Immunolocalization of plasma-membrane H+-ATPase and tonoplast-type pyrophosphatase in the plasma membrane of the sieve element-companion cell complex in the stem of Ricinus communis L.

Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L. and, for comparison, in stems of Cucurbita pepo L. and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue. The plasma-membrane (PM) H+-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies. Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM. Different PM H+-ATPase antibody clones, raised against the PM H+-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types. Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H+-ATPase isoforms. The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K+ and amino acids, as one of the unknown roles of ATP found in SEs. The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes.

Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth.

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H+ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 microM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H+ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H+ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen.

Simulations on the number of entanglements of a polymer network using knot theory.

Polymer networks, created on the computer using the Bond-Fluctuation-Algorithm, offer the possibility to count the number of entanglements. We generated networks consisting of 5000 chains that were cross linked at their end groups via tetra-functional cross linkers. The analysis of the topology was performed by computing the Homfly polynomial of the entanglements offering a much more precise determination of the knot and entanglement type than the Gaussian linking number. It also allows us to determine the influence of Brunnian links. Results concerning the connection between the chain length and the number of entanglements are shown.

Phytotropin-binding sites and auxin transport in Cucurbita pepo: evidence for two recognition sites.

Two properties of phytotropins, their ability to bind to 1-N-naphthylphthalamic acid (NPA) receptors located on microsomal vesicles isolated from Cucurbita pepo L. hypocotyls, and to stimulate auxin (indol-3-yl acetic acid, IAA) accumulation into such vesicles by blocking its efflux from them, were assessed in double labelling experiments using [2,3,4,5-(3)H]1-N-naphthylphthalamic acid and 3-indolyl-[2-(14)C]acetic acid. Two sites of differing affinities and activities on IAA accumulation were found. 1-N-Naphthylphthalamic acid was found to have high affinity (KD at 10(-8)mol·l(-1)) for one site and low affinity (KD at 10(-6) mol·l(-1)) for the other, whereas 2-(1-pyrenoyl)benzoic acid displaced NPA with high efficiency (KD below 10(-8) mol·l(-1)) from both sites. Other phytotropins had intermediate affinities for either site. Occupation of the site with low affinity for NPA stimulated auxin accumulation, while occupation of the high-affinity site with a phytotropin did not interfere with auxin accumulation into vesicles.

Membranes markers in highly purified clathrin-coated vesicles from Cucurbita hypocotyls.

The two plasma-membrane (PM) markers: 1,3-ß-glucan synthase and naphthylphthalamic-acid-binding capacity are localized in two peaks of activity on isopycnic Ficoll/D2O gradients prepared from a zucchini (Cucurbita pepo L.) hypocotyl post-microsomal fraction. The denser peak overlaps with the major distribution of clathrin and represents a region of the gradient enriched in coated vesicles (cv). Further purification of the pooled cv-fractions has shown that these PM marker activities are not borne by the cv, but are instead carried by smooth membrane fragments also present in these fractions. As judged from the results of Western blotting with polyclonal antibodies prepared against the 100-kilodalton (kDa) subunit of a PM H(+)-ATPase and the 70-kDa subunit of a tonoplastic H(+)-ATPase, these contaminants are of both PM and endomembrane origin. The PM contaminants however, differ from phase-partitioning- and free-flow-electrophoresis-purified PM prepared from microsomal fractions of zucchini hypocotyls in terms of their bouyant density in Ficoll/D2O gradients. Moreover, they do not appear to be present as sealed, outside-out, vesicles. Highly purified cv fractions from zucchini hypocotyls cross-react with subunit antibodies from both vacuolar and PM H (+)-ATPases. These results are discussed in terms of recent findings on cv ATPases from bovine brain.

Ghosts of chromaffin granules accumulate biogenic amines according to a "pump and leak system" without contribution of carrier-mediated efflux.

