Cell surface-localized alkaline phosphatase of Escherichia coli as visualized by reaction product deposition and ferritin-labeled antibodies.

When cells of a wild-type Eschericia coli O8 strain bearing a complete lipopolysaccharide were incubated for alkaline phosphatase reaction product and examined by electron microscopy, the depostion of lead salts was to be observed primarily within the periplasmic space. A similar treatment of cells derived from this strain, which bears a highly abbreviated lipopolysaccharide, showed a mixed cell surface and periplasmic localization of reaction product, suggesting a surface association of a portion of the enzyme. To further explore this possibility, ferritin-antibody conjugates against the active enzyme and its irreversibly dissociated subunits were prepared and allowed to react with cells of both strains. The results obtained from these experiments revealed the presence of both the active enzyme and inactive subunits of the enzyme at the cell surface of the mutant strain. The evidence obtained offers further proof of the validity of the reaction product deposition technique and indicates that alkaline phosphatase may be associated with some component of the outer membrane in this organism. The observation of enzyme subunits at the cell surface further suggests that an association of these subunits with structural components of the cell envelope may provide a locus at which they may dimerize to form active enzyme.     

Interaction of water with poly-α-amino acids. II. Relationships between counter-ion and relaxation processes

The effect of hydration on the solid-state conformation and relaxation behavior of the alkaline earth salts of poly (L -glutamic acid) has been examined. The calcium and strontium salts take the β-conformation and the magnesium salt takes the α-form. No hydration-dependent conformational transitions were observed. The amount of bound water, as measured by differential scanning calorimetry, appears to depend on the size of the cation, while conformation has little or no effect. A relative measure of the barrier to side-chain reorientation was obtained from the temperature of the side-chain (β₁) relaxation. It was found that the barrier is determined by electrostatic forces and increases with increasing cation surface charge density. Side-chain motion also appears to be more hindered in the β-conformation than in the α-conformation.     

DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) has been explored widely for DNA sequencing. The major requirement for this method is that the DNA sequencing fragments must be free from alkaline and alkaline earth salts as well as other contaminants for accurately measuring the masses of the DNA fragments. We report here the development of a novel MS DNA sequencing method that generates Sanger-sequencing fragments in one tube using biotinylated dideoxynucleotides. The DNA sequencing fragments that carry a biotin at the 3′-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Only correctly terminated biotinylated DNA fragments are subsequently released and loaded onto a mass spectrometer to obtain accurate DNA sequencing data. Compared with gel electrophoresis-based sequencing systems, MS produces a very high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and completely eliminates the compressions associated with gel electrophoresis. The high resolution of MS allows accurate mutation and heterozygote detection. This optimized solid-phase DNA-sequencing chemistry plus future improvements in detector sensitivity for large DNA fragments in MS instrumentation will further improve MS for DNA sequencing.     

Measuring oxygen diffusion coefficients with polarographic oxygen electrodes. II. Fermentation Media

Fermentation media consist of a large number of chemicals which composition undergoes alteration during the course of fermentations. In consequence, the conventional methods and correlations for gas diffusion coefficient measurement and prediction cannot be easily applied to such systems. Oxygen diffusion coefficients have been measured in simulated chemical systems as well as in complex solutions of nutrient broth, using the polarographic technique introduced in a previous article. It is identified that sugars and salts are the major factors influencing oxygen diffusion coefficients in these aqueous fermentation media. The effect of salts on oxygen diffusion coefficients in electrolyte solutions has been found to be well correlated with the square root of total ionic strength of electrolyte solutions. The individual effect of glucose and its combined effect with salts are explored in order to reach rational correlations capable of predicting oxygen diffusion coefficients in synthetic fermentation media. For aqueous solutions of glucose plus salts, it is observed that the log-additive relationship can be used to account for the combined effect. Finally, a linear correlation has been established in measuring oxygen diffusion coefficients in aqueous solutions having different concentrations of nutrient broth.     

Inhibition of Leuconostoc mesenteroides P-60 by D-tyrosine and its relationship with sodium and potassium content of the medium

Evidence for a growth inhibition of Leuconostoc mesenteroides P-60 by the d-isomer of tyrosine has been obtained by comparison of the relative activities of l- and dl-tyrosine for this organism, and by comparison of tyrosine assay results obtained on acid and alkaline hydrolysates of casein and beef round. It has been further found that this inhibition is significantly greater on a conventional medium buffered with both sodium and potassium salts than on one containing all of the buffer salts in the potassium form. Similar studies with Leuconostoc citrovorum 8081 and Lactobacillus delbrueckii 3 indicate that these organisms are neither inhibited nor stimulated significantly by the presence of d-tyrosine, and are therefore to be recommended as more satisfactory assay organisms for tyrosine whenever the d-isomer is involved in the assays.     

