Effects of high concentration of salts on the esterase activity and structure of a kiwifruit peptidase, actinidain

Effects of salts on the activity and stability of actinidain were examined. With increasing salt concentration up to 0.5 M, the activity (kcat/Km) for N-alpha-Cbz-L-lysine p-nitrophenyl ester decreased to 40% of that in the absence of salt. The inhibitor constant Ki of LiCl, NaCl, and KCl was 0.16-0.43 M. With 3 M KCl and NaCl, the specificity constant kcat/Km recovered to 110 and 75%, respectively. No re-activation was observed with LiCl. The inhibition and re-activation were dependent on the changes in both Km and kcat, whereas no CD change was observed. The tryptophan fluorescence of actinidain was not affected by 0-0.5 M salt, but a considerable decrease in its intensity was observed with increasing salt concentration from 0.5 to 3.0 M. These results suggest that the inhibition observed with the lower salt concentration (<0.5 M) is due to attenuation of the electrostatic interaction between the enzyme and substrate, and the higher concentration (0.5-3.0 M) induces structural change in the states of tryptophan residues, which is associated with the re-activation. Actinidain keeps considerably high activity and stability even in the presence of 3 M salts.     

Salt effects on beta-glucosidase: pH-profile narrowing

Salts inhibit the activity of sweet almond beta-glucosidase. For cations (Cl(-) salts) the effectiveness follows the series: Cu(+2), Fe(+2)>Zn(+2)>Li(+)>Ca(+2)>Mg(+2)>Cs(+)>NH(4)(+)>Rb(+)>K(+)>Na(+) and for anions (Na(+) salts) the series is: I(-)>ClO(4)(-)>(-)SCN>Br(-) approximately NO(3)(-)>Cl(-) approximately (-)OAc>F(-) approximately SO(4)(-2). The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k(cat)/K(m) for the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic pK(a)s and not to an effect on the pH-independent second-order rate constant, (k(cat)/K(m))(lim). For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent pK(a1)increases (from 3.7 to 4.9) and the apparent pK(a2)decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent (k(cat)/K(m))(lim) (=9 x 10(4) M(-1) s(-1)) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the pK(a)s when the LiCl concentration is increased to 1.0 M. The results of these salt-induced pK(a) shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a pK(a) of 3.7 and dissociated from the group with a pK(a) of 7.2) is very small ( approximately 0.03%). At higher salt concentrations, where the two pK(a)s become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, (k(cat)/K(m))(lim), of the monoprotonated species. For other enzymes which may show such salt-induced pK(a) shifts, this provides a convenient test for the role of reverse protonation.     

Effects of salts on thermolysin: activation of hydrolysis and synthesis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester, and a unique change in the absorption spectrum of thermolysin.

It has been reported that neutral salts such as NaCl activate the thermolysin-catalyzed hydrolysis of substrates containing glycine at the P1 position (carboxylic side of the cleavage bond) [Holmquist, B. & Vallee, B.L. (1976) Biochemistry 15, 101-107]. In this paper, we demonstrate that high concentrations (1-4 M) of neutral salts greatly enhance the thermolysin activity in both hydrolysis and synthesis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZAPM), a precursor of a peptide sweetener, aspartame, in which the L-aspartyl residue is the P1 residue. The enzyme activity is enhanced with an increase in salt concentration in a pseudo-exponential fashion. The degree of activation by salts was in the order LiCl > NaCl > KCl. The rate of ZAPM hydrolysis in the presence of 3.8 M NaCl was 6-7 times higher than that in its absence, and 50 times or more activation is expected in saturated NaCl solution. The activation is brought about solely through an increase in the catalytic constant (kcat), and the Michaelis constant (Km) is not affected at all by the presence of NaCl. On mixing thermolysin with NaCl, a unique absorption difference spectrum suggesting a conformational change of the enzyme was observed. The intensity increased in a pseudo-exponential fashion with increase of NaCl concentration up to 3 M, and this dependence is similar to that of the enzyme activity.     

Reaction of lysyl oxidase with soluble protein substrates: effect of neutral salts.

