potassium-thiocyanate and sodium-thiocyanate

To understand the effects of specific ions on protein-water interactions and the thermodynamic stability of proteins in salt solutions, we use a molecular dynamics (MD) simulation to examine the water structure, orientational distribution, and dynamics near the surface of ubiquitin. In particular, we consider NaCl, NaBF

Topics: Anions; Hydrogen Bonding; Molecular Dynamics Simulation; Salts; Surface Properties; Thiocyanates; Ubiquitin; Water

To seek a mechanism linking tobacco smoking with the increased incidence and severity of rheumatoid arthritis, deduced from many retrospective surveys, by studying arthritis/fibrosis development in rats.. Rats (>300) received low levels of sodium/potassium thiocyanate (10 or 25 mmol/l) in their drinking water to raise their blood thiocyanate levels, mimicking the elevated levels of blood, salivary and urinary thiocyanate found in smokers.. Thiocyanate supplements increased the severity of experimental arthritis induced by tailbase injection of (1) Freund's complete adjuvants (mycobacteria plus various adjuvant-active oils), (2) collagen type-II with Freund's incomplete adjuvant (no mycobacteria), (3) the synthetic lipid amine, avridine in an oil and (4) the natural hydrocarbons squalene (C(30)H(50)) and pristane (C(19)H(40)). This pro-arthritic effect was independent of sex, rat strain or changing diet and housing facilities. Thiocyanate supplements also amplified the acute/persisting inflammatory responses to paw injections of pristane, zymosan and microcrystalline hydroxyapatite. Iodide salts also mimicked some of these effects of thiocyanate.. Thiocyanate, a detoxication product of HCN present in tobacco smoke, increased (or even induced) inflammatory responses to several agents causing arthritis or fibrotic inflammation in rats. It, therefore, can act as a co-arthritigen, or 'virulence factor' and could be a therapeutic target to reduce arthritis expression and morbidity.

Topics: Administration, Oral; Animals; Arthritis, Experimental; Collagen; Diamines; Disease Models, Animal; Female; Hydrocarbons; Hydrogen Cyanide; Inflammation; Male; Mycobacterium; Rats; Rats, Wistar; Severity of Illness Index; Smoking; Thiocyanates

Raman spectra of water+N,N-dimethylformamide (DMF) mixtures and their solutions with NaNCS, KNCS and NH(4)NCS were obtained. The bands of nu(CO) stretching, delta(OCN) bending, r(CH(3)) rocking and nu(N-CH)(3)) stretching of the DMF molecule with and without salts were studied. The dependence of the vibration frequencies and Raman intensities of the bands on the composition of the mixed solvent was discussed. The change of the band frequencies as a result of the presence of the salts and the solvation of the cations by the solvent molecules was examined. The stronger cation solvation by the aprotic solvent molecules instead of the water molecules in DMF concentrated solutions was discussed. The nu(CN) and nu(CS) vibrations of the SCN(-) ions were observed as a function of the cation present and the solvent composition. The presence of the SCN(-) ions as "free", contact ion pairs, or solvent separated pairs, was discussed.

Topics: Dimethylformamide; Formamides; Solutions; Solvents; Spectrum Analysis, Raman; Thiocyanates; Water

The purification of biological macromolecules by affinity chromatography is a widespread technique used to separate a protein from other biological components. However, this method may destroy the protein's physiological activity because elution conditions aimed to dissociate the protein of interest from the high-affinity matrix often irreversibly denature it. In the present work, we have developed a solid-phase assay to determine the optimal elution conditions for any buffer (in two steps) by determining (i) the lowest buffer concentration yielding maximum dissociation from the immobilized component and (ii) the highest buffer concentration that can be used without the loss of the protein's binding activity. Any buffer that can be reasonably used between these defined concentrations is suitable for elution within this interval. The screen is easily performed within a few hours and only requires nanograms to a few micrograms of protein. As an example, we demonstrate that more than 95% of the human transferrin receptor bound to a transferrin-sepharose ligand affinity column can be eluted with full binding activity at KSCN concentrations between 232 and 414 nM, whereas elution with urea is not suitable to purify fully functional protein.

