Differential effects of magnesium on tubulin-nucleotide interactions

Magnesium-depleted 2-(N-morpholino)ethanesulfonate (Mes), glutamate, tubulin and microtubule-associated proteins were prepared and used to study the effects of exogenously added MgCl2 on tubulin-nucleotide interactions in 0.1 M Mes with microtubule-associated proteins and in 1.0 M glutamate. Endogenous levels of Mg2+ in the systems studied were approximately stoichiometric with the tubulin concentrations and largely derived from the tubulin. We examined the effects of added Mg2+ on tubulin polymerization, GDP inhibition of polymerization, binding of GDP and GTP to tubulin, and GTP hydrolysis. Exogenously added Mg2+ had markedly different effects on these reactions. The order of their sensitivity for a requirement for added Mg2+ was as follows: GTP binding greater than GTP hydrolysis greater than polymerization greater than GDP binding. Inhibition of polymerization by GDP varied inversely with the Mg2+ concentration and was greatest in the absence of the cation. These results indicate that GDP and GDP-Mg2+ interact with similar affinity at the exchangeable site, while GTP-Mg2+ has a higher affinity for tubulin than does free GTP. Nevertheless, under appropriate conditions, free GTP can interact sufficiently well with tubulin to permit both nucleation and elongation reactions.     

Examination of tubulin-nucleotide interactions by protein fluorescence quenching measurements

The fluorescence emission spectrum of bovine brain tubulin is quenched upon binding of GTP, GMPP(NH)P, or GMPP(CH₂)P to the tubulin·GDP complex. At saturating levels of GTP or its nonhydrolyzable β-γ analogues, the partially quenched spectrum is virtually identical, suggesting that a similar conformational state is attained in each case. Titrations with each ligand yielded dissociation constants of 0.8, 3, and 3μM for GTP, GMPP(NH)P, and GMPP(CH₂)P; in all cases the stoichiometry is essentially one molecule of nucleotide per dimer. It is concluded that GDP and GTP stabilize different conformations and that the nonhydrolyzable analogues mimic the binding of GTP. This may be related to the ability of GMPP(NH)P and GMPP(CH₂)P to maintain a pre-assembly conformation similar to tubulin·GTP.     

Effects of pH on protein-protein interactions and implications for protein phase behavior

The effects of pH on protein interactions and protein phase behavior were investigated by measuring the reduced second osmotic virial coefficient (b2) for ovalbumin and catalase, and the aggregate and crystal solubilities for ovalbumin, beta-lactoglobulin A and B, ribonuclease A and lysozyme. The b2 trends observed for ovalbumin and catalase show that protein interactions become increasingly attractive with decreasing pH. This trend is in good agreement with ovalbumin phase behavior, which was observed to evolve progressively with decreasing pH, leading to formation of amorphous aggregates instead of gel bead-like aggregates, and spherulites instead of needle-like crystals. For both acidic and basic proteins, the aggregate solubility during protein salting-out decreased with decreasing pH, and contrary to what is commonly believed, neither aggregate nor crystal solubility had a minimum at the isoelectric point. beta-Lactoglobulin B was the only protein investigated to show salting-in behavior, and crystals were obtained at low salt concentrations in the vicinity of its isoelectric point. The physical origin of the different trends observed during protein salting-in and salting-out is discussed, and the implications for protein crystallization are emphasized.     

Modulation of tubulin-nucleotide interactions by metal ions: comparison of beryllium with magnesium and initial studies with other cations.