Soluble proteins released into the medium of aortic tissues in culture behave as substrates for the enzyme lysyl oxidase. The reaction shows an unusual dependence on the concentration of neutral salts in the assay medium. Practically no enzyme activity was observed in Tris-HCl, 0.005 m, pH 7.6 buffer. However, supplementing the buffer with high concentrations of KCl, KBr, NaCl, and (NH₄)₂SO₄ (in decreasing order of effectiveness) accelerated velocities as much as 10-fold. CaCl₂, KSCN, and KI at increasing concentrations became strongly inhibitory. β-Aminopropionitrile, a specific inhibitor of lysyl oxidase, effectively blocked the catalysis in low and high KCl. The salt-stimulated effects on lysyl oxidase activity were not as noticeable when insoluble proteins were used as substrates. Kinetic studies employing double reciprocal plots revealed that high KCl concentrations (2.0 m) raised the maximum velocity of the reaction but did not alter the apparent K_m. Thus high salt concentrations did not affect the binding of the soluble substrate to the enzyme. In high salts, however, more radioactive substrate proteins appeared to bind to the enzyme, suggesting that the high salt environment increases the fraction of the total enzyme potentially capable of binding to and catalyzing a reaction with the substrate.     

The radiation response of cultured mammalian V79-S171 cells exposed to a wide concentration range of sulphate salt solutions.

The radiation response of Chinese hamster cells (V79) exposed to a wide concentration range of Li2SO4, Na2SO4 or K2SO4 has been examined and compared with the radiation response of cells treated in an identical manner with LiCl, NaCl, or KCl solutions. At hypotonic salt concentrations, cells were radiosensitized by both the chloride and sulphate salts. At high salt concentrations, approximately greater than 0.9 M, a radioprotective effect was observed with both chloride and sulphate salts. At intermediate salt concentrations from about 0.2 to 0.9 M, the cells that were treated with the sulphate salt solutions were radioprotected; cells treated with chloride salt solutions were radiosensitized. The difference in radiation response was attributed to the difference in anions for the two types of salts used.     

Effect of lithium salts on lactate dehydrogenase,adenylate kinase,and 1-phosphofructokinase activities

Inhibitions of 30?nM rabbit muscle 1-phosphofructokinase (PFK-1) by lithium, potassium, and sodium salts showed inhibition or not depending upon the anion present. Generally, potassium salts were more potent inhibitors than sodium salts; the extent of inhibition by lithium salts also varied with the anion. Li₂CO₃ was a relatively potent inhibitor of PFK-1 but LiCl and lithium acetate were not. Our results suggest that extents of inhibition by monovalent salts were due to both cations and anions, and the latter needs to be considered before inhibition can be credited to the cation. An explanation for monovalent salt inhibitions is proffered involving interactions of both cations and anions at negative and positive sites of PFK-1 that affect enzyme activity. Our studies suggest that lithium cations per se are not inhibitors: the inhibitors are the lithium salts, and we suggest that in vitro studies involving the effects of monovalent salts on enzymes should involve more than one anion.     

Effects of divalent cations and sodium taurocholate on pancreatic lipase activity with gum arabic-emulsified tributyrylglycerol substrates.

The effects of Ca2+ and/or sodium taurocholate on lipase activity with gum arabic-emulsified tributyrylglycerol substrates were investigated. Calcium was found to slightly increase lipase activity while bile salts showed marked inhibition except at very low concentrations. Calcium eliminated inhibition seen with low concentrations of bile salts and reduced the inhibition seen at higher bile shift of the enzyme from the alkaline region in the absence of bile salt to the slightly acidic region in the presence of bile salt. Calcium was shown to eliminate the time lag periods between enzyme addition and maximum rate of hydrolysis seen at low substrate concentrations and the time lag noted when bile salts were included with normal (substrate concentration not limiting) assay concentrations of substrate. Zeta potential measurements indicated that Ca2+ reduced the negative charge on the gum arabic-emulsified particle while bile salts did not increase the negative charge. Commercial preparations of gum arabic were found to have significant concentrations of Ca2+ and Mg2+.     

Effect of neutral salts on the interaction of rat brain hexokinase with the outer mitochondrial membrane.

