ATP-dependent Ca2+ transport and Ca2+-ATPase activities in an enriched plasma membrane fraction from gypsy moth larval midgut tissue

A subcellular fraction enriched in plasma membranes was obtained from gypsy moth (Lymantria dispar) larval midgut tissue. Using [⁴⁵Ca]²⁺ as a tracer, Ca²⁺ transport activity by membrane vesicles in the enriched fraction was measured and shown to be ATP-dependent, with a very high affinity for Ca²⁺ (apparent K_m for [Ca²⁺ _free]

1 Abbreviations used: [Ca²⁺free] = concentration of free (unbound) calcium ion;CaM = calmodulin; F = fraction; IOV = inside-out membrane vesicles; W-5 = N-(6-aminohexyl)-1-naphthalenesulfonamide; W-7 = N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.

= 22 nM). Ca²⁺ transport was abolished upon addition of the calcium ionophore, A23187. Ca²⁺-stimulated, Mg²⁺-dependent ATPase activity peaked between 100 and 200 nM Ca²⁺_free. Ca²⁺-Mg²⁺-ATPase activity was inhibited by vanadate, 2 phenothiazine drugs (trifluoperazine and chlorpromazine), and the naphthalene sulfonamide, W-7; the related compound, W-5, and ouabain had a negligible effect. These results suggest the presence of a high affinity plasma membrane Ca²⁺ pump in gypsy moth larval midgut cells and are discussed in light of earlier work involving calcium transport in isolated midguts of larval Hyalophora cecropia. Ionic and other conditions that characterize the midgut physiology of larval Lepidoptera (e.g., luminal pH; electrochemical gradient for Ca²⁺; effect of certain ions and inhibitors on Ca²⁺ transport) contrast significantly with those found in adult Diptera. The implications that these differences may have for calcium regulation are discussed. © 1992 Wiley-Liss, Inc.     

The pH profile for acid-induced elongation of coleoptile and epicotyl sections is consistent with the acid-growth theory

The acid-growth theory predicts that a solution with a pH identical to that of the apoplast of auxintreated tissues (4.5–5.0) should induce elongation at a rate comparable to that of auxin. Different pH profiles for elongation have been obtained, however, depending on the type of pretreatment between harvest of the sections and the start of the pH-incubations. To determine the acid sensitivity under in vivo conditions, oat (Avena sativa L.) coleoptile, maize (Zea mays L.) coleoptile and pea (Pisum sativum L.) epicotyl sections were abraded so that exogenous buffers could penetrate the free space, and placed in buffered solutions of pH 3.5–6.5 without any preincubation. The extension, without auxin, was measured over the first 3 h. Experiments conducted in three laboratories produced similar results. For all three species, sections placed in buffer without pretreatment elongated at least threefold faster at pH 5.0 than at 6.0 or 6.5, and the rate elongation at pH 5.0 was comparable to that induced by auxin. Pretreatment of abraded sections with pH-6.5 buffer or distilled water adjusted to pH 6.5 or above gave similar results. We conclude that the pH present in the apoplast of auxin-treated coleoptile and stems is sufficiently low to account for the initial growth response to auxin.Abbreviations FS free space - IAA indole-3-acetic acid This research was supported by a grant from the National Adonautics and space Administration (NASA), NAGW 1394 to R.E.C., NASA grant NAGW-297 to M.L.E., and NASA grant NAG 1849 to D.L.R.     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

In vivo pH measurement in the xylem of broad-leaved trees using ion-sensitive field-effect transistors

Summary A new method of in vivo pH determination in the xylem of broad-leaved trees using ion-sensitive field effect transistors is developed and its suitability for use is studied. In the first few hours after the sensor had been implanted in the xylem signals could be detected which were generated in response to mechanical damage; particularly strong signal changes are detectable in Populus balsamifera L., Tilia cordata Mill, and Aesculus hippocastanum L. The pH values of the xylem sap extracted from branches corresponded to the values measured by the in vivo method only at certain times. Due to sensor drift the measuring accuracy of long-term experiments lasting up to 3 weeks is restricted. The in vivo measurement of pH in the xylem of poplar branches revealed the ability of the living xylem to buffer the pH of the sap to its own characteristic value.Dedicated to Prof. Dr. O. L. Lange to his 65th birthday     

Effect of pH on lipid accumulation by an oleaginous yeast:Rhodotorula glutinis IIP-30

Maximum lipid production (66% w/w dry wt) inRhodotorula glutinis IIP-30 utilizing glucose in a fed-batch fermentation under N-limiting conditions at 30°C, was at pH 4. At pH 3, 5 and 6, the lipid contents were 12%, 48% and 44%, respectively. There was only a small change in the fatty acid profile over the pH range examined, although the ergosterol content decreased by a third as the pH increased.     

