THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : X. AN EXPERIMENTAL TEST OF SOME ASPECTS OF THE TEORELL AND MEYER-SIEVERS THEORIES OF ELECTRICAL MEMBRANE BEHAVIOR

1. The Teorell, Meyer-Sievers theory characterizes the electrochemical behavior of membranes by their selectivity constant "A_p" which is derived conventionally from concentration potential measurements at various concentration levels. The selectivity constant may, however, be derived also from entirely independent, different experimental data, namely base exchange studies. The constants arrived at in this second way are designated as "A_b." The selectivity constants derived by these two methods must be in reasonable, at least semiquantitative agreement if the basic assumptions of the theory are correct. 2. The selectivity constants A_p and A_b were determined for eleven different sets of membranes of different electrochemical activity and of different (8.2 to 80 volume per cent) water content. 3. The potentiometric selectivity constants A_p are in most cases several orders of magnitude greater than the corresponding A_b values. With membranes of great porosity and high electrochemical activity the A_b values approach at least in order of magnitude the A_p values. 4. It is concluded that the unexpectedly large discrepancy between the A_p and A_b values is due to some inherent weakness of the Teorell, Meyer-Sievers theory, most likely to its neglect of any structural factors.     

Stabilization of glucoso-6-phosphate dehydrogenase by its substrate and cofactor in an ultrasonic field

The inactivation kinetics of glucoso-6-phosphate dehydrogenase (GPDH) and its complexes with glucoso-6-phosphate and NADP⁺ was characterized in aqueous solutions at 36–47°C under treatment with low frequency (27 kHz, 60 W/cm²) and high frequency ultrasound (880 kHz, 1 W/cm²). To this end, we measured three effective first-order inactivation rate constants: thermal k _in ^* , total (thermal and ultrasonic) k _in, and ultrasonic k _in(US). The values of the constants were found to be higher for the free enzyme than for its complexes GPDH-GP and GPDH-NADP⁺ at all temperatures, which confirms the enzyme stabilization by its substrate and cofactor under both thermal and ultrasonic inactivation. Effective values of the activation energies (E _a) were determined and the preexponential factors of the rate constants and thermodynamic activation parameters of inactivation processes (ΔH*, ΔS*, and ΔG*) were calculated from the temperature dependences of the inactivation rate constants of GPDH and its complexes. The sonication of aqueous solutions of free GPDH and its complexes was accompanied by a reduction of E _a and ΔH* values in comparison with the corresponding values for thermal inactivation. The E _a, ΔH*, and ΔS* inactivation values for GPDH are lower than the corresponding values for its complexes. A linear dependence between the growth of the ΔH* and ΔS* values was observed for all the inactivation processes for free GPDH and its complexes.     

A nondetergent sulfobetaine improves protein unfolding reversibility in microcalorimetric studies

A nondetergent sulfobetaine (NDSB) was found to improve unfolding reversibility of several proteins by inhibiting heat-induced aggregation. As a consequence, ΔH_cal/ΔH_vH ratios were also improved to values close to 1 for a two-state unfolding. NDSB is effective in a wide range of pH values and especially at acidic pH generally used to calculate ΔC_p values by the Kirchhoff relation. The sulfobetaine also allows recording protein refolding by protecting the heat-induced unfolded state against aggregation.     

Effect of phospholipid on the redox potential and enzymic properties of cytochromes b.

The effects of phospholipid on the redox behavior of b cytochromes in succinate-cytochrome c reductase, the cytochrome b-c₁ complex, and an isolated cytochrome b preparation were investigated by the oxidative and reductive titrations. Three E_m values of cytochrome b were observed in the phospholipid-sufftcient and -depleted succinate-cytochrome c reductase. Their midpoint potentials at pH 7.4 are 75, 75, and ?100 mV for the sufficient and 10, ?30, and ?160 mV for the depleted reductase. The molar distribution of the b cytochromes of these E_m values correspond to 30, 30, and 40%, respectively. The E_m values of the isolated cytochrome b preparations were not affected by addition of phospholipids. The isolated b preparation contained two components of equal concentration with E_m values of ?85 and ?200 mV. No direct correlation between enzymic activity and the amount of high potential b cytochromes present in the systems was demonstrated. Very little difference was observed in redox behavior of b cytochromes between the aged inactive preparations of phospholipid-depleted reductase and that of freshly prepared reconstitutively active enzyme.     