The glucose 6-phosphate (Glc-6-P)-induced solubilization of mitochondrial hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) from rat brain can be reversed by low concentrations (ionic strength <~0.02 m) of neutral salts. When compared to the original particulate enzyme (i.e., enzyme found on the particles prior to solubilization by Glc-6-P), the rebound enzyme is similar in distribution on sucrose gradients, K_m for ATP, inhibition by antiserum to purified brain hexokinase, and resistance to removal by exhaustive washing of the particles. The effectiveness of chloride salts at promoting rebinding increases in the order Cs⁺< Rb⁺< K⁺≤ Na⁺< Li⁺< Mg²⁺. This salt-induced rebinding is attributed to the screening of negative charges on the enzyme and/or membrane by cations, thereby decreasing repulsive forces and enhancing attractive interactions between enzyme and membrane. Solubilization of the enzyme, both in the presence and absence of Glc-6-P, is increased at alkaline pH, as would be expected due to increasing repulsive interactions between negative charges on membrane and enzyme as the pH is increased beyond the pI of the enzyme (pI = 6.3). In contrast to previous interpretations, P_i displayed no special efficacy at reversing Glc-6-P-induced solubilization, being comparable to other neutral salts on an ionic strength basis. However, P_i and its structural analog, arsenate, were shown to counteract specifically the Glc-6-P-induced inhibition and conformational change in the enzyme. At higher concentrations (ionic strength >~ 0.02 m) neutral salts themselves lead to reversible dissociation of the enzyme from the mitochondria. The efficacy of the salts depends primarily on the pH and on the position of the anion in the Hofmeister series, with salts of chaotropic anions (SCN^?, I^?, Br^?) being most effective. At pH 6, both chaotropic and nonchaotropic salts solubilize the enzyme, while at pH 8.5, only the chaotropes retain this ability. Neutral salts also have a reversible effect on the conformation of the enzyme, as reflected by enzymatic activity, with chaotropic salts again being most effective; there is no pronounced influence of pH (in the range of pH 6–8.5) on the ability of the salts to cause conformational change in the enzyme. Based on a lack of correlation between saltinduced solubilization and conformational changes affecting activity, it is concluded that the latter are not directly responsible for release of the enzyme from the membrane. In the presence of KSCN, the extent of solubilization decreased with increase in temperature, indicating a negative enthalpy for solubilization. In contrast, in the absence of salt, the enthalpy for solubilization was positive. These temperature effects and the effects of neutral salts on the hexokinase-membrane interaction are interpreted in terms of a model in which electrostatic forces are considered to be of major importance. At low ionic strength, repulsive forces between negative charges on enzyme and membrane predominate; screening of these charges by cations diminishes the repulsion, effectively enhancing attractive electrostatic forces between enzyme and membrane and thus promoting their interaction. At higher ionic strengths, the attractive electrostatic forces are themselves disrupted, resulting in dissociation of the enzyme from the membrane. It is proposed that the greater effectiveness of chaotropic salts at disrupting these attractive forces is due to their increased ability to penetrate through hydrophobic regions of enzyme and membrane to relatively inaccessible sites of electrostatic-interaction.     

Effect of free and conjugated bile salts on alpha-amylase activity.

The effect of individual bile salts on alpha-amylase hydrolysis of Cibachron Blue starch was studied at pH 6.0. With sodium cholate, taurocholate and taurodeoxycholate, enzyme activity was increased to 150-160 percent of the control value, at a concentration of similar to 1 mmol/l bile salt. The increased activity extended up to 4 mmol/l. The bile salts sodium deoxycholate and taurochenodeoxycholate exerted activation and inhibition depending on the concentration. With deoxycholate (0.75 mmol/l), activation (150 percent) was evident, while inhibition was apparent above 2.5 mmol/l. With taurochenodeoxycholate maximum activity (135 percent) was observed at 0.25 mmol/l, while inhibition was evident above 1.5 mmol/l. Chenodeoxycholate and lithocholate exerted marked inhibition at concentrations as low as 0.5 mmol/l. Inhibition of alpha-amylase by chenodeoxycholate was competitive with both soluble and insoluble starch substrates. Since the pH of the jejunum is in the region of 6.0 the phenomenon of activation and inhibition of alpha-amylase by bile salts at this pH could be of physiological significance.     

The denaturation of ribonuclease A by combinations of urea and salt denaturants.

When urea is added to ribonuclease A that has already been denatured by salt (CaCl₂, LiClO₄ or LiCl were used), a second co-operative transition occurs, supporting the previous demonstration that these salts cause only partial denaturation. Also we have studied the effect of the salts on the urea denaturation, and the effect of urea on the salt denaturation. At low concentrations urea makes the salt transitions occur at lower concentrations, but at higher concentrations it changes the transition so that the completely disordered protein found in urea is produced by the salt. At low concentrations the salts actually stabilize the protein against denaturation by urea, but at higher concentrations they destabilize it. The data are presented in “phase diagrams” which are found to be very useful for such three-component systems.     

Purification and characterization of a mesohalic catalase from the halophilic bacterium Halobacterium halobium.