Cellulase production and activity in a species ofCladosporium

ACladosporium species produced large amounts of cellulase enzyme components when grown in shake-culture with medium containing carboxymethylcellulose. There was significantly less activity when Avicel, filter paper or cotton were used as substrates. KNO₃ was better than NH₄Cl or urea for the production of cellulase. Tween 80 at 0.1% (w/v) increased the production of cellulase by 1.5 to 4.5-fold. All the cellulase components were optimally active in the assay at pH 5.0 and 60°C.     

Measurements of transmembrane pH differences of low extents in bacterial chromatophores

In chromatophores from photosynthetic bacteria the interaction of the fluorescent monoamine, 9-amino, 6-chloro, 2-methoxyacridine (ACMA), with the membrane is evaluated and described by an S-shaped adsorption isotherm. This phenomenon is hysteretic, as indicated by the difference between the adsorption and desorption branches of the binding isotherm. Maximal saturation of adsorption is reached at one ACMA per one to four lipid molecules, indicating that the probe binds in its neutral form. Adsorption of the probe on the membrane causes a large quenching of its fluorescence, which is explaind as being due to hypochromic effects following stacking and aggregation in a medium of low dielectric constant. A further quenching of fluorescence is brought about by imposing artificially induced transmembrane pH's. This latter phenomenon titrates in at increasing pH values and approaches saturation when pH is 2. The dependence of pH on the observed quenching of fluorescence is predicted by considering a model based on the equilibrium distribution of the amine between two phases at different pH's, in which adsorption of the probe on the membrane is used to evaluate its free concentration in the inner and outer compartments of the chromatophore vesicle. It is proposed that the equation thus obtained should be used to measure pH from the quenching of ACMA fluorescence.Abbreviations pH transmembrane pH difference between the inner and outer compartments - Q quenching of fluorescence - BChl Bacteriochlorophyll - ACMA 9-amino-6-chloro-2-methoxyacridine - 9AA 9-aminoacridine - Tricine N-tris-(hydroxymethyl)methylglycine - MES 2-morpholinoethanesulfonic acid - FCCP carbonylcyanide-p-trifluoro-methoxy-phenylhydrazone - CCCP carbonylcyanide-m-chloro-phenylhydrazone     

Binding protein dependent transport of C4-dicarboxylates in Rhodobacter capsulatus

The characteristics of malate transport into aerobically grown cells of the purple photosynthetic bacterium Rhodobacter capsulatus were determined. A single transport system was distinguished kinetically which displayed a K_t value of 2.9 ± 1.2 μM and V_max of 43 ± 6 nmol · min^-1 · mg^-1 protein. Competition experiments indicated that the metabolically related C4-dicarboxylates succinate and fumarate are also transported by this system. Malate uptake was sensitive to osmotic shock and evidence from the binding of radiolabelled malate and succinate to periplasmic protein fractions indicated that transport is mediated by a dicarboxylate binding protein. The activity of the transport system was studied as a function of external and internal pH and it was found that a marked activation of uptake occurred at intracellular pH values greater than 7. The use of a high affinity binding protein dependent system to transport a major carbon and energy source suggests that Rhodobacter capsulatus would be capable of obtaining growth sustaining quantities of C4-dicarboxylates even if these were present at very low concentrations in the environment.     

The relationship between surface sediment diatom assemblages and pH in 33 Galloway lakes: Some regression models for reconstructing pH and their application to sediment cores

The species composition of surface sediment diatom assemblages in 33 Galloway lakes, pH range c. 4.5 to 7.4, is related in a statistically significant manner to water acidity (or to factors closely associated with pH). Predictive models of summer mean pH, using simple regression equations of Index B (Renberg & Hellberg, 1982) with both Scandinavian and Galloway data sets, and multiple regression equations using diatom pH preference groups and individual species, are described and applied to fossil diatoms in six sediment cores. Although multiple regression of individual species gives the highest correlation coefficient (r²=0.87) in the modern data set this method is least appropriate for reconstructing pH values from fossil material where predictor species are often absent. Of the four methods examined here it is suggested that multiple regression of diatom preference groups is probably the most suitable for pH reconstructions from sediment cores.     

Transplant experiments. Growth-limiting factors for diatoms and bluegreen algae in Norwegian lakes

Population densities and total phosphorus concentrations in samples from different lakes of south-eastern Norway were determined. In addition some transplant experiments with dilute phytoplankton populations were carried out. A laboratory batch culture method was used.The diatoms studied may be divided into three ecological groups based on their cell densities and total phosphorus concentrations in the samples. This classification was supported by the experimental results. Cyclotella spp., Asterionella formosa and Tabellaria fenestrata did not grow or had low growth rates above pH 9. Synedra cf. acus and Fragilaria crontonensis had often high growth rates within the pH 9–10 range, but were not able to grow at pH values above 10. High pH-values had no effect on the growth rate of Oscillatoria. Oscillatoria, Synedra and Stephanodiscus were severely growth-limited in filtered water from oligotrophic lakes. Maximum growth rates of all the populations studied were often obtained after addition of phosphate and chelated iron (FeEDTA) in combination to filtered water samples from oligotrophic/mesotrophic lakes.