Dissociation constants of phenothiazine drugs incorporated in phosphatidylcholine bilayer of small unilamellar vesicles as determined by carbon-13 nuclear magnetic resonance spectrometric titration

The dissociation constants (pK_ms) of the phenothiazine drugs promazine, chlorpromazine, and triflupromazine, incorporated in the phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUV), were investigated by a ¹³C nuclear magnetic resonance (NMR) titration method employing their N-¹³CH₃ (ionizable group) labelled derivatives. Use of the labelled drugs enabled direct observations of the ionization equilibrium of the N-dimethyl group. A second derivative spectrophotometric study proved that 95-98% of the phenothiazine species in the sample solutions (200 μM phenothiazine in the presence of 27 mM PC SUV) were incorporated into the PC bilayer, which simplified the calculation of pK_m values by allowing that the phenothiazines in the aqueous phase could be neglected. The pK_m values were calculated from the chemical shift dependence of the N-dimethyl ¹³C NMR signal on the pH value of sample solutions. The pK_m values obtained were smaller than those measured in aqueous solutions by about one unit. The existence of cholesterol (30 mol%) in the PC bilayer showed little effect on the pK_m values, suggesting that cholesterol in the bilayer does not largely affect the interfacial region where the N-dimethyl group of the incorporated phenothiazines is located. The results offered clear evidence for the pK_m decrease and provided their precise values.     

Properties of extracellular polymer having an effect on expression of activated sludge

Extracellular polymer in activated sludge was found to affect expression of these sludge by raising the value of the average specific resistance (α_av) during filtration and lowering the value of the modified consolidation coefficient (C_e) during consolidation. Further, the addition of extracellular polymer to the sludges caused higher values of α_av and lower values of C_e. The logarithmic plots of α_av and C_e against the quantity of extracellular polymer was linear. When these two values (α_av and C_e) of each activated sludge were plotted against the quantity of extracellular polymer extracted from each sludge, they were correlated by a single straight line with two exceptions. These were the return sludge of a textile factory and the anaerobically digested sludge of a sewage treatment plant. These exceptions indicate that the values of α_av of these sludges are higher than those of other sludges containing the same amount of extracellular polymer and that of C_e are lower than those of other sludges. It was confirmed that the extracellular polymer of these two sludges contains a lot of polysaccharides of the molecular weight 600–100,000 (with a standard of polyethylene glycol) by gel chromatography. The effects of the flocculant on dewatering were investigated. The effects of the cationic polymer flocculant addition to the extracellular polymer were analyzed using an ultrafiltration cell (molecular weight cut-off 10,000). The ultrafiltration rate of extracellular polymer was much improved by adding a cationic polymer flocculant. The removal of extracellular polymer by adding cationic polymer flocculant was ascertained by measuring the molecular weight distribution of the polysaccharides. On the other hand, no flocculations of extracellular polymer and sludge by adding anionic and nonionic polymer flocculant could be observed.     

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b561 enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b₅₆₁ proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b₅₆₁-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b₅₆₁ paralogs from Arabidopsis thaliana (Acytb₅₆₁-A, Acytb₅₆₁-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb₅₆₁-A resembles the best characterised member of the CYBASC family, the cytochrome b₅₆₁ from adrenomedullary chromaffin vesicles, and that Acytb₅₆₁-B is atypical compared to other CYBASC proteins. Haem oxidation–reduction midpoint potential (E_M) values were found to be fully consistent with ascorbate oxidation activities and Fe^3 +-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b₅₆₁ from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E_M values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E_M values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe^3 +-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E_M values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b₅₆₁ paralogs exist as homodimers.     

Indole alkaloids as systematic markers of the Apocynaceae

Indole alkaloids can be characterized by skeletal specialization (S), determined upon consideration of their relative position on a biogenetic map and the number of their naturally occurring substitutional derivatives, as well as by oxidation level (O). The mean (S) and (O) for contained alkaloids of a given plant taxon are taken to represent evolutionary advancement parameters, respectively EA_s and EA_o.A correlation of these EA_s/EA_o values for tribes of the Apocynaceae-Plumerioideae reveals a chemical gradient, given by gradually increasing EA_s and EA_o values, to link Carisseae-Alstonieae-Rauvolfieae-Tabernaemontaneae.     

Horse liver alcohol dehydrogenase SS: purification and characterization of the homogenous isoenzyme.

