Activities of Ca2+ pump and low affinity Ca2+/H+ antiport in plasma membrane vesicles of corn roots

ATP-dependent Ca₂₊-uptake was investigated in sealed plasmamembrane vesicles isolated from corn roots (Zea mays L. cv.Hybrid-3352/Palma-Pioneer). In a chloride-containing medium,at high calcium concentrations, about 30% of the total Ca²⁺accumulation ({small tilde}4 nmol Ca²⁺ mg^–1 protein) wasshown to be protonophore-sensitive and corresponded to the fractionof Ca²⁺ not accumulated in a sulphate-containing medium. Furthermore,vesicles in the presence of nitrate, which stimulates H⁺ transport,or vesicles preloaded with H⁺, take up Ca²⁺ more rapidly, suggestingthat, at high calcium concentrations, there is a mechanism forCa²⁺ transport which depends on the magnitude of the protongradient across the membrane. The fraction of Ca²⁺ uptake shownto be sensitive to the protonophore CCCP increased by about150–200% as the Ca²⁺ concentration in the medium increasedfrom 50µM to 250µM. Under the same conditions, theCCCP-insensitive fraction of Ca²⁺ accumulated was reduced byabout 25–30% suggesting that different Ca²⁺ affinitiesexist in the two Ca²⁺ uptake processes. Although calmodulinstimulation was not observed, the sensitivity to Ca²⁺ and externalpH indicates that H⁺ gradient-independent Ca²⁺ accumulationreflects activity of the Ca²⁺–pump. These results indicatethat the plasma membrane of corn roots contain two distinctmechanisms of Ca²⁺ transport: a high Ca²⁺ affinity, proton gradient-independentCa²⁺ pump and a low Ca²⁺ affinity, proton gradient-dependentCa²⁺/H⁺ antiport, which have greatest activity at concentrationsof Ca²⁺ below and above 50+M, respectively. Key words: Ca²⁺/H+ antiport, Ca²⁺ pump, plasmalemma, roots, Zea mays L.     

The influence of plants on concentration and fractionation of Zn, Pb, and Cu in salt marsh sediments (Tagus Estuary, Portugal)

Sediment cores were collected from two sites of the Tagus estuary salt marshes which differed in degree of metal contamination. At each site, six 60-cm-long cores were taken, three from a non-vegetated intertidal zone, and one from each of areas colonized by salt marsh plants, Spartina maritima, Halimione portulacoides and Arthrocnemum fruticosum, respectively. Total concentrations and concentrations in sequential extractions of Zn, Pb, and Cu were determined in several sediment layers. Sediment slices containing most of the roots (5–15-cm depth) were enriched in metals in comparison with other depths in the core and with non-vegetated cores. Additionally, metals in sediment slices with roots were preferentially linked to the residual fraction. These results are evidence that aquatic plant roots can have a strong influence on metal concentration and speciation in sediments. Since metals become immobilized in vegetated sediments, the preservation of salt marshes or the creation of artificial wetlands could be considered as an efficient natural means for maintaining ecosystem health or restoring ecosystem quality.     

Soil charcoal as long‐term pyrogenic carbon storage in Amazonian seasonal forests

Forest fires (paleo + modern) have caused charcoal particles to accumulate in the soil vertical profile in Amazonia. This forest compartment is a long‐term carbon reservoir with an important role in global carbon balance. Estimates of stocks remain uncertain in forests that have not been altered by deforestation but that have been impacted by understory fires and selective logging. We estimated the stock of pyrogenic carbon derived from charcoal accumulated in the soil profile of seasonal forest fragments impacted by fire and selective logging in the northern portion of Brazilian Amazonia. Sixty‐nine soil cores to 1‐m depth were collected in 12 forest fragments of different sizes. Charcoal stocks averaged 3.45 ± 2.17 Mg ha^?1 (2.24 ± 1.41 Mg C ha^?1). Pyrogenic carbon was not directly related to the size of the forest fragments. This carbon is equivalent to 1.40% (0.25% to 4.04%) of the carbon stocked in aboveground live tree biomass in these fragments. The vertical distribution of pyrogenic carbon indicates an exponential model, where the 0–30 cm depth range has 60% of the total stored. The total area of Brazil's Amazonian seasonal forests and ecotones not altered by deforestation implies 65–286 Tg of pyrogenic carbon accumulated along the soil vertical profile. This is 1.2–2.3 times the total amount of residual pyrogenic carbon formed by biomass burning worldwide in 1 year. Our analysis suggests that the accumulated charcoal in the soil vertical profile in Amazonian forests is a substantial pyrogenic carbon pool that needs to be considered in global carbon models.     

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.     

