Protein diffusion in crowded electrolyte solutions

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.     

Computational Oral Absorption Simulation for Low‐Solubility Compounds

Bile micelles play an important role in oral absorption of low‐solubility compounds. Bile micelles can affect solubility, dissolution rate, and permeability. For the pH–solubility profile in bile micelles, the Henderson–Hasselbalch equation should be modified to take bile‐micelle partition into account. For the dissolution rate, in the Nernst–Brunner equation, the effective diffusion coefficient in bile‐micelle media should be used instead of the monomer diffusion coefficient. The diffusion coefficient of bile micelles is 8‐ to 18‐fold smaller than that of monomer molecules. For permeability, the effective diffusion coefficient in the unstirred water layer adjacent to the epithelial membrane, and the free fraction at the epithelial membrane surface should be taken into account. The importance of these aspects is demonstrated here using several in vivo and clinical oral‐absorption data of low‐solubility model compounds. Using the theoretical equations, the food effect on oral absorption is further discussed.     

Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest: I. The Influence of Solvent

The electrochemical properties of three nitroimidazoles, a nitropyrazole, a nitrofuran and three nitroben-zenoid compounds have been extensively investigated in a range of solvents. The reduction pathway for the nitro group is independent of the cyclic function to which it is attached, but is strongly influenced by the nature of the solvent. In aqueous media, generally, a single, irreversible 4-electron reduction occurs to give the hydroxylamine. In aprotic media (dimethylformamide, methylene chloride or dimethylsulphoxide), a reversible one-electron reduction takes place to form a stable nitro radical anion. At more negative values, a further 3-electron reduction occurs, irreversibly to give the hydroxylamine. In mixed aqueous-organic systems, intermediate behaviour is found, with the reversibility of the RNO₂/RNO₂^- couple increasing with addition of organic medium. The control of the reduction pathway, by changing the electrolytic medium is discussed in relation to the biological activities of the drugs and identification of the short-lived reduction intermediate responsible for DNA damage.     

A study of light-induced conductivity in bacteriorhodopsin

Measurements have been made of light-induced conductivity changes and the associated kinetics of the relaxation processes in aqueous suspensions and sonicated liposomes containing bacteriorhodopsin (bR). Aqueous suspensions exhibit a single relaxation time of 1 to 2 ms. The addition of D₂O to the aqueous suspension slows down the relaxation time, fourfold. Similar behaviour is seen in sonicated liposomes with a relaxation time of 2 to 3 ms. Activation energies of approximately 14 and 6 kJM^-1 are obtained for the effect in sonicated liposomes and aqueous suspension containing bR, respectively. These relaxation processes with lifetime of 1 to 2 ms suggest conformational changes in the protein moiety of bR which most probably may be associated with protonation-deprotonation processes or less likely the release and binding of small ions.     

Surface heterogeneity of bovine sperm revealed by aqueous two-phase partition

Ejaculated, bovine sperm have been subjected to multiple partition in aqueous two-phase systems. This partition, carried out in a countercurrent fashion, reveals heterogeneity of the sperm population with respect to surface properties. The sperm, when partitioned in phase systems that detect non-change associated surface properties (change-insensitive) are largely distributed as two distinct populations. In charge-sensitive phase systems (which principally detect cell surface molecules carrying charge) the sperm do not show any obvious surface heterogeneity. Considerable heterogeneity is revealed in affinity-ligand phase systems containing palmitic acid coupled to one of the phase components-poly(ethylene glycol). There is a difference in surface heterogeneity between sperm which have been washed in buffer or left unwashed, direct from the ejaculate. This is indicative of weak adsorption of proteins to the sperm surface in seminal fluid. These results show that bovine ejaculated sperm is a heterogeneous cell population having unequal distributions of a number of different surface molecules.     

Partition coefficients of plant cuticles for monoterpenes

Summary Cuticle/water partition coefficients (K_c/w) for d-limonene, -pinene and -pinene were determined by an extrapolation and a desorption method. The sorption experiments were carried out with isolated angiosperm and gymnosperm cuticles and with [¹⁴C]-labelled monoterpenes, which were obtained biosynthetically. Both methods were suitable for the determination of the K_c/w of volatile hydrophobic compounds. For the angiosperm cuticles the partition coefficients are of the order of 10⁴, which indicates a high accumulation of monoterpenes in the cuticle. The values of the conifer cuticles of Picea abies (L.) Karst. and Abies alba Mill., however, are lower due to their high lignin content. This is proved by the increase of the partition coefficients after removal of polar and phenolic components. The K_c/w can be estimated with good accuracy from the octanol/water partition coefficient, which was determined experimentally.     

