New evidence of the nonequilibrium nature of the "slow modes" of diffusion in polyelectrolyte solutions

The time-dependent behavior of the dissolution of polyelectrolyte powders in pure water and moderate ionic strength aqueous solvent was monitored by flowing dissolving material through an online filter, and then through a multiangle light scattering unit, a refractometer, and a capillary viscometer. When the polyelectrolytes were dissolved in solutions of moderate ionic strength, their dissolution behavior was similar to that of neutral polymers. When dissolved in pure water, however, there was consistently a small population of aggregates that appeared at the beginning of the dissolution process, which then rapidly diminished. For large pore filtration, the aggregates reached a final low level, and slowly disappeared over the span of many days, whereas for small pore filtration the aggregates disappeared completely over a scale of minutes. The real-time data, together with size exclusion chromatography analysis, shed light on previously unanswered questions concerning the nonequilibrium nature of this small population of polyelectrolyte aggregates in low ionic strength solutions, and its relation to the "extraordinary phase" of diffusion (or "slow modes"). Further evidence is also provided that both angular scattering maxima due to interpolyion correlations and the maximum of reduced viscosity vs polyion concentration ("electroviscous" effect) at low ionic strength are equilibrium properties that are unrelated to these aggregates.     

THE INFLUENCE OF IONIC STRENGTH ON THE INTERACTION OF QUATERNARY DRUGS WITH MAMMALIAN ACETYLCHOLINESTERASE IN RELATION TO POSSIBLE REGULATORY EFFECTS

Abstract— The effects of inorganic salts, gallamine triethiodide and (+)-tubocurarine chloride on mammalian acetylcholinesterase (AChE) were examined. The results were obtained mainly from soluble erythrocyte AChE; particle-bound and detergent-solubilized rat brain enzymes were also used. Three aspects of AChE were examined, namely direct effects on activity, the recovery of activity of the diethylphosphorylated enzyme and thirdly the aggregation of the enzyme at low ionic strength as shown by chromatography on columns of Sepharose 6B. The action of gallamine on AChE was controlled by the substrate concentration and the ionic strength of the medium. Both inhibition and activation by gallamine could be observed, depending on the particular conditions used. All effects of gallamine disappeared when the ionic strength was raised to 015. The action of gallamine closely resembled the result of increasing ionic strength by adding NaCl, for in both cases the apparent affinity of AChE for substrate decreased and concomitantly the maximum velocity of hydrolysis increased. The phosphorylated enzyme recovered activity more rapidly when gallamine or tubocurarine were present, or when the ionic strength was increased. Aggregation of all enzyme forms was observed at low ionic strength; an increase to I = 015 dissociated AChE to the single molecular form. It was concluded that the mammalian enzymes closely resembled electric eel AChE. The possible methods by which regulation of AChE activity could occur are discussed in relation to these results.     

Changes in Glucose, Fructose and Saccharose Metabolism in Tobacco Plants Infected with Potato Virus Y

The content of glucose, fructose and saccharose as well as changes in the activities of enzymes involved in their biosynthesis and degradation were studied in tobacco plants infected with potato virus Y (necrotic strain) during the acute-infection period. Over the first part of this period, accumulation of saccharose, glucose and fructose was observed concurrently with decreased activities of the enzymes metabolizing saccharose, glucose and fructose (saccharases, saccharose synthase and hexokinases) and enhancement in the activities of enzymes synthesizing these carbohydrates (saccharosephosphate synthase, glucose-6-phosphate and/or fructose-6-phosphate phosphatases). The subsequent period was characterised by a reduction in both phosphatases that (together with just slightly raised saccharosephosphate synthase) could hardly produce enough sugars for the highly stimulated enzymes such as saccharases, saccharose synthase, and both kinases. Presumably for this reason, the previously increased content of sugars was considerably reduced to the level of control plants. The activities of glucokinase, fructokinase, saccharases and saccharose synthase were strongly increased at the culmination of virus multiplication and negatively correlated with the content of free glucose, fructose and saccharose. This revised version was published online in June 2006 with corrections to the Cover Date.     