Effects of cationic micelles on the rate of reaction of p-nitrophenyl esters with dihydrolipoic acid and dihydrolipoylamido derivatives

Dihydrolipoic acid (DHLA) 2a and its surfactant derivatives, trialkyl(2-lipoylamidoethyl) ammonium salts 2b-c, have been investigated, mainly in micellar solutions of cetyltrimethyl-ammonium bromide (CTABr), as esterolytic reagents toward p-nitrophenyl esters. The origins of the observed kinetic effects are discussed, and the reactivity of these reagents are compared with that of other thiolytic systems. The results indicate that DHLA, although not a surfactant, is effectively comicellized by CTABr, and micelles of CTABr and DHLA are among the most effective esterolytic systems, at moderately alkaline pHs, so far reported.     

Lipase from Candida paralipolytica

The extracellular lipase from Candida paralipolytica required alkaline earth metal ion as the cofactor*² in the reaction mixture not emulsified but dispersed by shaking, contradicting the fact that it required bile salt or anionic surfactant as the essential activator*³ in the systems emulsified with polyvinyl alcohol, as previously reported.¹) The two kinds of factors necessary to activate both reaction systems respectively were unexchangeable for each other. These facts would be direct evidences of the difference of interfacial nature between two substrate forms prepared from the same substrate.

The zero-order reaction has been observed under the non-emulsified conditions and the activation mechanism by alkaline earth metal ions has been studied partly.     

9-Amino-acridine as a probe of the electrical double layer associated with the chloroplast thylakoid membranes

Chloroplasts washed with monovalent cations are found to quench 9-amino-acridine fluorescence after resuspension in a cation-free medium. This quenching occurs in the absence of a high energy state and can be reversed by the addition of salts. The effectiveness of these salts is related to the charge carried by the cations and appears to be essentially independent of the associated anions. The order of effectiveness is polyvalent > divalent > monovalent, and virtually no variation is found within the groups of monovalent cations and divalent cations tested. Furthermore, choline and lysine are as effective as alkali metal cations, and lysyl-lysine is almost as effective as alkaline earth metal cations. These results are consistent with an effect mediated by the electrical double layer at the membrane surface rather than chemical bonding, and can be qualitatively explained in terms of the Gouy-Chapman theory.It appears that 9-amino-acridine acts as a diffusible monovalent cation which increases its fluorescence when displaced from the diffuse layer adjacent to the negatively charged membrane surface. The 9-amino-acridine fluorescence changes have been experimentally correlated with the cation-induced chlorophyll a fluorescence changes also observed with isolated chloroplasts.     

Yttrium-89 magnetic resonance study of simple coordination complexes

⁸⁹Y FT NMR studies of some simple salts and complex ions have been carried out on a Varian FT80A spectrometer at 3.895 MHz. Corresponding gadolinium salts with the matching counterion have been used as relaxation reagents. Shifts dependent on complexation equilibria have been observed for the carboxylate salts.     

Cytochemical demonstration of phosphatases in membrane-recycling structures of endodermal cells

We applied cytochemical procedures to demonstrate the presence of acid and alkaline phosphatase in the visceral yolk-sac endoderm of rats using frozen, aldehyde-fixed tissue with cerium as the capture agent. This procedure allowed more detailed topochemical localization than was possible using unfrozen tissue or with lead as the capture agent. Acid phosphatase was found to be present in lysosomes as well as in a small number of apical canaliculi, which are thought to be recycling structures of the cell membranes in endodermal cells. Reaction products of alkaline phosphatase were observed on the outer surface of apical, lateral, and basal cell membranes. In addition, some apical vacuoles contained alkaline phosphatase, and more apical canaliculi were positive for alkaline phosphatase than for acid phosphatase. However, most of the apical canaliculi were negative for both enzymes. It is suggested that acid and alkaline phosphatase are taken up by different numbers of apical canaliculi during the detachment of apical canaliculi from lysosomes and resorption vacuoles.     