When subjected to the stress of growth in a relatively low-salt environment (1.25 M NaCl), the halophilic bacterium Halobacterium halobium induces a catalase. The protein has been purified to electrophoretic homogeneity and has an M(r) of 240,000 and a subunit size of approximately 62,000. The enzyme is active over a broad pH range of 6.5 to 10.0, with a peak in activity at pH 7.0. It has an isoelectric point of 4.0. This catalse, which is not readily reduced by dithionite, shows a Soret peak at 406 nm. Cyanide and azide inhibit the enzyme at micromolar concentrations, whereas maleimide is without effect. The addition of 20 mM 3-amino-1,2,4-triazole results in a 33% inhibition in enzymatic activity. The tetrameric protein binds NADP in a 1:1 ratio but does not peroxidize NADPH, NADH, or ascorbate. Although the enzymatic activity is maximal when assayed in a 50 mM potassium phosphate buffer with no NaCl, prolonged incubation in a buffer lacking NaCl results in inactive enzyme. Moreover, purification must be performed in the presence of 2 M NaCl. Equally as effective in retaining enzymatic function are NaCl, LiCl, KCl, CsCl, and NH4Cl, whereas divalent salts such as MgCl2 and CaCl2 result in the immediate loss of activity. The catalase is stained by pararosaniline, which is indicative of a glycosidic linkage. The Km for H2O2 is 60 mM, with inhibition observed at concentrations in excess of 90 mM. Thus, the mesohalic catalase purified from H. halobium seems to be similar to other catalases, except for the salt requirements, but differs markedly from the constitutive halobacterial hydroperoxidase.     

Monovalent cation-induced conformational change in glucose oxidase leading to stabilization of the enzyme

Glucose oxidase (GOD) from Aspergillus niger is an acidic dimeric enzyme having a high degree of localization of negative charges on the enzyme surface and dimer interface. We have studied the effect of monovalent cations on the structure and stability of GOD using various optical spectroscopic techniques, limited proteolysis, size exclusion chromatography, differential scanning calorimetry, and enzymic activity measurements. The monovalent cations were found to influence the enzymic activity and tertiary structure of GOD, but no effect on the secondary structure of the enzyme was observed. The monovalent cation-stabilized GOD was found to have a more compact dimeric structure but lower enzymic activity than the native enzyme. The enzyme's K(m) for D-glucose was found to be slightly enhanced for the monovalent cation-stabilized enzyme (maximum enhancement of about 35% for LiCl) as compared to native GOD. Comparative denaturation studies on the native and monovalent cation-stabilized enzyme demonstrated a significant resistance of cation-stabilized GOD to urea (about 50% residual activity at 6.5 M urea) and thermal denaturation (Delta T(m) maximum of 10 degrees C compared to native enzyme). However, pH-induced denaturation showed a destabilization of monovalent cation-stabilized GOD as compared to the native enzyme. The effectiveness of monovalent cations in stabilizing GOD structure against urea and thermal denaturation was found to follow the Hofmeister series: K(+) > Na(+) > Li(+).     

Effect of Salts on Growth and Pectinase Production by Halotolerant Yeast, <Emphasis Type="Italic">Debaryomyces nepalensis</Emphasis> NCYC 3413

In this study, Debaryomyces nepalensis NCYC 3413 isolated from rotten apple was studied for its halotolerance and its growth was compared with that of Saccharomyces cerevisiae in high salt medium. The specific growth rate of D. nepalensis was not affected by KCl even up to a concentration of 1 M, whereas NaCl and LiCl affected the growth of D. nepalensis. Among all tested salts, LiCl showed maximum inhibition on growth. At all conditions, halotolerance of D. nepalensis was much higher than that of S. cerevisiae. D. nepalensis showed maximum viability (80–100%) when grown in KCl, which was higher than with NaCl and LiCl. Pectinase production by D. nepalensis was noted at all high salt concentrations, namely, 2 M NaCl, 2 M KCl, and 0.5 M LiCl, and the maximum specific activity was observed when the strain was grown in 2 M NaCl.     