Recovery from insulin-induced hypoglycemia after saccharose or glucose administration

This study compared the effects of saccharose and glucose on the recovery from insulin hypoglycemia. 17 normal volunteers (12 men, 5 women, 25-40 years old) received the same dose (0.1 IU i.v.) of semisynthetic rapid-acting human insulin on two different days after an overnight fast. Blood glucose and C peptide were measured in venous blood samples before as well as at regular time intervals after insulin administration. 30 min after the injection, 20 g saccharose or 20 g glucose p.o. (diluted in water) were given. The mean glucose values were at most time intervals higher after glucose than after saccharose administration. In addition, glucose ingestion resulted in an earlier and steeper blood glucose rise (mean recovery rates during the first 5 min 3.10 and 1.38 mg/dl/min for glucose and saccharose, respectively). The C peptide values decreased progressively and did not achieve baseline levels even at 120 min in spite of blood sugar normalization. It is concluded that glucose acts faster than saccharose in insulin-induced hypoglycemia. Exogenous insulin results in a prolonged depression of C peptide which lasts longer than the hypoglycemic effect.     

Leakage of internal markers from erythrocytes and lipid vesicles induced by melittin, gramicidin S and alamethicin: a comparative study.

The membrane-disruptive capacities of melittin, derivatised melittins, alamethicin and gramicidin S have been compared for the human erythrocyte membrane and lipid vesicles of three different compositions (phosphatidylcholine, 85% phosphatidylcholine/15% phosphatidylserine, and a lipid analogue of the outer leaflet of the human erythrocyte membrane). The sensitivity to ionic strength, divalent metal ions and polylysine of release of fluorescent markers from liposomes and of haemoglobin from intact erythrocytes has been assayed. Acetyl melittin was found to he more effective than melittin in lysing phosphatidylcholine and phosphatidylcholine/phosphatidylserine vesicles, somewhat less effective in the lipid analogue and markedly less effective in lysing erythrocytes. Succinyl melittin was non-haemolytic, but was able to lyse lipid vesicles at a high concentration. Ca2+ inhibited melittin haemolysis at high ionic strength (150 mM NaCl), but produced a more complex response of stimulation followed by inhibition at low ionic strength. In lipid vesicles, Ca2+ either stimulated melittin lysis or was ineffective. Zn2+ exerted effects similar to Ca2+ with lipid vesicles at approx. 10-fold lower concentration except that a weak inhibition was observed for the erythrocyte membrane lipid analogue at high ionic strength. Polylysine strongly inhibited haemolysis by melittin at low ionic strength, but was ineffective or stimulatory in lipid vesicle lysis. High phosphate concentration also inhibited melittin haemolysis, but again no corresponding effect could he found in any of the lipid vesicle systems. These disparities between effects of melittin on erythrocytes and lipid vesicles support the proposal that melittin-protein interactions are of consequence to its haemolytic action. Similar experiments were performed with gramicidin S and alamethicin in order to compare their lytic properties with those of melittin. It was found that each lysin exhibited its own individual pattern of sensitivity to lipid composition, ionic strength and inhibition by cations. It thus appears likely that the detailed molecular interactions responsible for lysis are significantly different for each of these three agents.     

Influence of protein net charge on the nucleic acid helix-destabilizing activity of various pancreatic ribonucleases

Summary Helix-destabilization of double-stranded poly[d(A-T)] induced by various homologous pancreatic ribonucleases which differ in their net charges has been studied under different ionic strength conditions.The response of the destabilizing activity of the various proteins to ionic strength is represented by bell-shaped curves, whose maxima are shifted to higher ionic strength values the higher the number of positive charges of the RNAase involved in the nucleic acid-protein complex.This observation is discussed, and a model proposed, that could explain the experimental results presented.     

Studies on the mechanism of action of the alkaline phosphatase from Dictyostelium discoideum

The Dictyostelium discoideum alkaline phosphatase was investigated kinetically in an attempt to elucidate its mechanism of action. Analysis of the hydrolysis of p-nitrophenyl phosphate by stopped-flow spectrophotometry revealed biphasic kinetics, suggesting a double displacement enzyme mechanism. Furthermore, Tris stimulated activity in an uncompetitive manner, a result that was consistent with this interpretation. The enzyme was inhibited reversibly by phosphate at low ionic strength, but the inhibition was irreversible at high ionic strength and the latter effect was enhanced at alkaline pH values. These results indicate that high ionic strength and alkaline pH conditions bring about a conformational change that renders the enzyme susceptible to irreversible inhibition by phosphate.     

