Involvement of Amino-Acid Side Chains of Membrane Proteins in the Binding of Glutathione to Pig Cerebral Cortical Membranes

Glutathione (GSH), a general antioxidant and detoxifying compound, is the most abundant thiol-containing peptide in the central nervous system. It has been earlier shown to regulate the functions of glutamate receptors and to possess specific binding sites in both neurons and glial cells. The possible involvement of disulfide bonds, cysteinyl, arginyl, lysyl, glutamyl, and aspartyl residues in the binding of tritiated GSH to specific sites in pig cerebral cortical synaptic membranes was now studied after covalent modification of membrane proteins. Treatment of synaptic membranes with the thiol-modifying reagents 5,5-dithio-bis(2-nitrobenzoate) (DTNB) and 4,4-dithiodipyridine (DDP) dramatically enhanced the binding of [³H]GSH in a dose-dependent manner. Dithiothreitol (DTT) alone reduced the binding, but pretreatment of the membranes with DTT potentiated the enhancing effect of DTNB. On the other hand, when the modification with DTNB was followed by treatment with DTT, the enhancement by DTNB was completely reversed. N-ethylmaleimide, a thiol alkylating agent, and phenylisothiocyanate, a thiol- and amino-group modifying compound, reduced the binding, and their effects were additive. The guanidino-modifying agent phenylglyoxal reduced the binding but the carboxyl-modifying reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide had no significant effect. The results indicate that cysteinyl side chains and disulfide bonds are essential in the binding of GSH to membrane proteins and that arginyl and lysyl side chains may also be directly involved in this process.     

Interaction between carbohydrate residues of alpha(1)-acid glycoprotein (orosomucoid) and progesterone. A fluorescence study

Interaction between progesterone and the carbohydrate residues of alpha(1)-acid glycoprotein was followed by fluorescence studies using calcofluor white. The fluorophore interacts with polysaccharides and is commonly used in clinical studies. Binding of progesterone to the protein induces a decrease in the fluorescence intensity of calcofluor white, accompanied by a shift to the short wavelengths of its emission maximum. The dissociation constant of the complex was found equal to 8.62 microM. Interaction between progesterone and free calcofluor in solution induces a low decrease in the fluorescence intensity of the fluorophore without any shift of the emission maximum. These results show that in alpha(1)-acid glycoprotein, the binding site of progesterone is very close to the carbohydrate residues. Fluorescence intensity quenching of free calcofluor in solution with cesium ion gives a bimolecular diffusion constant (k(q)) of 2.23 x 10(9) M(-1) s(-1). This value decreases to 0.19 x 10(9) M(-1) s(-1) when calcofluor white is bound to alpha(1)-acid glycoprotein. Binding of progesterone does not modify the value of k(q) of the cesium. Previous studies have shown that the terminal sialic acid residue is mobile, while the other glycannes are rigid [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94]. Red-edge excitation spectra and Perrin plot experiments performed on sialylated and asialylated alpha(1)-acid glycoprotein show that binding of progesterone to alpha(1)-acid glycoprotein does not modify the local dynamics of the carbohydrate residues of the protein.     

Folding and oxidation of the antibody domain C(H)3

The non-covalent homodimer formed by the C-terminal domains of the IgG1 heavy chains (C(H)3) is the simplest naturally occurring model system for studying immunoglobulin folding and assembly. In the native state, the intrachain disulfide bridge, which connects a three-stranded and a four-stranded beta-sheet is buried in the hydrophobic core of the protein. Here, we show that the disulfide bridge is not required for folding and association, since the reduced C(H)3 domain folds to a dimer with defined secondary and tertiary structure. However, the thermodynamic stability of the reduced C(H)3 dimer is much lower than that of the oxidized state. This allows the formation of disulfide bonds either concomitant with folding (starting from the reduced, denatured state) or after folding (starting from the reduced dimer). The analysis of the two processes revealed that, under all conditions investigated, one of the cysteine residues, Cys 86, reacts preferentially with oxidized glutathione to a mixed disulfide that subsequently interacts with the less-reactive second thiol group of the intra-molecular disulfide bond. For folded C(H)3, the second step in the oxidation process is slow. In contrast, starting from the unfolded and reduced protein, the oxidation reaction is faster. However, the overall folding reaction of C(H)3 during oxidative folding is a slow process. Especially, dimerization is slow, compared to the association starting from the denatured oxidized state. This deceleration may be due to misfolded conformations trapped by the disulfide bridge.     