Design of biologically active, conformationally constrained GnRH antagonists

The introduction of conformational constraints into a flexible peptide hormone can be exploited to develop models for the conformation required for receptor binding and activity. In this review, we illustrate this approach to analog design using our work on antagonists of gonadotropin-releasing hormone (GnRH). Design of a conformationally constrained, competitive antagonist of GnRH, cyclo[delta 3,4 Pro-D4ClPhe-DTrp-Ser-Tyr-DTrp-NMeLeu-Arg-Pro-bet a Ala] led to the prediction of its bioactive conformation. Template forcing experiments show that this conformation is accessible to other active GnRH analogs. Two-dimensional NMR studies verified the predicted conformation in solution. The predicted binding conformation has recently been used to design two new analogs incorporating side chain-side chain linkages suggested by the conformational model: Ac-delta 3,4Pro-D4FPhe-DTrp-Dap-Tyr-DTrp-Leu-Arg-Asp-Gly- NH2 and Ac-delta 3,4Pro-D4FPhe-DTrp-Dap-Tyr-D2Nal-Leu-Arg-Pro-Asp -NH2. These analogs were synthesized and the one predicted to be most similar to the parent conformation had equivalent potency while the second, designed to refine the conformational hypothesis, was found to exhibit enhanced potency, thus confirming the original binding conformation hypothesis. These compounds and their derivatives now provide a new class of GnRH antagonists possessing both high biological potency and limited conformational flexibility, thus making them ideal for both biophysical and structure-activity studies.     

Potassium influx into maize root systems : influence of root potassium concentration and ambient ammonium

Potassium influx into roots of dark-grown decapitated maize seedling (Zea mays L., cv Pioneer 3369A) was examined in presence and absence of ambient ammonium and at various root potassium concentrations. Six-day old seedlings which were dependent on the endosperm reserves for their energy source were exposed to KCl (labeled with ⁸⁶Rb) ranging from 5 to 200 micromolar. At both low (13 micromoles per gram fresh weight) and high (100 micromoles per gram fresh weight) root potassium concentration, isotherms indicated two potassium influx systems, one approaching saturation at 50 to 100 micromolar potassium and an additional one tentatively considered to be linear. A mixed-type inhibition by ammonium for the low-concentration saturable system was indicated by a concomitant decrease in V_max and increase in K_m. High root potassium concentration decreased V_max but had little effect on K_m of this system. The rate constant for the second quasilinear system was decreased by ambient ammonium and by high root potassium status. Transfer of high potassium roots to potassium-free solutions resulted in an increase in influx within 2 hours; by 24 hours influx significantly exceeded that of roots not previously exposed to potassium. In roots of both low and high root potassium concentrations, potassium influx was restricted progressively as ambient ammonium increased to about 100 micromolar, but there was little further inhibition as ammonium concentrations increased beyond that to 500 micromolar. The data imply that potassium influx has two components, one subject to inhibition by ambient ammonium and one relatively resistant.     

Interaction of chemical probes with sarcoplasmic reticulum membranes

The labelling of the sarcoplasmic reticulum membranes by the chemical probes, trinitrobenzenesulfonate (TNBS) and fluorodinitrobenzene (FDNB) has been investigated. The incorporation of TNBS, but not of FDNB, depends on the binding of Ca2+ or Mg2+ to the membranes. The labelling of lipids and of the various reticulum proteins by TNBS is increased by those agents, but the effect is not uniform for all membrane proteins. The Ca2+ -ATPase contributes only 2.2% for the total labelling of the sarcoplasmic reticulum proteins, whereas the proteins of molecular weight 90 000 and 30 000 contribute about 34 and 56%, respectively. However, the Ca2+-ATPase isolated from the membrane reacts with an amount of TNBS 5-fold higher than that which reacts with the enzyme in situ. Both probes, TNBS and FDNB, inhibit the Ca2+-ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum, whereas the Mg2+-ATPase remains unaffected. The results indicate that FDNB is maximally incorporated into the sarcoplasmic reticulum membrane, whereas only some of the membrane amino groups are accessible to TNBS in the absence of Ca2+, Mg2+ or ATP which, when present, make additional amino groups available to TNBS. The highest degree of TNBS incorporation takes place into proteins, other than the ATPase, but sufficient reaction occurs with the enzyme to inhibit its activity.     

Effects of follicle-stimulating hormone on inhibin release by different testicular compartments in the adult ram.

The aim of this study was to determine the bidirectional release of immunoreactive inhibin-alpha (irINH-alpha) by different testicular compartments in the adult ram and to assess the effects of FSH on the distribution of inhibin in the testis. Immunoreactive INH-alpha was measured by RIA in fluid samples collected concurrently from the three testicular compartments--the seminiferous tubules, the interstitium, and the vascular system--through catheters inserted surgically into the rete testis, testicular lymphatic duct system, and spermatic veins, respectively. Overall, the concentration of irINH-alpha in rete testis fluid was 25 times the level in testicular lymph and over 500 times the concentration in peripheral blood. The pattern of irINH-alpha concentration in rete testis fluid was inversely related to that in testicular lymph, but i.v. administration of FSH had a decoupling effect on this relationship by depressing inhibin concentration in testicular lymph without affecting inhibin levels in rete testis fluid. Nevertheless, increased flow of testicular lymph more than compensated for the transient fall in irINH-alpha concentration so that, overall, the total output of inhibin via the testicular lymphatic duct system (and the vascular system) increased significantly. No persistent or significant changes were observed in the flow rate of rete testis fluid or concentration of irINH-alpha in the fluid after administration of FSH. The time frame for the response of the testis to FSH is indicative of the involvement of a mediator. Electrophoretic analysis of serially collected testicular lymph samples consistently revealed an FSH-induced release of a series of proteins in the M(r) range of 30,000-32,000.(ABSTRACT TRUNCATED AT 250 WORDS)