Isolation of the individual structural elements of bacteria of the genus Bordetella and a study of their properties. I. The formation of mureinoplasts and true protoplasts from B. pertussis]

B. pertussis suspension was tested by De Voe et al. method (1970) and its modification with the solutions of a definite ionic composition and a lysozyme. The best results were obtained by the following modification elaborated by the authors. The microbes were grown on the casein-carbon agar for 36 hours and were washed with chilled 0.5 M NaCl. The suspension was washed 4 times with the same solution and then the precipitate was suspended in saccharose solution (0.5 M). In 2 hours the saccharose was replaced by a solution of salts with lysozyme. After a 2-hour incubation at 35 degrees C the substance was centrifugated for 20 minutes and the precipitate suspended in the tris-buffer at pH 7.8. The following changes were observed: after the washing and incubation with saccharose there was seen a strong stretching and separation of the cell wall (CW) from the cytoplasmic membrane (CPM); cells without the CW were rarely revealed; 2) after the lysozyme treatment there were many cells of spherical shape (phasic-contrast microscopy) without any CW, limited by the CPM only. Morphologically they were no different from the true protoplasts of the Gram-positive bacteria. The chemical analysis also confirmed a possibility of obtaining the true protoplasts of the Gram-negative bacteria.     

Stimulation of L-tryptophan transport by di- and polysaccharides in the small intestine of chicks

In in vivo and in vitro experiments the effect of various carbohydrates on the absorption of L-tryptophan in the chick small intestine was investigated. On feeding the chicks with semisynthetic diet containing 58.5% of carbohydrates a stimulatory effect of glucose, particularly of starch and saccharose, on the entry of L-tryptophan into the portal vein from the chick gastrointestinal tract has been found. Using an in vitro technique the activating effect of starch and disaccharides (maltose, saccharose) on the intestinal transport of L-tryptophan was detected while monosaccharides (glucose, fructose) at different concentrations had no effect on this process or inhibited it. The possibility that energy of disaccharide hydrolysis is used to stimulate transport process is discussed.     

Sigmoid kinetics of human erythrocyte glucose-6-phosphate dehydrogenase

Several disagreements and inconsistencies have appeared regarding whether human erythrocyte glucose-6-phosphate dehydrogenase exhibits sigmoid or classical kinetics with respect to NADP+ binding. The latest report is that the purified enzyme exhibits classical kinetics while the intracellular enzyme exhibits sigmoid kinetics (H. N. Kirkman, and G. F. Gaetani (1986) J. Biol. Chem. 261, 4033-4038). The various investigations were carried out at fixed pH, ionic strength, and temperature. The steady-state kinetics of crude and purified erythrocyte glucose-6-phosphate dehydrogenase are reported here at various temperatures, ionic strengths, and pH values and as a function of glucose 6-phosphate concentration. Sigmoid kinetics were observed for both purified and crude enzyme samples at high pH, temperature, ionic strength, and concentration of glucose 6-phosphate with Hill coefficients varying between 1.40 and 1.90. In contrast, at low pH, temperature, and ionic strength, the crude enzyme samples exhibit sigmoid kinetics while the purified samples exhibit classical kinetics despite the high concentration of glucose 6-phosphate. High concentrations of glucose 6-phosphate and factors favoring the enzyme in the dimeric form are necessary conditions for the observation of sigmoid kinetics in human erythrocyte glucose-6-phosphate dehydrogenase. These factors are high pH, ionic strength, and temperature. The observed sigmoid kinetics in this enzyme is explained as arising from tetramer-dimer transitions.     

Yeast hexokinase: substrate-induced association--dissociation reactions in the binding of glucose to hexokinase P-II.

A method is described for the purification of native hexokinases P-I and P-II from yeast using preparative isoelectric focussing to separate the isozymes. The binding of glucose to hexokinase P-II, and the effect of this on the monomer--dimer association--dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation. Association constants for the monomer-dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupired in the dimer. At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme-glucose complex was 3 X 10(-4) mol 1(-1) and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I=0.02 mol 1(-1), pH less than 7.5) was also independent of enzyme concentration (in the range 10-1000 mug ml-1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non-cooperative; values of the intrinsic dissociation constant varied from 2.5 X 10(-3) mol 1(-1) at pH 7.0 to 6 X 10(-3) at pH 6.5. Under intermediate conditions (pH 7.0, ionic strength=0.15 mol 1(-1)), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperatively (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations. The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.     