Conformational and electrostatic properties of unoccupied and liganded estrogen receptors determined by aqueous two-phase partitioning

The technique of aqueous two-phase partitioning (ATPP) has been used to characterize conformational and electrostatic properties of unoccupied and liganded rat uterine estrogen receptors. The adaptation of the hydroxylapatite receptor assay with ATPP systems has permitted estrogen receptor (ER) partition coefficients to be accurately determined, even when the partitioning process results in significant loss of ER binding capacity. The pH and salt dependences of estrogen receptor partition coefficients indicate that the theory governing partitioning behavior can be accurately applied to partitioning data obtained with crude cytosols. This technique has revealed a ligand-induced change in the properties of the unoccupied receptor that precedes the process of heat-induced transformation in vitro. The difference in partitioning behavior between unoccupied and nontransformed estrogen receptor is observed in all combinations of buffers and salts tested and is of equal magnitude as the difference between partition coefficients of nontransformed and transformed ER. The partition coefficients of both unoccupied and nontransformed ER are constant over the ER concentration range in which binding cooperativity has been previously demonstrated. The combined effects of salt and pH on ER partition coefficients indicate a pI of approximately 5.5 for both unoccupied and nontransformed estrogen receptors. However, the partition coefficients at the pI differ. It is concluded that estradiol binding to its unoccupied receptor results in a change in surface properties of the ER monomer that is independent of receptor transformation and makes the receptor less hydrophobic.     

Effect of weaning terms and protein deficit in rat pup nutrition on activities of digestive enzymes

Restriction of protein in nutrition of rat pups weaned at different terms has been found to produce changes in activities of digestive enzymes (maltase, alkaline phosphatase, aminopeptidase M, and glycyl-L-leucine dipeptidase) in the small and large intestine both at once after cessation of nutrition with low-protein diet for 10 days and 4 months later. In adult animals after the earlier or later weaning there are observed not only a decrease or increase of the enzyme activities, but also a different type of distribution of the alkaline phosphatase activity along the small intestine, which is more pronounced in the lately weaned rats. Thus, disturbance of metabolic programming of enzyme systems of the small and large intestine due to a change of quality of nutrition in early ontogenesis depends on terms of weaning of animals.     

SYSTEMIC INSECTICIDAL ACTION OF NICOTINE AND CERTAIN OTHER ORGANIC BASES

Nicotine and nicotine salts are taken up by the roots of plants from solutions, and when 0.01–0.001 % nicotine is used the plants become toxic to Aphis fabae and to Pieris brassicae larvae and can be shown to contain nicotine. The results with Phaedon cochleariae adults and larvae are less satisfactory. No systemic action is observed when the nicotine is watered on to soil in which plants are growing and no nicotine can be detected in the plants. Apparently the nicotine is decomposed in the soil.
When applied several times to the upper surface of a bean leaf nicotine kills aphids on the underside. There is some evidence that nicotine can be translocated further through the plant following leaf applications, but the toxic action at any distance is very weak in the plants used in the present experiments and can only be produced by frequent applications of rather concentrated nicotine solutions. Leaf absorption and subsequent translocation has not been observed with nicotine salts.
The various organic bases, including some piperidine phosphonites and allied compounds tested, are of very little interest as contact or systemic insecticides against aphids.     

Interrelation between thrombin structure and its stability

Data concerning peculiarities of fermentative nature and structure of thrombin in water-salt solution have been generalized; regularities of stabilizing effect made on thrombin by various polyols and other substances have been analyzed. It has been shown that formation of thrombin optimum macrostructure is one of the methods of its stabilization. Presence of different dissolving additives changes this enzymes hydration and this affects its stability and activity. There exist some systems to stabilize thrombin solutions. The systems consist of various salts, low-molecular and high-molecular polyols, surfactants, protein chain, composition buffer, etc. It has been shown that optimal concentrations of polyols, buffer salts and surfactants, as well as protein interaction increase considerably thrombin stability, preserving secondary structure even under its low concentration in the solution.     

Effect of weaning terms and protein deficit in the rat pup nutrition on activities of digestive enzymes

Restriction of protein in nutrition of rat pups weaned at different terms has been found to produce changes in activities of digestive enzymes (maltase, alkaline phosphatase, aminopeptidase M, and glycyl-L-leucine dipeptidase) in the small and large intestine both at once after cessation of nutrition with low-protein diet for 10 days and 4 months later. In adult animals after the earlier or later weaning there are observed not only a decrease or increase of the enzyme activities, but also a different type of distribution of the alkaline phosphatase activity along the small intestine, which is more pronounced in the lately weaned rats. Thus, disturbance of metabolic programming of enzyme systems of the small and large intestine due to a change of quality of nutrition in early ontogenesis depends on terms of weaning of animals.     