Down-regulation of protein L-isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

Protein L-isoaspartyl methyltransferase (PIMT) repairs the damaged proteins which have accumulated abnormal aspartyl residues during cell aging. Gene targeting has elucidated a physiological role for PIMT by showing that mice lacking PIMT died prematurely from fatal epileptic seizures. Here we investigated the role of PIMT in human mesial temporal lobe epilepsy. Using surgical specimens of hippocampus and neocortex from controls and epileptic patients, we showed that PIMT activity and expression were 50% lower in epileptic hippocampus than in controls but were unchanged in neocortex. Although the protein was down-regulated, PIMT mRNA expression was unchanged in epileptic hippocampus, suggesting post-translational regulation of the PIMT level. Moreover, several proteins with abnormal aspartyl residues accumulate in epileptic hippocampus. Microtubules component beta-tubulin, one of the major PIMT substrates, had an increased amount (two-fold) of L-isoaspartyl residues in the epileptic hippocampus. These results demonstrate that the down-regulation of PIMT in epileptic hippocampus leads to a significant accumulation of damaged tubulin that could contribute to neuron dysfunction in human mesial temporal lobe epilepsy.     

Characteristic moisture curves and maximum water content of two crop residues

Limited data are available relating water potential () to crop residue water content (), although this relationship is important to study decomposition and moisture retention of the residue layer in no-till systems and other agricultural situations where residues are used. The objectives of this study were (i) to determine the characteristic moisture curves of rye (Secale cereale L.) and clover residues (Trifolium incarnatum L.), and (ii) to determine residue characteristics that can predict maximum water content of crop residues. Air-dried residues were separated into leaves and stems, cut into 0.5 cm length pieces and saturated with distilled water. Pieces of the drained residues were dried to various water contents in the laboratory and then transferred into thermocouple psychrometer chambers. Characteristic moisture functions of the type = a ^–b, where a and b are empirical constants, were fitted to the data. The characteristic moisture curves had a similar shape to that of a Cecil sandy loam soil used as an example; however, while plant residues were able to retain up to 4.3 g H₂O g^–1, the mineral soil retained only 0.22 g H₂O g^–1. Soluble carbohydrate concentration can be used as a practical index to estimate maximum water content of residues, given the good relationship between both variables (R ² = 0.92).     

Effect of organic and mineral fertilizers on N-use by wheat under different irrigation frequencies

A field trial was established in Errachidia, southern Morocco, to investigate the interaction between wheat residue management and mineral 15N-labelled ammonium sulphate, under different irrigation treatments, applied to wheat (Triticum durum var. Karim). In treatments I1, I2, I3 and I4, plots were irrigated every 10, 15, 21 and 30 days. Each plot contained three sub-plots that received three fertilization treatments: T1 received 42 kg N ha-1 of ammonium sulphate before seedling, 42 kg N ha-1 of ammonium sulphate labelled with 9.764 at % 15N excess at tillering and 84 N kg ha-1 of ammonium sulphate at flowering; T2 received 42 kg N ha-1 of ammonium sulphate labelled with 9.764 at % 15N excess at seedling, 42 kg N ha-1 at tillering and 42 kg N ha-1 at flowering; T3 received 4800 kg ha-1 of wheat residue labelled with 1.504 at % 15N excess and 42 kg N ha-1 of ammonium sulphate before seedling and 42 kg N ha-1 of ammonium sulphate at flowering. Nitrogen fertilization with 168 kg N ha-1 did no significantly increase grain and straw yields in comparison to the 126 kg N ha-1 application. The combination of the organic input and supplementary application of mineral fertilizer N has been found as a more attractive management option. For all irrigation treatments, the % recovery of N in the whole plant was higher in plants that received 15N at tillering (63%, 49% respectively for irrigation intervals between 10 and 30 d) than in plants that received 15N just after seeding (28% for irrigation each 10- and 30-d intervals). For the irrigation treatment each 10 and 15 days, the 15N was mainly recovered by the grain for all fertilization treatments, whereas for irrigation treatment each 30 days, the grain and straw recovered nearly equal amounts of fertilizer. For grain and straw of wheat, nitrogen in the plant derived from the fertilizer was low, while most of the N was derived from the soil for all irrigation and fertilization treatments. The % nitrogen in the plant derived from the fertilizer values showed no significant difference between the different plant parts. The results suggested a dominant influence of moisture availability on the fertilizer N uptake by wheat. Under dry conditions the losses of N can be allotted to denitrification and volatilisation.     