Implication of a tyrosine residue in the unspecific bile salt binding site of human pancreatic carboxylic ester hydrolase

Tyrosine residues of the human pancreatic carboxylic-ester hydrolase (EC 3.1.1.1) (also referred to as cholesterol-ester hydrolase, EC 3.1.1.13) were nitrated in the ortho-position by the use of tetranitromethane. The specificity of the reaction has been verified and the inhibition observed was shown to be unrelated to the weak polymerization of the protein. Among the 27 tyrosines present in the enzyme, seven or eight were nitrated but only one residue, with a pK of 8.3, seems to be responsible for the loss of activity. This decrease in enzyme activity appears only in assays which were performed in the presence of bile salts, suggesting that of the two bile salt binding sites postulated on the enzyme, only one, referred to the as the 'unspecific site' (Lombardo, D. and Guy, O. (1980) Biochim. Act 611, 147-155), was modified. This is in agreement with the similar loss of enzyme activity observed on emulsified and soluble substrate. The most important result is the difference observed in experiments of the protective effects of bile salts. The protection with sodium taurodeoxycholate is independent of its critical micellar concentration, showing that monomers protect this site, whereas the protection observed in experiments with sodium cholate appears only for supramicellar concentrations of bile salt. Since this latter bile salt promotes the dimerization of the enzyme, we can conclude that a premicellar bile salt binding site (protected by monomers) is transformed in a functional micellar binding site (protected by micelles). This conformational transformation seems to be consecutive to the dimerization, as has been recently proposed.     

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei. Effects of pH, salts, temperature, and source of NADPH on enzyme activity and substrate specificity studies.

Activity analyses of pure dihydrofolate reductase from amethopterin-resistant Lactobacillus casei conducted with commercial sources of NADPH yielded a progression of nonlinear assay tracings whose shapes were both pH dependent and reminiscent of classical product inhibition. The extent of curving of the assay tracings was dependent on the source and age of the commercial NADPH and was enhanced as the pH was decreased from 7.5 to 5.0. Under these conditions a “pseudo”-pH-activity profile, exhibiting a maximal specific activity of 9 units/mg of protein between pH 7.0 and 7.5, was found. In contrast, freshly prepared NADPH provided strictly linear assay tracings over the pH range of 8.5 to 5.0, yielding uniformly higher specific activities than those observed with commercial NADPH. The new pH-activity profile was characterized by a broad optimum between pH 5.0 and 6.0, with a maximal specificity activity of 24.9 units/ mg in 0.1m potassium phosphate in the absence of added salt. The curving phenomenon and pseudo-pH optimum observed with commercial NADPH is attributed to the presence of minor but potent inhibitory impurities in these coenzyme preparations. Optimal concentrations of monovalent (~0.1 m) and divalent (~0.05 m) salts activated the enzyme between 1.5- and 1.7-fold, resulting in maximal specific activities in the range of 34 to 39 units/mg. A similar extent of activation was observed in 0.8 m Tris-acetate buffer, pH 5.5. At concentrations of monovalent salts above 0.5 m and of divalent salts above 0.2 m a reduction in salt-dependent activation and, in some cases, inhibition of activity were obtained. Substrate specificity studies indicated that the V for folate at saturating levels is 1% of that for dihydrofolate. Deamino-NADPH yielded V values 1.4-fold higher than that for NADPH, while acetylpyridine-NADPH and thio-NADPH provided values 6.5- and 235-fold lower, respectively, than the value with the natural coenzyme. Gel electrophoresis studies reflected a similar trend of selectivity in the interaction of NADPH and its analogs to form stable binary complexes. Stable ternary complexes of enzyme and amethopterin were formed with NADPH, deamino-NADPH, thio-NADPH, and acetylpyridine-NADPH. Although neither dihydrofolate nor NADP⁺ and its analog form stable complexes with L. casei dihydrofolate reductase, both NADP⁺ and deamino-NADP⁺ interact with enzyme and dihydrofolate to generate stable ternary complexes.     

The inhibition and activation of polyamine oxidase from oat seedlings

In a homologous series of di-guanidines (NH₂C(–NH)NH(CH₂)_xNHC(–NH) NH₂) where x=2–12, greatest inhibition of polyamine oxidase was found with x=8. The synthetic fungicide guazatine269-1 was particularly effective as an inhibitor of polyamine oxidase, with Ki of ca 10^-8 M. Inhibition due to the tri-amine derived from guazatine by hydrolysis was less effective by a factor of ca 200. Comparison of various inorganic salts at 1 M showed that polyamine oxidase activity was enhanced in the order RbCl>KCl>KBr>NH₄Cl>NaNO₃>LiCl>LiCl=NaCl> control (no salt) >CaCl₂=MgCl₂. Activity in RbCl was about 4 to 5 times greater than in the salt-free control. Enzyme activity is rapidly lost during assay. This loss of activity could not be attributed to inhibition by aminopropylpyrroline or diaminopropane. Moreover the superoxide scavenger copper salicylate had no protective effect on enzyme activity.     