Adsorption of biopolymers at hydrophilic cellulose-water interface

The extent of adsorption (Gamma2(1)) of bovine serum albumin (BSA), beta-lactoglobulin, lysozyme, gelatin, and DNA from aqueous solution onto the hydrophilic surface of cellulose has been measured as function of biopolymer concentration at different temperatures, pHs, and ionic strengths, and in the presence of a high concentration of inorganic salts and denaturants. In all cases, the value of Gamma2(1) increases with the increase of biopolymer concentration (X2) in bulk and it attains a maximum value at a critical mole fraction concentration X2m. The value of Gamma2m depends upon the nature of protein, temperature, pH, and ionic strength, as well as the nature of neutral salts present in excess. Gamma2m for proteins at a fixed physicochemical condition stands in the following order: Gelatin>betalactoglobulin>lysozyme>BSA. The isotherms for adsorption of DNA nucleotides on cellulose surface at pH 4.0 have been compared at different temperatures and ionic strengths, and in the presence of high concentration of inorganic salts LiCl, NaCl, KCl, and Na2SO4. Values of Gamma2m for different systems have been evaluated and critically compared. At pH 6.0 and 8.0, Gamma2(1) values of DNA nucleotides on cellulose are all negative due to the excess positive hydration of cellulose. At pH 4.0, adsorption of nucleotides of acid, alkali, and heat-denatured DNA widely differ from each other and in the presence of excess concentration of urea becomes negative. The probable mechanisms of biopolymer-cellulose adsorption in terms of polymer hydration, steric interaction, London-van der Waals, hydrophobic, and other types of interactions have been discussed qualitatively. The standard free energy change for the adsorption of protein and DNA nucleotides on the cellulose surface at the state of adsorption saturation has been calculated in kJ per kg of cellulose using an integrated form of the Gibbs adsorption equation. The relation between DeltaG degrees and maximum affinities between biopolymers and the polysaccharide interface have been discussed for various systems.     

Use of chemically modified proteins to study the effect of a single protein property on partitioning in aqueous two-phase systems: Effect of surface charge

A series of charge-modified thaumatins with different values of surface charge were partitioned in aqueous two-phase systems (ATPS) to study the effect of surface charge as a single property on partitioning. Electrophoretic mobility of the proteins in titration curves was used as a measure of surface charge. Four modified proteins derived from thaumatin with the following values of isoelectric point: 8.70, 8.15, 5.60, and 4.50 were used for partitioning. The resolution of the systems in terms of protein surface charge was calculated. Partitioning of modified thaumatins in PEG 4000/dextran systems with phosphate buffer, Tris buffer, NaCl, KCl, and sulfate salts was carried out. Among the sulfate salts tested, the addition of 50 mM Li(2)SO(4) to the system buffered with phosphate gave the highest value of resolution for differences in surface protein charge (RSPC). It shows a decrease in the value of K (partition coefficient) with an increase in the protein's charge. The addition of 100 mM KCl to the system promoted the opposite effect on the RSPC value. Charge-modified proteins were partitioned in PEG/salt systems to investigate the ability of these systems for resolving differences in surface charge. The PEG/citrate system seemed to have almost no ability for resolving proteins on the basis of surface charge differences; PEG/phosphate systems had some capability for resolving differently charged proteins. The more negative proteins tended to have higher values of K than the more positively charged fractions. The use of charge-modified proteins allowed the investigation of the effect of protein surface charge on partitioning in aqueous two-phase systems independently from other protein parameters as they were prepared from a common parent protein thaumatin. This technique provides an interesting novel tool to investigate the effect of protein surface charge on partitioning in ATPS taking protein charge as an independent parameter. (c) 1996 John Wiley & Sons, Inc.     

Effect of bile salts on the interfacial inactivation of pancreatic carboxylester lipase

Pig pancreatic carboxylester lipase (cholesterol esterase, E.C. 3.1.1.13) was inactivated at a tributyrin/water interface. The apparent rate constant for inactivation increased with increase in the particle surface area of the tributyrin emulsion. The large energy of activation and entropy change for inactivation (33.7 Kcal.mol-1 and 35.8 cal.mol-1.deg-1, using 5 mM sonicated tributyrin at 37 degrees C, respectively) suggest that the observed inactivation reflects denaturation of the enzyme at the tributyrin/water interface. Bile salts protected the enzyme from irreversible inactivation at the tributyrin/water interface. The protection by bile salts was related both to their concentration and to the tributyrin concentration (substrate surface area). The protection by bile salts was not related to their concentration below or above their critical micellar concentration; the binding of bile salts to enzyme was probably the dominant protection factor. Similar stabilization was observed with other detergents such as Brij-35, Triton X-100, and sodium dodecyl sulfate. These results suggest that inactivation of carboxylester lipase occurs at a high-energy lipid-water interface and that an important role of bile salts in vivo is to stabilize carboxylester lipase at interfaces.     

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.