Alcohol dehydrogenase SS was prepared from horse liver by salt fractionation, ion-exchange chromatography, and affinity chromatography. The purified isoenzyme is free from extraneous protein and other alcohol dehydrogenase isoenzyme contaminants and contains four Zinc atoms per molecule. The substrate specificity with saturated aliphatic alcohols and aldehydes of two to six carbon chain lengths has been investigated. The K_m values and turnover numbers at maximal velocity (k_cat) are presented. Values of k_cat are constant within a substrate category and independent of the substrate chain length, while the K_m values decrease with the increase of the substrate chain length. The K_m values for two- and three-carbon substrates are large, that for ethanol (40 mm) is two orders of magnitude larger than that reported for classical preparations of horse liver alcohol dehydrogenase. At pH 7, the k_cat values for alcohol oxidation are almost 30 times smaller than for aldehyde reduction. The enzyme has been characterized with regard to specific activity with several nonsteroidal substrates and with two steroids: 3-oxo-5β-androstan-17β-ol and 5β-pregnan-21-ol-3,20-dione hemisuccinate. NAD(H) is the preferred coenzyme. Values of K_m for NADH with constant steroidal substrates are an order of magnitude smaller than the corresponding K_m values with nonsteroidal substrates. A possible explanation for this observation is presented.     

Effect of Temperature on Heterotrophic Glucose Uptake, Mineralization, and Turnover Rates in Lake Sediments

The V_max and turnover rates (TR) of [U-¹⁴C]glucose uptake and mineralization of Lake Kinneret (Israel) sediment are temperature dependent. The following activation energies were determined: glucose uptake, ~15,000 cal (62,760 J); TR of glucose uptake, ~10,000 cal (41,840 J); glucose mineralization, 7,500 to 15,000 cal (31,380 to 62,760 J); and TR of glucose mineralization, ~15,000 cal. Q₁₀ values varied as follows: glucose uptake, ~2.3; TR of glucose uptake, ~1.8; and glucose mineralization, ~2.5. K + S_n values increased slightly with temperature and might reflect an increased K with increased temperatures. Glucose respiration/uptake ratios were low (9.5 to 12%) and were apparently not greatly influenced by the presence or absence of oxygen or by different assay temperatures. Aerobic or anaerobic sediments assayed under either aerobic or anaerobic conditions did not exhibit greatly different V_max, TR, or K + S_n values.     

Characteristics of α-glucan phosphorylase from Chlorella vulgaris

α-Glucan phosphorylase from Chlorella vulgaris has been partially purified. In the direction of glucan phosphorolysis the apparent K_m for Pi was ca 2.4 mM at pH 7.1. In the direction of glucan synthesis the K_m for G1P was ca 0.12 mM at pH 6.2. The enzymic activity was inhibited by physiological concentrations of ADP, ATP, ADPG and UDPG. In the direction of starch degradation in the presence of 2.4 mM Pi the I_0.5 values for ADP and ATP were ca 1.6 and 2.9 mM, respectively, while in the direction of synthesis in the presence of 0.12 mM G1P the values were ca 0.23 and 1.4 mM, respectively. The Hill plots for starch degradation showed n values of 2.2 for ADP and 2.2 for ATP and values of 1.5 and 1.2, respectively, for starch synthesis. Both ADPG and UDPG were linear competitive inhibitors either with respect to Pi or with respect to GIP. The K_i values for ADPG and UDPG in the direction of phosphorolysis were shown to be ca 0.11 and 0.51 mM, respectively, and those in the direction of synthesis 0.033 and 0.15 mM, respectively.     

Reexamination of the interaction between ferredoxin:NADP reductase and NADP

A comparison was made of graphical and subtractive methods for the determination of the dissociation constant of a complex between ferredoxin:NADP reductase and NADP. The subtractive method gave K_d values near 10 μm which are consistent with recently determined values for K_m,NADP in assays of NADP photoreduction by chloroplast membranes. The graphical method gave values which were considerably higher. The difference between the two methods is due to the failure of the graphical method to correct for the amount of each component present in the complex at the low NADP/ flavoprotein ratios necessary for binding studies. A second NADP binding site of much lower affinity (K_d approx 1 mm) was also detected.     