Unidirectional inward transport into, accumulation by and loss of biogenic amines from ghosts of bovine chromaffin granules were studied to determine whether a carrier-mediated process contributes to the outward passage of amines across the granule membrane. In the presence of ATP-Mg2+, incubated ghosts (30 degrees C; pH 7.3) showed a reserpine-sensitive (IC50 2-5 nmol/l), unidirectional, inward transport of catecholamine (CA = 70% adrenaline/30% noradrenaline), 5-hydroxytryptamine (5-HT) and tyramine with different Km values (mumol/l: tyramine 2; 5-HT 5; CA 8) but with the same Vmax [20 nmol/(mg protein.min)] during the first 3 min of incubation. During longer incubation, the rate of unidirectional inward transport declined rapidly with time to about 30 to 40%, at which level it stayed nearly constant from 50 to 100 min of incubation. As the decline of unidirectional transport was independent of the amine accumulated in the ghosts, it is concluded that it reflects ageing of the membrane vesicles. During incubation for up to 100 min with CA, 5-HT or tyramine (10- to 30-fold Km) in the presence of ATP-Mg2+, the amine content of the ghosts increased, approaching a steady-state content (nmol amine/mg protein: CA 400-500, 5-HT 250, tyramine 60), which was negatively correlated with the lipid solubility of the amine (tyramine greater than 5-HT greater than CA), whereas the rate of approach to steady state (t1/2 in min: CA 20-30, 5-HT 7-10, tyramine less than 5) was positively correlated. Low concentrations of reserpine (greater than or equal to 25 nmol/l) caused net loss of amine from amine-loaded ghosts by inhibition of inward transport. However, reserpine did not reduce the fractional rate of loss (FRL) of CA-loaded ghosts induced by NH+4 or uncoupling agents. Accelerated exchange diffusion was not found to occur at the granule membrane, as addition of high concentrations of 5-HT or dopamine to CA-loaded ghosts did not result in a higher FRL of CA than did blockade of inward transport by reserpine. Analysis of steady-state kinetics revealed the following features of the granule transport. The approach to steady state (t1/2) was independent of the rate of inward transport. The steady-state amine content of ghosts approached a limiting value as the external concentration of the amine was increased; it was determined by the same kinetic constants (Km and the time-dependent Vmax) as found for the rate of carrier-mediated inward transport.(ABSTRACT TRUNCATED AT 400 WORDS)

Bisulfite interacts with binding sites of the auxin-transport inhibitor N-1-naphthylphthalamic acid.

The affinity of the auxin-transport inhibitor N-1-naphthylphthalamic acid (NPA) for membrane particles as well as for solubilized binding sites from Cucurbita pepo L. hypocotyls was reduced by low concentrations of bisulfite (half-maximal inhibition at 2·10(-3)-3·10(-3) M). Two membrane fractions obtained by sedimentation aided with polyethylene glycol showed differential sensitivity to bisulfite. Other oxidizing or reducing substances tested at 1 mM had no effect, except for N-ethylmaleimide (80% inhibition) and iodine (complete inhibition), both of which reduced the number of binding sites but not their affinity. Addition of bisulfite to either the isoalloxane ring of flavoproteins or to pyridoxal phosphate or quinones is proposed as a possible mechanism of action. Sulfur dioxide, at concentrations measured in polluted air, can lead to bisulfite concentrations in plant tissue sufficient to interfere with NPA-binding sites and hence with auxin transport.

Kinetic characterization of N-1-Naphthylphthalamic acid binding sites from maize coleoptile homogenates.

The association of NPA with particulate material from corn coleoptile homogenates and the dissociation of the complexes formed are characterized. Using a filtration method, a single rate constant for the association at 0° C was determined as 2.7×10(-6) M(-1) min(-1), and for the dissociation of the complex a rate constant of 0.086 min(-1) was obtained with the same method. From these a dissociation constant of the complex of 3.4×10(-8) M can be calculated, which is in good agreement with the dirct determination of this constant in equilibrium measurements. This is taken as an indication for a simple reaction schema describing the reaction of NPA with its particle-bound receptor, and it can be inferred that membrane permeation does not impose a limitation on this reaction under the conditions used in this investigation. This view is strengthened by the absence of any indication for a carrier mechanism in "counterflow" experiments. A few minor deviations from quantitative predictions of the simple binding reaction schema are discussed.

Fractionation of particulate material from maize coleoptile homogenates with polyethylene glycol.

Polyethyleneglycol (PEG) has been used to sediment particulate material from maize coleoptile homogenates at low centrifugal forces. The resuspended sediments were used for N-1-naphthylphthalamic acid (NPA)-binding studies. Binding activity was influenced by monovalent cations in the resuspension medium, but even at concentrations of up to 1.2 M NaCl or 0.5 M LiCl or CsCl, half of the binding activity was still recovered. Binding activity was influenced by divalent cations, because it decreased when Ca(2+) and Mg(2+) ions in the medium were complexed with EDTA. Fractionated sedimentation using increasing concentrations of PEG resulted in two peaks of NPA-binding activity at about 3% and 6% PEG. The 3% peak cintained enzymatic markers for mitochondria and endoplasmatic reticulum while the 6% peak contained NPA-binding activity only. Possible explanations for the bimodal distribution of NPA binding after fractionated PEG precipitation are discussed.

A gradient of diffusible substance in a monolayer of cultured cells.

Monolayers of hypoxanthine phosphoribosyl transferase-deficient and their corresponding wildtype cells have been placed adjacent to each other with a newly described method. Autoradiographs from such preparation after incubation with 3H-hypoxanthine allow the direct visualization of gradients of incorporated radioactivity at the border between the two cell types. The gradients can be described by an exponential function, and the amount of radioactivity incorporated decreases to less than 1% at a distance of 1 mm from the wild-type cells. A possible mechanism to convert exponential gradients to linear ones over a certain concentration range is discussed.