Kinetic Theory Model for Ion Movement through Biological Membranes: II. Interionic Selectivity

The equation presented in the previous paper for steady-state membrane ionic current as a function of externally applied electric field strength is numerically analyzed to determine the influence of ionic and membrane molecule parameters on current densities. The model displays selectivity between different ions. A selectivity coefficient S_i, defined as the ratio of current carried by an ionic species i at a given field strength to the current carried by a reference species at the same field strength, has the following properties: (a) S_i is a function of electric field strength except for ion-membrane molecule interactions yielding velocity independent collision frequencies; (b) for ion-membrane molecule interactions characterized by a collision frequency that is a decreasing (increasing) function of increasing ionic velocity, ions whose S_i > 1 (<1) at zero field strength will show maxima (minima) (minima[maxima]) in their S_i vs. electric field strength curves.     

Interaction between humic substances and carbohydrates in a Pampasic pond (Argentina)

Interaction between humic substances from a Pampasic pond and carbohydrates was studied. Determinations were performed by gel chromatography using radiolabeled glucose. The results showed that upon lowering the pH the interaction decreases in relation to the steric arrangement that causes the diminution of the accessibility of the available sites. Results of displacement experiments carried out by addition of unlabeled glucose, galactose, saccharose and starch, confirmed that the interaction occurs mainly through labile non specific bonds and is, to a certain degree, independent of the carbohydrate molecular weight involved. This revised version was published online in July 2006 with corrections to the Cover Date.     

Binding of complement component C1q by spectrin

125I-labelled human C1q was found to bind to human spectrin. Scatchard plots for the binding process were non-linear, indicating the possible presence of multiple classes of binding sites for C1q on spectrin. The binding was ionic-strength-dependent; the extent of binding decreased with increasing ionic strength. Chemical modification of arginine and histidine residues on C1q as well as pretreatment of C1q at pH 4.45 or at 56 degrees C reduced its spectrin binding activity. The amount of 125I-labelled C1q bound to immune complexes was reduced by the presence of spectrin. Spectrin was also able to deplete the complement haemolytic activity of human serum in a dose-dependent manner.     

Characterization of the tryptophan environments of interleukins 1 alpha and 1 beta by fluorescence quenching and lifetime measurements

The tryptophan environments of interleukins 1 alpha and 1 beta, immunomodulatory proteins with similar biological activities but only 25% sequence homology, were characterized by steady-state and dynamic fluorescence measurements. Both proteins exhibited similar emission maxima, but the emission intensity of IL-1 beta was greatly enhanced by increasing the ionic strength of the medium, whereas that of IL-1 alpha was unaffected. The two cytokines were also similarly quenched by the polar quencher acrylamide, but differences were observed for the ionic quenchers iodide and cesium. The fluorescence intensity decays of both cytokines were characterized by two (long and short) component lifetimes. However, the average lifetime of IL-1 beta (4.4 ns) was much longer than that of IL-1 alpha (1.93 ns). Taken together with the results of steady-state measurements, we suggest that the single tryptophan of IL-1 beta is statically quenched by neighboring charged residues, whereas the tryptophan fluorescence of IL-1 alpha is unaffected by ionic strength, and that the tryptophans of the two proteins have different accessibilities to ionic quenchers. The results are discussed in terms of similarities and differences in the tryptophan environments of the two proteins.     

Charge modification of the endothelial surface layer modulates the permeability barrier of isolated rat mesenteric small arteries

We hypothesized that modulation of the effective charge density of the endothelial surface layer (ESL) results in altered arterial barrier properties to transport of anionic solutes. Rat mesenteric small arteries (diameter approximately 190 microm) were isolated, cannulated, perfused, and superfused with MOPS-buffered physiological salt solutions. MOPS-solutions were of normal ionic strength (162 mM, MOPS), low ionic strength (81 mM, LO-MOPS), or high ionic strength (323 mM, HI-MOPS), to modulate ESL charge density (normal, high, or low ESL charge, respectively). Osmolarity of MOPS, LO-MOPS, and HI-MOPS was kept constant at 297 mosmol/l, using additional glucose when necessary. Perfusate solutions were supplemented with 1% BSA. Arteries were cannulated with a double-barreled theta-pipet on the inlet side and a regular pipet on the outlet side. After infusion of FITC-labeled dextran of 50 kDa (FITC-Delta50) and the endothelial membrane dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, the dynamics of arterial dye filling were determined with confocal microscopy. ESL thickness, as determined from the initial exclusion zone for FITC-Delta50 on the luminal endothelial surface, was 6.3 +/- 1.4 microm for LO-MOPS, 2.7 +/- 1.0 microm for MOPS, and 1.1 +/- 1.3 microm for HI-MOPS. At low ionic strength, FITC-Delta50 permeated into the ESL with a total ESL permeation time (tauESL) of 26 min, and at normal ionic strength with a tauESL of 20 min. No apparent exclusion of FITC-Delta50 from the ESL could be observed at high ionic strength. In conclusion, we demonstrate that the modulation of solvent ionic strength influences the thickness and barrier properties of the ESL.     