Topics: Apoproteins; Buffers; Chromatography, Affinity; Drug Stability; Ferric Compounds; Humans; Indicators and Reagents; Microchemistry; Receptors, Transferrin; Sepharose; Thiocyanates; Transferrin; Urea

Heterogeneity of intestinal D-glucose transport is demonstrated using pig jejunal brush-border membrane vesicles in the presence of 100/0 (out/in) mM gradients each of NaCl, NaSCN, and KSCN. Two D-glucose transport systems are kinetically distinguished: high-affinity, low-capacity system 1, which is equivalent to the symporter SGLT1; and low-affinity, high-capacity system 2, which is not a member of the SGLT family but is a D-glucose and D-mannose transporter exhibiting no preference for Na(+) over K(+). A nonsaturable D-glucose uptake component has also been detected; uptake of this component takes place at rates 10 times the rate of components characterizing the classical diffusion marker L-glucose. It is also shown that, in this kinetic work, 1) use of D-glucose-contaminated D-sorbitol as an osmotic replacement cannot cause the spurious appearance of nonexistent transport systems and 2) a large range (>/=50 mM) of substrate concentrations is required to correctly fit substrate saturation curves to distinguish between low-affinity transport systems and physical diffusion.

Topics: Animals; Biological Transport; Coloring Agents; Diffusion; Glucose; Intestinal Absorption; Intestinal Mucosa; Intracellular Membranes; Kinetics; Mannose; Membrane Glycoproteins; Monosaccharide Transport Proteins; Osmosis; Sodium Chloride; Sodium-Glucose Transporter 1; Sorbitol; Swine; Thiocyanates

The influx of L-lysine into apical vesicles from the chicken jejunum occurs through two systems, one with low Michaelis constant (K(m)) and features of system b0,+ and the other with relatively high K(m) for L-lysine and with properties of system y+. In the present study the effect of a lysine-enriched diet (Lys, containing 68 g L-lysine/kg dietary protein, control animals 48 g/kg) on L-lysine uptake through both transport systems was investigated. Results show that 1) lysine enrichment had no effect on either body weight or the efficiency of food utilization. 2) In Lys-fed animals, the mediated L-lysine influx was best fitted to the two-system model with y+ and b0,+ activity. 3) In the presence of an Na+ gradient, total L-lysine uptake is significantly higher in Lys-fed animals than in control birds (about 40% increase). 4) Lys diet increases K(m)b0,+ 6-fold (KSCN gradient) and 12-fold (NaSCN gradient) and maximum velocity (Vmax) by 6- and 20-fold, respectively. The effects of Lys enrichment on the y(+)-like system are only observed on the Vmax and in the presence of a Na+ gradient (30% increase). 5) Na+ is involved in the activation of the transport process in the Lys-fed chickens, but there is no correlation between external Na+ concentration and L-lysine influx. In conclusion, both b(0,+)- and y(+)-like transport systems are upregulated by dietary lysine but with different kinetic profiles; the high-capacity y(+)-like carrier shows a Vmax increase without changes in K(m), whereas the low-capacity b(0,+)-like system shows an increase in Vmax as well as in the K(m).

Topics: Animals; Biological Transport; Body Weight; Chickens; Diet; Energy Metabolism; Ethylmaleimide; Glucose; Intestinal Mucosa; Jejunum; Kinetics; Lysine; Male; Microvilli; Sodium-Potassium-Exchanging ATPase; Sucrase; Thiocyanates

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (KmA = 2.4 +/- 0.7 micromol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na+ (l-methionine inhibition constant KiA, NaSCN = 21.0 +/- 8.7 micromol/L and KSCN = 55.0 +/- 8. 4 micromol/L); second, a low-affinity transport mechanism (KmB = 164. 0 +/- 13.0 micromol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na+ (KiB for L-methionine, NaSCN = 1.7 +/- 0.3 and KSCN = 3.4 +/- 0.9 mmol/L). L-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b0,+ and y+, respectively.

