an important signal controlling transferrin receptor number.