Nitrogen metabolism inErica and soybean,two species differing by their sensitivity to inorganic N source

Growth of soybean was not altered, whatever the inorganic N-source (NO₃, NH₄ or a NO₃/NH₄ mixture); conversely, growth of Erica x darleyensis plants in vitro decreased more in. NH₄ medium than in a NO₃ medium, compared to a NO₃/NH₄ medium. The GS/GOGAT pathway (in NH₄ medium), the nitrate and nitrite reductase activities (in NO₃ medium), as the contents in free nitrogenous forms and total nitrogen (in NO₃ and NH₄ media) were not more altered in Erica than in soybean, compared to a NO₃/NH₄ medium. PEPCase activity was the highest in soybean irrespective of the N-treatments; the involvement of PEPCase in N-metabolism could be explained by its function in ionic and osmotic balances rather than its function in supplying carboxylates as acceptors for NH₄-assimilation.     

Optimization of the coating of octyl-CALB with ionic polymers to improve stability and decrease enzyme leakage

Abstract

Lipase B from Candida antarctica (CALB) immobilized on octyl-agarose (OC) was submitted to coating with polyethylenimine (PEI) and dextran sulfate (DS). Using lowly loaded enzyme preparations, the properties of OC-CALB preparations hardly improved, suggesting too large the distance between enzyme molecules. However, using OC-CALB preparations with maximum loading, CALB stability was greatly improved in different conditions after PEI coating. Moreover, the CALB release from the OC support in the presence of detergents, or during thermal or organic solvent inactivations was greatly reduced after this treatment (PEI plus DS coating). The results pointed that the main positive effect of this coating could be derived from the physical intermolecular crosslinking of the CALB molecules with the polymers that reduce the enzyme desorption from the support. The coating of OC-CALB-PEI with DS only produced a minimal improvement on enzyme performance. Even though the enzyme release was much more difficult after physical crosslinking, all enzyme molecules could be released from the OC support combining an ionic detergent (SDS), high buffer concentration, pH 3 and 45?°C, while using the OC-CALB just 2% SDS at pH 7 and 25?°C was enough to release all enzyme. The support could be reused several cycles. Thus, this strategy permitted to greatly reduce the enzyme desorption during operation and to improve enzyme stability while keeping the enzyme immobilization reversibility.     

Triazavirine supramolecular complexes as modifiers of the peptide oligomeric structure

In this study, we present molecular dynamics simulations of the antiviral drug triazavirine, that affects formation of amyloid-like fibrils of the model peptide (SI). According to our simulations, triazavirine is able to form linear supramolecular structures which can act as shields and prevent interactions between SI monomers. This model, as validated by simulations, provides an adequate explanation of triazavirine’s mechanism of action as it pertains to SI peptide fibril formation.     

羊骨酶解液的植物乳杆菌发酵

为进一步提高羊骨酶解液的营养价值和生物利用度,探讨乳酸菌发酵对钙离子释放、短肽形成以及酶解液抗氧化活性的影响。首先从7株乳酸菌中筛选出了产蛋白酶能力最强的植物乳杆菌,接种羊骨酶解液,以发酵液中Ca~(2+)浓度为指标,响应面法优化得到发酵工艺:酶解液中添加1%的麦芽糖,调节起始pH为5.5,以4%接种量接种驯化好的植物乳杆菌,37℃摇床发酵14 h。发酵过程中,植物乳杆菌活菌数与Ca~(2+)含量(R=0.495,P0.01)和短肽得率(R=0.655,P0.01)呈极显著正相关,而多肽生成量与短肽得率呈负相关(R=–0.013)。发酵液中的Ca~(2+)浓度(P0.05)、水解度(P0.01)、短肽得率(P0.01)、羟脯氨酸含量(P0.01)均显著高于酶解液(P0.01),植物乳杆菌活菌数达到94.6×10~8 CFU/m L。发酵还可使酶解液对DPPH、·OH、O_2~-·三种自由基的清除能力显著提高(P0.01,P0.05)。综上所述,以植物乳杆菌发酵羊骨酶解液可进一步促进骨钙的转化和胶原短肽的生成并显著提高其体外抗氧化能力,短肽和Ca~(2+)反过来促进植物乳杆菌的繁殖,增强了酶解液的益生功能,乳酸菌产生的维生素、胞外多糖等物质使羊骨酶解液更富营养。     

Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A.

Synthetic peptides patterned after the predicted transmembrane sequence of botulinum toxin A were used as tools to identify an ion channel-forming motif. A peptide denoted BoTxATM, with the sequence GAVILLEFIPEIAI PVLGTFALV, forms cation-selective channels when reconstituted in planar lipid bilayers. As predicted, the self-assembled conductive oligomers express heterogeneous single-channel conductances. The most frequent openings exhibit single-channel conductance of 12 and 7 pS in 0.5 M NaCl, and 29 and 9 pS in 0.5 M KCl. In contrast, ion channels are not formed by a peptide of the same amino acid composition as BoTxATM with a scrambled sequence. Conformational energy calculations show that a bundle of four amphipathic alpha-helices is a plausible structural motif underlying the measured pore properties. These studies suggest that the identified module may play a functional role in the ion channel-forming activity of intact botulinum toxin A.     