Topics: Animals; Arginine; Biological Transport; Chickens; Ethylmaleimide; Glucose; Jejunum; Kinetics; Lysine; Male; Methionine; Microvilli; Models, Biological; Sodium-Potassium-Exchanging ATPase; Sucrase; Thiocyanates

1. In the present study we have examined the sensitivity of A and ASC amino-acid-carrier activities in rat liver plasma-membrane vesicles to the thiol-group modifying reagents N-ethylmaleimide (NEM) and iodoacetamide (IA). To this end, the different Na(+)-dependent entities involved in alanine transport were assessed. 2. NEM inactivated Na(+)-dependent alanine transport as a result of the inhibition of both system A and ASC transport activities. The functional sensitivity of system A to NEM was greater than that of system ASC. 3. The presence of L-alanine (10 mM) during the exposure of vesicles to NEM afforded partial protection to system A, but not to the ASC, carrier. This effect was specific, since the presence of L-phenylalanine (10 mM) did not cause any protection. 4. Na+ did not protect A or ASC carriers against NEM inactivation; however, the presence of Na+ (100 mM-NaCl) and L-alanine (10 mM) during the exposure of the vesicles to NEM protected against inactivation of system A and ASC transport activities. The extent of protection was greater in the case of the system ASC transport activity than in the case of the A carrier. 5. IA also diminished Na(+)-dependent alanine transport by inhibition of A and ASC transport activities. Sodium and L-alanine afforded protection to both A and ASC transport activities from the inhibitory action of IA. The extent of protection induced by substrates was similar for both carriers. 6. It is concluded that there is one, or several, free thiol groups in A and ASC carriers, the integrity of which is essential for transport activity. Sensitivity to thiol-group-specific reagents and the pattern of protection with substrates against inactivation is different in A and ASC carriers. That suggests the existence of topological dissimilarities regarding the thiol-group containing site(s) in A and ASC amino acid carriers.

Topics: Alanine; Amino Acid Transport Systems; Aminoisobutyric Acids; Animals; Biological Transport; Carrier Proteins; Cell Membrane; Ethylmaleimide; Iodoacetamide; Liver; Male; Rats; Rats, Inbred Strains; Sodium; Thiocyanates

The transport of L-histidine, a selective substrate for system N, across liver plasma membrane vesicles has been studied. The amino acid is accumulated sevenfold within the intravesicular space in the presence of a sodium gradient. Lithium can replace sodium to some extent in the cotransport mechanism. The amino acid translocation is not influenced by the transmembrane electrical potential difference. Histidine kinetics involves a saturable component plus a linear one both in the presence and in the absence of sodium. Sodium affects mainly the affinity of the translocator and to a lesser extent its mobility. Histidine uptake is competitively inhibited by glutamine and it is affected by alanine in a noncompetitive manner.

Topics: Alanine; Animals; Biological Transport; Cell Membrane; Glutamine; Histidine; Kinetics; Liver; Male; Membrane Potentials; Potassium; Rats; Rats, Inbred Strains; Sodium; Thiocyanates

The uptake of cytidine, of thymidine and of uridine in brush border vesicles prepared from the cortex of rat kidney has been studied by the technique of rapid filtration. The nucleosides were not metabolized in the vesicles. The time-courses of uptake in the presence of inwardly directed gradients of Na+ and of K+ showed an overshoot, indicating uphill transport. The overshoot was much more pronounced with Na+ than with K+; it was not observed when Na+ was at equilibrium across the membrane. The uptake of the nucleosides was stimulated by an inside negative potential in the presence of Na+. These results provide evidence for a cotransport of pyrimidine nucleosides with Na+. The apparent Km's for the uptake of cytidine, of thymidine and of uridine were 3.76 mumol X 1(-1), 4.18 mumol X 1(-1) and 7.21 mumol X 1(-1) respectively. The uptake of the pyrimidine nucleosides was insensitive to 6-nitrobenzylthioinosine. This insensitivity as well as the high affinity for the nucleosides and the capacity for uphill transport indicate that the nucleoside carrier(s) is renal brush border is (are) different from the carriers found in most other cell types.

Topics: Animals; Biological Transport, Active; Cytidine; In Vitro Techniques; Kidney; Male; Microvilli; Pyrimidine Nucleosides; Rats; Rats, Inbred Strains; Thiocyanates; Thymidine; Time Factors; Uridine

Topics: Body Fluids; Body Water; Extracellular Fluid; Humans; Thiocyanates; Water