A CD study of the role of metal ions in the conformation of poly(L-lysine)

The effect of LiCl, NaCl, and CsCl as univalent salts, and of CaCl₂, ZnCl₂, and MgCl₂ as divalent salts, on the α and antiparallel β-sheet, and random conformations of poly(L-lysine) (PLL), in water at room temperature were examined by means of CD and compared quantitatively on the basis of elliptical strength at the maximal peak. Changes in the α-helical and antiparallel β-sheet helical conformations of PLL were markedly dependent on the salt concentrations of LiCl, NaCl, and CsCl, which induced decreases in negative intensity in that order. The CD spectrum of the random conformation, the most disordered form, displayed positive cotton effect in concentrations of these salts up to 3.0M and a negative peak in concentrations of 6.0M. The effect of these salts on the random conformation of PLL was stronger than that on the α- and β-conformations in higher concentrations. The CD spectrum of the random conformation in the presence of CaCl₂, ZnCl₂, and MgCl₂, on the other hand, showed negative cotton effect in salt concentrations as low as 3.0M. It was impossible, however, to measure the effect on α- and β-conformations of ZnCl₂ and MgCl₂ above concentrations of 10 mM because of a solubility problem with salts in alkaline solution.     

Effect of salts on the activity of carrot phosphofructokinase

Phosphofructokinase (EC 2.7.1.11) from carrot roots was activated by a number of salts. Increase in salt concentration beyond the optimum generally led to a decrease in enzyme activity. Salts of the multivalent anions sulfate and phosphate were very effective activators and inhibitors. Potassium acetate and potassium succinate were also activators. Potassium tartrate and potassium citrate produced a small stimulation at low concentration but with further increase they became inhibitory. The results suggested that the salt effect was largely due to anions rather than cations. Salts such as NaCl, KCl, and in particular potassium phosphate, relieved the inhibition of carrot phosphofructokinase by phosphoenolpyruvate. KCl and potassium phosphate also reversed the inhibition of carrot phosphofructokinase by citrate. The possible significance of these observations in the regulation of glycolysis and carbohydrate metabolism, and in salt respiration is discussed.     

Electrophysiological responses of galeal contact chemoreceptors of Apis mellifera to selected sugars and electrolytes

Using conventional electrophysiological methods, the galeal sensilla chaetica of the honey bee, Apis mellifera, responded linearly to the log of solute concentrations of sucrose, glucose, fructose, NaCl, KCl, and LiCl but not to CaCl₂ or MgCl₂, which failed to give consistent responses. These sensillae had much higher firing rates for sugar than salt solutions; their relative responses to lower concentrations being NaCl < KCl < LiCl ? fructose < glucose ? sucrose. At higher concentrations NaCl < LiCl < KCl ? glucose < fructose ? sucrose. Four different spike types were seen. The first type had the highest amplitude and resulted from sugar stimulation. The second type had a lower height and occurred in the first 30 sec of salt stimulation. A third type with the lowest height appeared with those of the second type after prolonged stimulation with KCl. A fourth type with a high amplitude resulted from mechanical stimulation. The sensilla adapted to sugar solutions linearly to the logarithm of time and non-linearly to the log of salt concentrations. Glucose-fructose mixed-sugar solution effected synergism of response while sucrose solutions caused inhibition when mixed with glucose and/or fructose. Responses of the sensilla to mechanical stimulation showed phasic-tonic characteristics. None of the sensilla tested responded to water.     

Modulation of concanavalin A stimulation of hamster lymphoid cells by lithium chloride.

Addition of LiCl (1–25 mM) to serum-free cultures of MHA hamster thymocytes, lymph node cells, or splenocytes stimulated with concanavalin A had a biphasic effect on [³H]thymidine incorporation. These concentrations of LiCl enhanced stimulation of [³H]thymidine incorporation by suboptimal levels of concanavalin A but inhibited stimulation of optimal and supraoptimal concentrations of concanavalin A. This effect was specific for Li⁺ since it was not observed when similar concentrations of Na⁺, K⁺, or Mg²⁺ were added to cultures stimulated by concanavalin A. The inhibitory effect of LiCl on concanavalin A stimulation was not reversed by addition of Na⁺, Ca²⁺, Mg²⁺, or Ca²⁺ + Mg²⁺ to the cultures. Significant reversal of LiCl inhibition of stimulation was observed when KCl was added to the cultures. However none of the ions tested blocked the Li-induced enhancement of [³H]thymidine incorporation in the presence of suboptimal concentrations of concanavalin A.