Mechanism for Cd2+ inhibition of (K++ Mg2+)ATPase activity and K+ (86Rb+) uptake join roots of sugar beet (Beta vulgaris)

Steady state kinetics were used to examine the influence of Cd²⁺ both on K⁺ stimulation of a membrane-bound ATPase from sugar beet roots (Beta vulgaris L. cv. Monohill) and on K⁺(⁸⁶Rb⁺) uptake in intact or excised beet roots. The in vitro effect of Cd²⁺ was studied both on a 12000–25000 g root fraction of the (Na⁺+K⁺+Mg²⁺)ATPase and on the ATPase when further purified by an aqueous polymer two-phase system. The observed data can be summarized as follows: 1) Cd²⁺ at high concentrations (>100 μM) inhibits the MgATPase activity in a competitive way, probably by forming a complex with ATP. 2) Cd²⁺ at concentrations <100 μM inhibits the specific K⁺ activation at both high and low affinity sites for K⁺. The inhibition pattern appears to be the same in the two ATPase preparations of different purity. In the presence of the substrate MgATP, and at K⁺ <5 mM, the inhibition by Cd²⁺ with respect to K⁺ is uncompetitive. In the presence of MgATP and K⁺ >10 μM, the inhibition by Cd²⁺ is competitive. 3) At the low concentrations of K⁺, Cd²⁺ also inhibits the 2,4-dinitrophenol(DNP)-sensitive (metabolic) K⁺(⁸⁶Rb⁺) uptake uncompetitively both in excised roots and in roots of intact plants. 4) The DNP-insensitive (non metabolic) K⁺(⁸⁶Rb⁺) uptake is little influenced by Cd²⁺. As Cd²⁺ inhibits the metabolic uptake of K⁺(⁸⁶Rb⁺) and the K⁺ activation of the ATPase in the same way at low concentrations of K⁺, the same binding site is probably involved. Therefore, under field conditions, when the concentration of K⁺ is low, the presence of Cd²⁺ could be disadvantageous.     

Response of potatoes to N fertilizer: Quantitative relations for components of growth

Summary Quantitative relationships for key processes influencing N response were derived from measurements of inorganic N in soil, the weights and N contents of foliage and tubers made at intervals during growth of maincrop potatoes in 11 N fertilizer experiments.Apparent mineralization rates (calculated from measurements of N uptake and inorganic N in the top metre and averaged over the growth period) were remarkably similar from site to site despite wide differences in the textures, water contents and organic matter contents of the soils. They were mostly about 0.78 kg N ha^–1 m^–1 d^–1.Inorganic N in the top 50 cm of soil was rapidly removed by the crop until it fell on all sites to a low value (about 4 g N cm^–3) which was maintained for the remainder of the growth period. When N fertilizer was applied, growth rate until at least the end of July was always well defined by a single coefficient in a previously derived equation. Average values of this coefficient for each of the soil types and for each of the years in which the experiments were carried out were within 20% of each other.The minimum %N in the dry matter needed to permit maximum growth rate declined with increase in plant weight in a similar manner to that previously found for other crops.Equations were found for the partition of assimilate and of nitrogen between the foliage and tubers. The coefficients in them were little affected by whether or not N fertilizer was applied.According to these relationships the maximum potential dry weight yield of tubers is 20 t ha^–1 and requires the crop to contain at least 290 kg N ha^–1.     

Extraction of chromatophores from Rhodospirillum rubrum in aqueous two-phase systems: A method for large-scale isolation of membrane particles

Chromatophores, membrane vesicles with the capacity of cyclic photophosphorylation, have been isolated from Rhodospirillum rubrum cells on a pilot plant scale. Results of disintegration in a glass bead mill and in a high pressure homogenizer were compared. The chromatophores were isolated from the crude extract by extraction in aqueous two-phase systems. In systems of polyethylene glycol (PEG) and dextran the chromatophores were partitioned to the upper PEG phase by the addition of PEG-palmitate. Most of the proteins and nucleic acids were forced to the bottom phase by addition of sodium chloride. Methods to prevent precipitation of the chromatophores were studied.     

Isolation and fractionation of CHO chromosomes in aqueous two phase systems using charged polymers and base specific macroligands

Summary Chromosomes were isolated in a preparative scale by synchronisation of CHO cells with a double Thymidine block followed by an arrest in the metaphase by addition of Colcemid. Under proper cultivation conditions a mitotic index of 77% total cells could be routinely achieved. Bulk chromosome preparations free of nuclei and other subcellular particles have been obtained by low speed centrifugation followed by a 60 transfer countercurrent distribution using aqueous two phase systems composed of polyethylenglycol and dextran. The partition of CHO chromosomes previously purified in aqueous two phase systems were studied further to develop a protocol for the separation and isolation of individual chromosomes. Partition experiments with chromosomes changing the electrostatic phase potential by addition of charged PEG-derivatives suggest the existence of relatively highly charged chromosome groups. Most promising results with regard to separation were obtained using two PEG-derivatives, which interact specifically with the bases in DNA. For this affinity partitioning a GC- and AT-specific macroligand were employed. Comparing CCD's using each of these ligands information on the GC and AT content of exposed DNA in the chromosomes groups could be derived, demonstrating that specific sequences of DNA are accessible at the surface of metaphase chromosomes.