With microtubule-associated proteins (MAPs) BeSO4 and MgSO4 stimulated tubulin polymerization as compared to a reaction mixture without exogenously added metal ion, while beryllium fluoride had no effect (E. Hamel et al., 1991, Arch. Biochem. Biophys. 286, 57-69). Effects of both cations were most dramatic at GTP concentrations in the same molar range as the tubulin concentration. We have now compared effects of beryllium and magnesium on tubulin-nucleotide interactions in both unpolymerized tubulin and in polymer. Polymer formed with magnesium had properties similar to those of polymer formed without exogenous cation, except for a 20% lower stoichiometry of exogenous GTP incorporated into the latter. In both polymers the incorporated GTP was hydrolyzed to GDP. Stoichiometry of GTP incorporation into polymers formed with beryllium or magnesium was identical, but much of the GTP in the beryllium polymer was not hydrolyzed. The beryllium polymer was more stable than the magnesium polymer. Beryllium also differed from magnesium in only weakly enhancing the binding of GTP in the exchangeable site of unpolymerized tubulin, while neither cation affected GDP exchange at the site. If both cations were present in a reaction mixture, polymer stability was little changed from that of the beryllium polymer, but most of the GTP incorporated into polymer was hydrolyzed. Six additional metal salts (AlCl3, CdCl2, CoCl2, MnCl2, SnCl2, and ZnCl2) also stimulated MAP-dependent tubulin polymerization, but enhanced polymer stability did not correlate with polymer GTP content. We postulate that enhanced polymer stability is a consequence of cation binding directly to tubulin and/or polymer while deficient GTP hydrolysis in the presence of beryllium, as well as aluminum and tin, is a consequence of tight binding of cation to GTP in the exchangeable site.     

Effect of pH on MHC class II-peptide interactions.

The effect of pH on class II-peptide interactions has been analyzed using several mouse (IAd, IAk, IEd, IEk) and human (DR1, DR5, DR7) MHC specificities, and eight different class II-restricted determinants. In direct binding assays, acidic conditions led to increased binding capacity for many class II-peptide combinations. IE molecules seemed to bind optimally around pH 4.5, whereas IA molecules displayed binding optima in the 5.5 to 6.5 range. In contrast, the DR molecules studied were, in most cases, affected only marginally by pH changes in the 4.5 to 7.0 range. Despite these apparent isotype-specific trends, no general rule could be formulated, because even for the same class II molecules, the binding capacity could be increased for many peptides when the binding was performed under acidic conditions, was unaffected for some, and even decreased for others. The mechanisms responsible for this complex behavior were analyzed in more detail by kinetic and equilibrium analysis of three different class II-peptide combinations (IAd/OVA 323-339, IAk/HEL 46-61, and DR1/HA 307-319). It was found that acidic pH conditions could affect both on and off rates for class II-peptide complexes. Depending on the net balance of these effects, either increases, decreases, or no effect on overall affinities at equilibrium were detected. In the case of IAd/OVA 323-339, it was also found that acidic conditions influenced the binding capacity of class II molecules by increasing the fraction of sites available for peptide binding, presumably by favoring dissociation of endogenously bound, acid-sensitive peptides.     

Effects of pH on benzimidazole fluorescence

Four benzimidazoles (unsubstituted, 5-methyl, 2-ethyl, and 2-ethyl-5-methyl) have been characterized by fluorescence spectroscopy. At low pH (<6), activation at 270 nm caused fluorescence at 305 nm; at high pH (<8), activation at 270 nm caused fluorescence at 365 nm. The relative proportion of peak fluorescence at either 305 or 365 nm was correlated with the pK_a values of the four benzimidazoles. It was concluded that the protonated specie of benzimidazole was fluorescent at 365 nm and the unprotonated specie was also fluorescent at 305 nm.     

Effects of pH on acetylcholine receptor function

Summary We have examined the effects of changing extracellular pH on the function of nicotinic acetylcholine receptors fromTorpedo californica using ion flux and electrophysiological methods. Agonist-induced cation efflux from vesicles containing purified, reconstituted receptors showed a monotonic dependence on external hydrogen ion concentration with maximal fluxes at alkaline pH and no agonist-induced efflux at pH's less than 5. A similar pH dependence was measured for the peak agonist-activated membrane currents measured in microelectrode voltage-clampedXenopus oocytes induced to expressTorpedo receptor through mRNA injection. Half-maximal inhibition occurred at a similar pH in both systems, in the range of pH 6.5–7.0. Single-channel currents fromTorpedo ACh receptors measured in patch-clamp recordings were also reduced in amplitude at acid pH with an apparent pK _a for block of <5. Measurements of channel kinetics had a more complicated dependence on pH. The mean channel open time determined from patch-clamp measurements was maximal at neutral pH and decreased at both acid and alkaline pH's. Thus, both channel permeability properties and channel gating properties are affected by the extracellular pH.     