Glutamate-induced polymerization of tubulin: characteristics of the reaction and application to the large-scale purification of tubulin

The multiple attack model of the α-amylase mechanism has been analyzed. Theoretical rate coefficients dependent on the degree of polymerization of substrate (n) have been applied. They are: effectiveness of hydrolysis (y_n) which affects V values, and the average number of unitary movements (ζ_n) which affects K_m values. The model explains the dependence of V and K_m on the degree of polymerization of substrates with n values higher than the number of subsites in the enzyme binding site. Distribution of the degree of polymerization of hog pancreas α-amylase products (n_p) and average n_p value has been found experimentally and applied to calculate the maximal number of unitary movements (z) of the enzyme during the single enzyme-substrate meeting. V and K_m values of hog pancreas α-amylase for amylose and different branched substrates have been determined and discussed in terms of the multiple attack theory. Their dependence on theoretic y_n and ζ_n values has been found in support of the model.     

S1 nuclease of Aspergillus oryzae: characterization of the associated phosphomonoesterase activity

S₁ nuclease (EC 3.1.30.1) of Aspergillus oryzae was found to catalyze the hydrolysis of 2′- or 3′-phosphomonoester groups from several mono- and oligonucleotides. The specificity of the enzyme for mononucleotide substrates was determined by steady-state kinetic measurements at pH 4.5. The values of V were similar for all ribonucleoside 3′-phosphates tested, and they were 50–400 times greater than those for the corresponding deoxyribonucleotides or ribonucleoside 2′-phosphates. Purine nucleotides had lower apparent K_m values than pyrimidine nucleotides. Apparent K_m values of mononucleotides were also strongly dependent on the type of sugar and the positions of phosphoryl groups. Substrate specificity, as expressed by $\text{V}\text{K}{}_{\mathrm{m}}$, occurred in the following order: ribonucleoside 3′,5′-bisphosphate > ribonucleoside 3′-phosphate > deoxyribonucleoside 3′,5'-bisphosphate > deoxyribonucleoside 3′-phosphate ≈ ribonucleoside 2′-phosphate. S₁ nuclease also catalyzed the dephosphorylation of the dinucleotide ApAp at a high rate and the release of PP_i from adenosine 3′-diphosphate 5′-phosphate at a low rate. The phosphomonoesterase activity of the enzyme was competitively inhibited by single-stranded DNA and 5′-nucleotides. Apparent K_i values for adenosine compounds occurred in the order ATP < ADP < AMP ? adenosine. Tests of S₁ nuclease for phosphotransferase activity at pH 4.5 and 7.0 were negative.     

Kinetic studies of the binding of inhibitors to thermolysin

The K_I values for inhibition of thermolysin activity by N-β-phenylpropionyl-aliphatic amino acids (Gly, Ala, Val, Leu, Ile) are correlated by π, the hydrophobic substituent parameter for the amino acid side chain (log K_I = ?0.73π ?1.80, correlation coefficient = 0.990). By contrast, the K_I values for the corresponding benzyloxycarbonyl amino acids are poorly correlated by π, but show a good correlation with the steric parameter E_s(log K_I = 0.880E_s ? 3.086, correlation coefficient = 0.985). Binding of β-phenylpropionyl-l-alanine is associated with an acidic residue of pK 7.3 and a basic residue of pK 8.0 in the E · I complex, and appears to raise the pK of Glu-143 by 2 units. Binding of benzyloxycarbonyl-Ala and -Phe is associated with an acidic residue of pK 8.0 and two basic residues, both with pK 8.3. Three similar pK values are observed with benzyloxycarbonyl-Phe. These results are interpreted in terms of different modes of binding of β-phenylpropionyl and benzyloxycarbonyl inhibitors.     

Evaluation of the toxicity of different phytoextracts of Ocimum basilicum against Anopheles stephensi and Culex quinquefasciatus

The larvicidal effect of the crude carbon tetrachloride, methanol and petroleum ether leaf extracts of a widely grown medicinal plant, Ocimum basilicum, against Anopheles stephensi and Culex quinquefasciatus was evaluated. Petroleum ether extract was found to be the most effective against the larvae of both mosquitoes, with LC₅₀ values of 8.29, 4.57; 87.68, 47.25 ppm and LC₉₀ values of 10.06, 6.06; 129.32, 65.58 ppm against A. stephensi and C. quinquefasciatus being observed after 24 and 48 h of treatment, respectively. The efficacy of petroleum ether was followed by that of the carbon tetrachloride and methanol extracts, which had LC₅₀ values of 268.61, 143.85; 446.61, 384.84 ppm and LC₉₀ values of 641.23, 507.80; 923.60, 887.00 ppm against A. stephensi after 24 and 48 h, respectively, and LC₅₀ values of 24.14, 17.02; 63.48, 53.77 ppm and LC₉₀ values of 295.38, 204.23; 689.71, 388.87 ppm against C. quinquefasciatus after 24 and 48 h of treatment, respectively. These extracts are highly toxic against mosquito larvae from a range of species; therefore, they may be useful for the management of mosquito larvae to control vector borne diseases.     