Complete assignment of 1H, 13C and 15N chemical shifts for bovine β-lactoglobulin: Secondary structure and topology of the native state is retained in a partially unfolded form

Although -lactoglobulin (-LG) has been studied extensively for more than 50 years, its physical properties in solution are not yet understood fully in terms of its three-dimensional (3D) structure. For example, despite a recent high-resolution crystal structure, it is still not clear why the two common variants of bovine -LG which differ by just two residues have different aggregation properties during milk processing. We have conducted solution-state NMR studies on a recombinant form of the A variant of -LG at low pH conditions where the protein is partially unfolded and exists as a monomer rather than a dimer. Using a¹³ C,¹⁵N-labelled sample, expressed in Pichia pastoris, we have employed the standard combination of 3D heteronuclear NMR techniques to obtain near complete assignments of proton, carbon and nitrogen resonances. Using a novel pulse sequence we were able to obtain additional assignments, in particular those of methyl groups in residues preceding proline within the sequence. From chemical shifts and on the basis of inter-residue NOEs, we have inferred the secondary structure and topology of monomeric -LG A. It includes eight antiparallel -strands arranged in a barrel, flanked by an -helix, which is typical of a member of the lipocalin family. A detailed comparison with the crystal structure of the dimeric form (for a mixture of A and B variants) at pH 6.5 reveals a close resemblance in both secondary structure and overall topology. Both forms have a ninth -strand which, at the higher pH, forms part of the dimer interface. These studies represent the first full NMR assignment of -LG and will form the basis for a complete characterisation of the solution structure and dynamics of this protein and its variants.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Near-ultraviolet circular dichroic activity of apomyoglobin: resolution of the individual tryptophanyl contributions by site-directed mutagenesis

The individual tryptophanyl contributions to the near-ultraviolet circular dichroic activity of apomyoglobin in its native conformation have been resolved by studying recombinant proteins with single tryptophanyl substitutions. Site-directed mutagenesis of sperm whale apomyoglobin was performed in order to obtain proteins containing only Trp A-5 or Trp A-12. These amino acid substitutions have very little effect on the overall globin fold as indicated by comparing the spectroscopic properties of the mutants with those of the wild type protein. The circular dichroism spectra of the two apomyoglobin mutants in the near ultraviolet were found to be significantly different, both indole residues having significant activity but of opposite sign. In particular, Trp A-5 shows the presence of a main positive peak centered near 294 – 295 nm with a marked shoulder at 285 nm, ascribed to the ¹L_Btransition. The spectrum of the mutant protein containing only Trp A-12 shows a large negative contribution with a minimum near 283 nm and a marked shoulder at 293 nm. The broadness of the negative contribution exhibited by Trp A-12 suggests that it may originate mainly from the ¹L_A transition. Received: 17 February 1997 / Accepted: 14 August 1997     

Can we produce carbon and climate neutral forest bioenergy?

Harvesting branches, stumps and unmercantable tops, in addition to stem wood, decreases the carbon input to the soil and consequently reduces the forest carbon stock. We examine the changes in the forest carbon cycle that would compensate for this carbon loss over a rotation period and lead to carbon neutral forest residue bioenergy systems. In addition, we analyse the potential climate impact of these carbon neutral systems. In a boreal forest, the carbon loss was compensated for with a 10% increase in tree growth or a postponing of final felling for 20 years from 90 to 110 years in one forest rotation period. However, these changes in carbon sequestration did not prevent soil carbon loss. To recover soil carbon stock, a 38% increase in tree growth or a 21% decrease in the decomposition rate of the remaining organic matter was needed. All the forest residue bioenergy scenarios studied had a warming impact on climate for at least 62 years. Nevertheless, the increases in the carbon sequestration from forest growth or reduction in the decomposition rate of the remaining organic matter resulted in a 50% smaller warming impact of forest bioenergy use or even a cooling climate impact in the long term. The study shows that carbon neutral forest residue bioenergy systems have warming climate impacts. Minimization of the forest carbon loss improves the climate impact of forest bioenergy.