Vanadate inhibition of stomatal opening in epidermal peels of Commelina communis

An H⁺ ATPase at the plasma-membrane of guard cells is thought to establish an electrochemical gradient that drives K⁺ and Cl^– uptake, resulting in osmotic swelling of the guard cells and stomatal opening. There are, however, conflicting results regarding the effectiveness of the plasma-membrane H⁺-ATPase inhibitor, vanadate, in inhibiting both H⁺ extrusion from guard cells and stomatal opening. We found that 1 mM vanadate inhibited light-stimulated stomatal opening in epidermal peels of Commelina communis L. only at KCl concentrations lower than 50 mM. When impermeant n-methylglucamine and HCl (pH 7.2) were substituted for KCl, vanadate inhibition was still not observed at total salt concentrations50 mM. In contrast, in the absence of Cl^–, when V₂O₅ was used to buffer KOH, vanadate inhibition of stomatal opening occurred at K⁺ concentrations as high as 70 mM. Partial vanadate inhibition was observed in the presence of the impermeant anion, iminodiacetic acid (100 mM KHN(CH₂CO₂H)₂). These results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect. In support of this hypothesis, an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially prevented vanadate inhibition of stomatal opening. Other anion-uptake inhibitors (1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid, 200 M Zn²⁺) were not effective. Decreased vanadate inhibition at high Cl^–/vanadate ratios may result from competition between vanadate and Cl^– for uptake. Unlike metabolic inhibitors, vanadate did not affect the extent of stomatal closure stimulated by darkness, further indicating that the observed action of vanadate represents a specific inhibition of the guard-cell H⁺ ATPase.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - FC fusicoccin - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid We thank Drs. R.T. Leonard (University of California, Riverside, USA) and K.A, Rubinson (Yellow Springs, Oh., USA) for helpful comments on the research, Janet Sherwood (Harvard University) for excellent plant care, and Angela Ciamarra, Anne Gershenson, Gustavo Lara (Harvard University) and Orit Tal (Hebrew University) for valuable technical assistance. This research was supported by a grant from the National Science Foundation (DCB-8904041) to S.M.A.     

Measures of cooperativity in the binding of ligands to proteins and their relation to non-additivity in protein-protein interactions

The analogy between cooperativity in the binding of ligands to proteins and non-additivity in protein-protein interactions is demonstrated and discussed in terms of the Wong and the Hill coefficients. A measure of non-additivity, the interaction constant, is rigorously derived for four thermodynamic cycles, involving the binding of small molecules to proteins and protein association. It is the reciprocal of the 'defect factor' of Laskowski et al. in Proteinase inhibitors: medical and biological aspects (ed. N. Katunuma et al.), pp. 55-68 (1983), and its logarithm is the Wong measure of cooperativity. These three measures are thus here given a common theoretical basis. The Hill coefficient for an asymmetric dimer that binds two different ligands which do not compete for the same site, at 50% saturation of each site, is derived. It is shown to be a function of the interaction constant and of the fraction of protein to which ligand is bound at both sites. These relations for protein-ligand interactions are then discussed in the context of non-additivity in protein-protein interactions.     

Strategy for analysing the co-operativity of intramolecular interactions in peptides and proteins

Double mutant cycles enable the measurement of pairwise interactions in proteins. This method is extended for mutations at any number of positions in the protein. This provides a way for determining the context dependence of pairwise interactions on other neighbouring residues.     

Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization

Ascorbic acid is the major factor in brain extract responsible for increasing the average acetylcholine receptor (AChR) site density on the cloned muscle cell line L5. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR alpha-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the beta-, gamma-, and delta-subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, alpha-actin, and creatine kinase. By in situ hybridization, we further show that the increase in alpha-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei. mRNA levels for the alpha-subunit in mononucleated cells are very low and do not significantly increase in response to ascorbic acid. The mononucleated cells are thus excluded as a possible source for the increase in alpha-subunit mRNA detected by Northern blot analysis. Our results indicate that there is a very specific action of ascorbic acid on the regulation of AChR alpha-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR alpha-subunit mRNA.     

Presence of telomeric and subtelomeric sequences at the fusion points of ring chromosomes indicates that the ring syndrome is caused by ring instability

In situ hybridization of a telomeric (TTA-GGG) _n sequence to metaphases from three cases of ring chromosome, involving respectively chromosomes 4, 16, and 20, showed the presence of the cognate sequences in all three rings. To investigate whether these ring chromosomes originated by telomere-telomere fusion, we determined, by in situ hybridization, whether telomere-associated sequences and/or specific distal sequences were still present in the ring chromosomes. The finding that these sequences were preserved in all the ring chromosomes strongly indicates that they originated by telomere-telomere fusion. All three subjects carrying the ring chromosomes are affected by the so-called ring syndrome, with failure to thrive, minor dysmorphic signs and no major anomalies. The r(4) patient has the ring in mosaic form with a normal cell line and has normal intelligence. The r(16) and the r(20) patients have moderate mental retardation and suffer from seizures. We conclude that the ring syndrome, even in its more severe manifestation, is caused by ring chromosome instability.     