The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics

A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a “buffering” effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (~pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0–7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination.     

Monitoring protein-small molecule interactions by local pH modulation

We have developed a technique for sensing protein-small molecule and protein-ion interactions in bulk aqueous solution by utilizing a pH sensitive dye, 5-(and-6)-carboxyfluorescein, conjugated to free lysine residues on the surfaces of designated capture proteins. The fluorescein intensity was found to change by about 6% and 15% for small molecule and ion binding, respectively. The assay works by modulating the local electric fields around a pH sensitive dye. This, in turn, alters the dye's apparent pK(A) value. Such changes may result directly from the charge on the analyte, occur through allosteric effects related to the binding process, or result from a combination of both. The assay was used to follow the binding of Ca(2+) to calmodulin (CaM) and thiamine monophosphate (ThMP) to thiamine binding protein A (TbpA). The results demonstrate a binding constant of 1.1μM for the Ca(2+)/CaM pair and 3.2nM for ThMP/TbpA pair, which are in excellent agreement with literature values. These assays demonstrate the generality of this method for observing the interactions of small molecules and ions with capture proteins. In fact, the assay should work as a biosensor platform for most proteins containing a specific ligand binding site, which would be useful as a simple and rapid preliminary screen of protein-ligand interactions.     

Effects of divalent cations on the activity of ribulosebisphosphate carboxylase: interactions with pH and with D2O as solvent

Maximum velocity-pH profiles have indicated that one ionizing group on the quinary complex of ribulosebisphosphate carboxylase must be dissociated and possibly a second protonated for catalytic activity. The pK values for the soybean enzyme are 7.5 and 9.1, respectively. A third group, identified from $\text{V}\text{K}\text{-}\text{pH}$ profiles for carbon dioxide as the variable substrate and having a pK of 8.1 for the soybean enzyme, must be dissociated for the binding of carbon dioxide. Only the pK at 7.5 is changed when manganese is substituted for magnesium, whereas only the pK at 8.1 is altered when deuterium oxide is substituted for water as solvent. Small but different solvent isotope effects are observed in the maximum velocity with the two cations.     

Effects of cytochalasin B on sperm-egg interactions

The effects of cytochalasin B (CB) on the interaction of sea urchin (Arbacia), molluscan (Spisula), and mammalian (Mus) gametes have been examined. Despite the absence of sperm incorporation and gamete membrane fusion, CB-treated Arbacia and Spisula eggs (1–10 μg/ml for 1–10 min) mixed with sperm activated. Unlike the situation observed in Arbacia and Spisula, mouse eggs treated with CB (1–50 μg/ml for 1 hr) are capable of sperm incorporation. These data are discussed with reference to possible mechanisms by which sperm may induce eggs to activate.     

Effects of pH on bacterial porin function.

Porin is a trimeric channel-forming protein in the outer membrane of Gram-negative bacteria. Functions of the porins OmpF, OmpC, and PhoE from Escherichia coli K12 were analyzed at various pHs. Preliminary results from bilayer lipid membrane and liposome swelling assays indicated that in vitro porin has at least two open-channel configurations with a small and a large size. The small channels were stabilized at low pH while the larger channels were detected under basic conditions. The size switch occurred over a very narrow range near neutral pH, and the two major open-channel configurations responded differently to variations in voltage. The presence of two or more pH-dependent substates of porin could explain the variability in pore diameter measured by others and suggests a more dynamic role for porin in the cell.     