Advantages of fungal enzyme production in solid state over liquid fermentation systems

The present paper attempts to explain why enzyme production in solid-state fermentation (SSF) is higher than in submerged fermentation (SmF). Recent work done in our laboratory [Biotechnol. Lett. 22 (2000) 1255; J. Ind. Microbiol. Biotechnol. 26 (5) (2001) 271; J. Ind. Microbiol. Biotechnol. 26 (5) (2001) 296] related to the production of invertase, pectinases and tannases, by Aspergillus niger grown by SSF and SmF is reviewed. To do such a comparative study, logistic and Luedeking–Piret equations are used in order to estimate the values of the following coefficients: maximal specific growth rate (μ_M), maximal biomass level (X_M), enzyme/biomass yield (Y_P/X) and secondary rate of production, or breakdown (k). It is shown that enzyme productivity is proportional to group, μ_MY_P/XX_M, corrected by a function of ν=k/Y_P/Xμ_M. In all three cases of enzyme production studied, productivity using a SSF system was higher than in SmF. Studies with invertase resulted in higher values of μ_MX_M. Studies with pectinases resulted in higher values of Y_P/XX_M. Studies with tannases resulted in higher Y_P/X and less negative values of k. Finally, a reaction–diffusion model is presented to try to explain such differences based on micrographic measurements of mycelial aggregates for each kind of fermentation system.     

Thermodynamics of complexation of lanthanides by methoxybenzoates

The thermodynamic parameters of complexation of lanthanide cations by ortho-, meta- and para- methoxybenzoates have been measured using potentiometric and calorimetric techniques at 25 °C and an ionic strength of 0.10 M (NaClO₄). The values of logβ₁₀₁ correlate well with the ligand acid values of pK_a, reflecting the strongly ionic nature of the metal-ligand interaction. No evidence is found for extra charge polarization in these aromatic ligands due to the lanthanide complexation.     

Using individual-muscle specific instead of across-muscle mean data halves muscle simulation error

Hill-type parameter values measured in experiments on single muscles show large across-muscle variation. Using individual-muscle specific values instead of the more standard approach of across-muscle means might therefore improve muscle model performance. We show here that using mean values increased simulation normalized RMS error in all tested motor nerve stimulation paradigms in both isotonic and isometric conditions, doubling mean simulation error from 9 to 18 (different at p?<?0.0001). These data suggest muscle-specific measurement of Hill-type model parameters is necessary in work requiring highly accurate muscle model construction. Maximum muscle force (F _max) showed large (fourfold) across-muscle variation. To test the role of F _max in model performance we compared the errors of models using mean F _max and muscle-specific values for the other model parameters, and models using muscle-specific F _max values and mean values for the other model parameters. Using muscle-specific F _max values did not improve model performance compared to using mean values for all parameters, but using muscle-specific values for all parameters but F _max did (to an error of 14, different from muscle-specific, mean all parameters, and mean only F _max errors at p?≤ 0.014). Significantly improving model performance thus required muscle-specific values for at least a subset of parameters other than F _max, and best performance required muscle-specific values for this subset and F _max. Detailed consideration of model performance suggested that remaining model error likely stemmed from activation of both fast and slow motor neurons in our experiments and inadequate specification of model activation dynamics.     

Differential susceptibility of parasitized and nonparasitized larvae of Trichoplusia ni to a nuclear polyhedrosis virus

Nonparasitized second-instar larvae of Trichoplusia ni were twice as susceptible (at the LD₅₀ level) to the singly enveloped T. ni nuclear polyhedrosis virus as those parasitized by Hyposoter exiguae (Hymenoptera: Ichneumonidae). The LD₅₀ values for nonparasitized and parasitized larvae were 1.58 × 10³ and 3.16 × 10³ polyhedra/ml of diet, respectively. The LD₉₅ value for parasitized larvae was approximateely 5 times higher than that for nonparasitized larvae. The slopes (b values) were 1.2 for parasitized larvae and 1.7 for nonparasitized larvae. The LT₅₀ values for parasitized larvae also were significantly longer than those for nonparasitized larvae. No significant difference was found between the food consumption of parasitized and nonparasitized T. ni larvae.