Hyphal walls of isolated lichen fungi

The hyphal walls of three mycobionts, isolated from the lichens Xanthoria parietina, Tornabenia intricata and Sarcogyne sp. were investigated by two techniques: microautoradiography of fungal colonies exposed to radioactive carbohydrate precursors; and binding, in vivo, of fluorescein conjugated lectins to hyphal walls of such colonies.N-[³H] acetylglucosamine was readily incorporated into tips, young hyphal walls and septa of the three mycobionts and the free-living fungus Trichoderma viride, but not into Phytophthora citrophthora, indicating that chitin is a major component of the mycobionts' hyphal walls. All three mycobionts, but neither of the free-living fungi, incorporated [³H] mannose and [³H] mannitol into their hyphal walls.Fluorescein-conjugated wheat germ agglutinin was bound to the hyphal walls of the three mycobionts and T. viride, but not to the walls of P. citrophthora; the binding pattern was similar to the grain pattern obtained in autoradiographs after short N-[³H] acetylglucosamine labelling. As wheat germ agglutinin binds specifically to chitin oligomers, the lectin binding tests further confirmed that chitin is a mycobiont hyphal wall component.Binding characteristics of several fluorescein-conjugated lectins to the three mycobionts indicated that this technique can yield useful information concerning the chemical composition of hyphal wall surfaces.List of abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - TCA trichloroacetic acid - PNA peanut agglutinin - LA lotus agglutinin - Glc NAc N-acetylglucosamine - ConA concanavalin A - SBA soybean agglutinin - WBA waxbean agglutinin Part of an M.Sc. thesis submitted by A. Braun to the Department of Botany, Tel Aviv University.     

CO2对离体欧芹叶片中蛋白质代谢的影响

以离体欧芹叶片为实验系统,研究了ＣＯ２对叶片中蛋白质降解的影响。对离体欧芹叶片中游离氨基酸含量及可溶蛋白含量的分析结果表明：ＣＯ２具有抑制离体欧芹叶片中蛋白质降解的作用,这为进一步了解ＣＯ２延缓叶片衰老的作用机理很有意义。     

Co-operative interactions during protein folding.

The theory for measuring co-operativity between interactions in proteins by protein engineering experiments is developed by introducing a procedure for analysing increasing orders of synergy in a protein with increasing numbers of residues. The (pairwise) interaction energy (delta 2Gint) between two side-chains may be measured experimentally by a double-mutant cycle consisting of the wild-type protein, the two single mutants and the double mutant. This procedure may be extended to three residues to give a value for delta 3Gint for a triple-mutant cube, and to higher orders using multi-dimensional mutant space. We now show that delta 3Gint is the excess energy of adding all three chains compared with the sum of all the pairwise values of delta 2Gint for each of the constituent double-mutant cycles and the sum of all the single addition energies. This physical interpretation extends to higher orders of mutation. delta nGint (i.e. the interaction energy for n residues), thus, reveals the layers of synergy in interactions as a protein is built up. This procedure is applied to measuring changes in synergy during the refolding of barnase for the triad of salt-linked residues Asp8, Asp12 and Arg110, which are mutated to alanine residues. The value of delta 3Gint in the folded structure is 0.77(+/- 0.06) kcal mol-1 (i.e. the triad is 0.77 kcal mol-1 more stable than expected from the sum of the individual pairwise interactions and single contributions). The value of delta 3Gint is still significant in the transition state for unfolding (0.60(+/- 0.07) kcal mol-1) and in the folding intermediate (0.60(+/- 0.13 kcal mol-1)). These results show that synergistic interactions exist in barnase, in its transition state for unfolding and in a refolding intermediate. A direct measurement of the change of co-operativity between the folded state and the transition state for unfolding shows a decrease of 0.17(+/- 0.04) kcal mol-1, suggesting that the initial stages of protein unfolding may be accompanied by some loosening of structure in parts that still interact. The similar extent of co-operativity in the transition state for unfolding and the intermediate in refolding suggests that the intermediate is homogeneous, at least in the region of the salt-linked triad, as heterogeneity would lower the co-operativity.     

COSMIC analysis of the major alpha-helix of barnase during folding

The structures of transition states and intermediates in protein folding may be analysed by protein engineering methods that remove simple interactions that stabilize the folded state. We have now extended the range and reliability of the procedure by using the COSMIC (Combination of Sequential Mutant Interaction Cycles) technique, in which a series of double-mutant cycles is constructed. In each cycle, the side-chains of two amino acid residues that interact in the folded state are mutated separately and together. Kinetic and equilibrium measurements on folding for each cycle show unambiguously whether or not two residues interact during protein folding. A series of such cycles has been constructed to leapfrog along the major alpha-helix of barnase, comprising residues 6 to 18. The helix is found to be intact from its C terminus to residue 12 but begins to unwind towards the N terminus in both the transition state for unfolding and in a folding intermediate.