Pig heart fumarase really does exhibit negative kinetic co-operativity at a constant ionic strength.

The kinetics of the action of fumarase on L-malate and fumarate were investigated at constant ionic strength. This was done to evaluate reports that fumarase follows simple Michaelis-Menten kinetics. However, when pH, buffer concentration and ionic strength are all maintained at constant values, the Lineweaver-Burk plots exhibit pronounced downward curvature, characteristic of negative kinetic co-operativity.     

Characterization and antifungal activity of gazyumaru (Ficus microcarpa) latex chitinases: both the chitin-binding and the antifungal activities of class I chitinase are reinforced with increasing ionic strength

Three chitinases, designated gazyumaru latex chitinase (GLx Chi)-A, -B, and -C, were purified from the latex of gazyumaru (Ficus microcarpa). GLx Chi-A,-B, and -C are an acidic class III (33 kDa, pI 4.0), a basic class I (32 kDa, pI 9.3), and a basic class II chitinase (27 kDa, pI > 10) respectively. GLx Chi-A did not exhibit any antifungal activity. At low ionic strength, GLx Chi-C exhibited strong antifungal activity, to a similar extent as GLx Chi-B. The antifungal activity of GLx Chi-C became weaker with increasing ionic strength, whereas that of GLx Chi-B became slightly stronger. GLx Chi-B and -C bound to the fungal cell-walls at low ionic strength, and then GLx Chi-C was dissociated from them by an escalation of ionic strength, but this was not the case for GLx Chi-B. The chitin-binding activity of GLx Chi-B was enhanced by increasing ionic strength. These results suggest that the chitin-binding domain of basic class I chitinase binds to the chitin in fungal cell walls by hydrophobic interaction and assists the antifungal action of the chitinase.     

Saccharide induced racemization of amino acids in the course of the Maillard reaction

Summary. The formation of D-amino acids on heating aqueous solutions of protein L-amino acids at pH 2.5 and pH 7.0 together with glucose, fructose or saccharose was investigated by enantioselective gas chromatography. The saccharide induced partial racemization (epimerisation) of L-amino acids is attributed to the Maillard reaction. Received October 1, 2001 Accepted October 2, 2001     

Sperm motility in turbot,Scophthalmus marimus: initiation of movement and changes with time of swimming characteristics

Synopsis Turbot sperm motility is observed using dark field microscopy and stroboscopic illumination combined with video recording. Sperm motility is triggered by dilution of spermatozoa in sea water or in non ionic media (glucose or saccharose), presenting osmotic pressure ranging from 300 to 2100 mOsmol. The percentage of motile spermatozoa reaches 100% under conditions of osmotic pressure of 300 to 1100 mOsmol and pH close to 8.0. In full sea water, glucose or saccharose solutions an agglutination of spermatozoa is observed; this is prevented by addition of bovine serum albumin (5 mg ml^–1). Immediately after transfer in activation solutions, 100% spermatozoa are motile in most samples freshly stripped. This percentage drops suddenly between 15 and 30% after 70 to 100 sec. The beat frequency remains at a constant value of 50 Hz during 40 s post activation and then drops suddenly between 15 and 30 Hz. The spermatozoa velocity is about 200 micrometers s^–1 during 30 to 40 s and then declines to a stable value of 100 micrometers s^–1 at 50 s post activation. After 1.20 mn, more and more spermatozoa become motionless. The minimum calculated and averaged distance covered during 1.20 min, is about 12 mm. The high performances of turbot spermatozoa motility are interpreted as a compensatory mechanism for the low sperm production.