Content and binding characteristics of the mitochondrial ATPase inhibitor,IF1 in the tissues of several slow and fast heart-rate homeothermic species and in two poikilotherms

We determined the IF₁ contents of pig, rabbit, rat, mouse, guinea pig, pigeon, turtle, and frog heart mitochondria and the effects of varying ionic strength upon the IF₁-mediated inhibition of the ATPase activity of IF₁-depleted rabbit heart mitochondrial particles (RHMP) by IF₁-containing extracts from these same eight species. The IF₁ binding experiments were run at both species-endogenous IF₁ levels and at an IF₁ level normalized to that present in rabbit heart mitochondria. When species-endogenous levels of rabbit heart IF₁ or either speciesendogenous or normalized levels of pig heart IF₁ were incubated with RHMP over a range of KCl concentrations, increasing the [KCl] to 150 mM had relatively little effect on IF₁-mediated ATPase inhibition. When either species-endogenous or normalized levels of guinea pig, pigeon, turtle, or frog heart IF₁ were incubated with RHMP under the same conditions, increasing [KCl] to 150 mM nearly completely blocked IF₁-mediated ATPase inhibition. While species-endogenous levels of rat and mouse heart IF₁ inhibited the ATPase activity of RHMP virtually not at all at any [KCl] examined, normalized levels of rat and mouse IF₁ inhibited the ATPase activity of RHMP to the same extents as species-endogenous levels of pig and rabbit heart IF₁, respectively, in the presence of increasing [KCl]. These experiments suggest that, while pig and rabbit heart mitochondria contain a full complement of higher-affinity IF₁, pigeon, guinea pig, turtle, and frog heart mitochondria cell contain essentially a full complement of a lower-affinity form of IF₁. In contrast, rat and mouse heart mitochondria contain only low levels of IF₁ which exhibit binding characteristics similar to those of the pig and rabbit heart inhibitor. The guinea pig is the only mammal thus far examined that contains a loweraffinity form of IF₁. In the present study we also determined the IF₁ contents and IF₁-to-F₁ ATPase activity ratios of cardiac muscle, skeletal muscle, liver, and brain mitochondria of rabbit, pigeon, and rat, species representative of the three homeothermic regulatory classes.     

Voltage-activated lonic currents in differentiating rat cerebellar granule neurons cultured from the external germinal layer

The electrical properties of the precursor cells of the external germinal layer of rat cerebellum were assessed during their differentiation in control medium (Dulbecco's modified Eagle's medium) supplemented or not with either basic fibroblast growth factor (bFGF) or 25 mM potassium chloride (KCI). Resting potential was shown to be –10 mV in all three conditions 3 hours after plating [days in vitro (DIV)0]. By DIV 5, it reached -63 mV for cells cultured in 25 mM KCI but only –28 mV in control and bFGF media. The main voltage-sensitive ionic current measured at DIV 0 under all conditions was a composite I_k consisting in a sustained K⁺ current blocked by tetraethylammonium (I_k(TEA)), plus a rapidly activating and inactivating TEA-insensitive I_k(A). Both currents increased with time in all conditions, but after 5 days I_K(A) became dominant in terms of density. I_K(TEA) is likely an I_K(Ca), since it was blocked by 67% in 1 mM TEA. On DIV O, I_Na and I_Ca were absent or small in amplitude. By DIV 3, 80% of the cells had currents able to generate a spike. Interestingly, I_Ca mean amplitude and current density measured at –10 mV in control condition on DIV 1 was singnificantly larger than those recorded in bFGF and 25 mM KCI. The order of appearance of the ionic currents, I_K, I_Ca, and I_Na, leads directly to fast spike activity allowing for poor calcium entry. Firing rate likely depends on I_K(A), which increased during the first 6 days of development but could be differentially regulated by bFGF. © 1995 John Wiley & Sons, Inc.     

Interpretation of ionic strength dependencies of the electron transfer in the Cytochrome c-Cytochrome b 5 system for the wild-type and lysine-mutated yeast Cytochrome c

Ionic strength dependencies of electron transfer between Cytochrome b ₅ and variants of yeast Cytochrome c were analyzed by curve fitting to the simple model of the electrostatic interaction between the two proteins assuming the process to be non-diffusion-controlled. Mutagenesis of Lys79, but not Lys72, leads to an increase of effective radius of the interacting charged species, suggesting that the mutation effects of the two residues on the electrostatic field distribution near the contact site are different, even within the crude electrostatic model used. Extrapolation of the ionic strength dependencies to infinite ionic strength resulted in similar values, around (2–3)×10^-6 for all Cyt c variants considered thus showing the lysine residue mutations to primarily affect protein association rather than the electron transfer directly. Based on the ionic strength dependencies of binding constants of the two proteins into an electrostatically stabilized complex, the monomolecular electron transfer rate constant was estimated to be 1.1×10⁴–1.6×10⁵ s^-1. The electrostatic part of the binding energy of the complex at I=0.19 was estimated to be-2.4 kcal/mol, strongly at variance with the values-13.0 and-6.4 kcal/mol reported for the two types of complexes identified using Brownian dynamics techniques. Possible reasons for this discrepancy are discussed.     

Role of metal cations in the regulation of NADP-linked isocitrate dehydrogenase from porcine heart

The regulatory role of divalent metal cations in the NADP-linked isocitrate dehydrogenase (EC 1.1.1.42) from porcine heart was analysed. Saturation curves with respect to the substrate threo-Ds-isocitrate complexed with the metals including manganous, cadmium, cobaltous and zinc ions showed sigmoid relationships characteristic of allosteric enzymes. The Hill's interaction coefficients were 1.90, 1.75, 1.28 and 1.12, respectively. Saturation kinetics of the substrate-metal complexes including magnesium, ferrous and nickel ions exhibited normal hyperbolic curves with Hill's coefficients of 1. The ionic radii of metal cations were closely correlated with the maximal velocity, the enzyme affinity and the Hill's n values for the substrate-metal complexes. Cooperative interactions of metal-substrate complexes with NADP-isocitrate dehydrogenase are discussed in relation to the sites of the enzyme for the binding of the metal-substrate complex.