Effects of plant hybridization on herbivore-parasitoid interactions

We studied the effects of host plant hybridization on the survival and mortality of the leaf-mining moth Phyllonorycter salicifoliella on hybrid and parental willow plants in the field and in a common garden experiment. P. salicifoliella survival differed significantly among three willow taxa in the field in 1994 but not in the field in 1995 or in the common garden. Parasitism by eulophid wasps differed significantly among taxa in 1994 and appeared to account for the variation in their survival. In the field in 1995, host feeding predation varied significant among taxa. The theory of tritrophic interactions predicts that plant genotype can affect natural enemy impact, and this study supports this prediction. Significant variation in survival and eulophid parasitism was also found among genotypes within taxa in the field in both years and in the common garden experiment. The common garden results show that genetic differences in plants affect the herbivore-parasitoid interaction. Variation among years in the patterns of survival and causes of mortality among field plants suggest that genotype by environment interactions may be important. Received: 1 March 1996 / Accepted: 4 November 1996     

Effects of TRH injection on hippocampal-hypothalamic-pituitary-thyroid interactions

The first experiment was conducted in an attempt to ascertain the effects of hippocampal lesions on plasma levels of total T₄, total T₃, free T₄, and TSH. Animals with hippocampal lesions had significantly lower levels of total T₄, free T₄ and TSH than cortical control and normal animals. There were no significant differences in total T₃ among the three groups. Since these results indicated that animals with hippocampal lesions manifested a dysfunction in the hypothalamic-pituitary-thyroid axis, a second experiment was undertaken to determine the site of mediation. A TRH injection test demonstrated that the dysfunction occurs at the level of the hypothalamus. It is hypothesized that in the normal animal the hippocampus exerts a facilitating effect on the hypothalamic-pituitary-thyroid axis.     

Effects of nitrogen fertilization on tritrophic interactions

Tritrophic interactions (plant—herbivore—natural enemy) are basic components of nearly all ecosystems, and are often heavily shaped by bottom-up forces. Numerous factors influence plants’ growth, defense, reproduction, and survival. One critical factor in plant life histories and subsequent trophic levels is nitrogen (N). Because of its importance to plant productivity, N is one of the most frequently used anthropogenic fertilizers in agricultural production and can exert a variety of bottom-up effects and potentially significantly alter tritrophic interactions through various mechanisms. In this paper, the potential effects of N on tritrophic interactions are reviewed. First, in plant-herbivore interactions, N availability can alter quality of the plant (from the herbivore’s nutritional perspective) as food by various means. Second, nitrogen effects can extend directly to natural enemies through herbivores by changes in herbivore quality vis-à-vis the natural enemy, and may even provide herbivores with a defense against natural enemies. Nitrogen also may affect the plant’s indirect defenses, namely the efficacy of natural enemies that kill herbivores attacking the plant. The effects may be expressed via (1) quantitatively and/or qualitatively changing herbivore-induced plant volatiles or other plant features that are crucial for foraging and attack success of natural enemies, (2) modifying plant architecture that might affect natural enemy function, and (3) altering the quality of plant-associated food and shelter for natural enemies. These effects, and their interactive top–down and bottom-up influences, have received limited attention to date, but are of growing significance with the need for expanding global food production (with accompanying use of fertilizer amendments), the widening risks of fertilizer pollution, and the continued increase in atmospheric CO₂.     

Effects of genotype-environment interactions on genetic correlations

The objective of the work presented here was to investigate the influence of genotype-environment interaction on genetic correlations. In our theoretical models we have considered plant populations consisting of random samples of lines from chromosome-doubled haploids produced from F ₁ gametes, highly inbred SSD-lines, and clones of randomly breeding populations grown in two and multiple environments. The results of our theoretical considerations are that if genotype-environment interaction exists, great differences are expected to occur in the estimates of genetic correlation coefficients obtained in different environments. Based on the variance and covariance components for genotype-environment interaction we suggest a new type of correlation coefficient, called genotype-environment correlation, r _ge . Our theory has been applied to several series of experiments. Estimates are presented from two series, both of which demonstrate clearly the consequences of genotype-environment interaction on the genetic correlations.     

Resolving the interactions of ocean acidification and temperature on coral calcification media pH

Coral Reefs - Ocean acidification typically reduces the calcification rates of massive Porites spp. corals, but increasing seawater temperatures (below the stress and bleaching threshold) can...