粘虫中肠α-淀粉酶活性的敏感性研究

研究了不同酶反应缓冲体系、pH值、氯离子浓度以及噁唑哒嗪对5龄2日粘虫 Pseudaletia separata Walker 中肠α－淀粉酶活性的影响。结果表明,乙酸-乙酸钠缓冲体系（pH 5.8）和磷酸氢二钠-磷酸二氢钠缓冲体系（pH 8.0）有利于增强α-淀粉酶活性,比活力最高分别达到4.49和4.97。在乙酸-乙酸钠缓冲体系（pH 5.8）中,5、10、20、40和80 mmol/L氯离子浓度引起α-淀粉酶活性呈现先减弱后增强的变化规律,而在磷酸氢二钠-磷酸二氢钠缓冲体系（pH 8.0）中仅呈现减弱的趋势。1.4 mmol/L噁唑哒嗪对α-淀粉酶活性的抑制率可达70%,但抑制程度随着反应体系中蛋白含量的增加而逐渐降低。     

Increase of electrical properties using a novel mixed buffer system in an enzyme fuel cell

Environment-friendly biocatalytic energy is considered to represent an attractive alternative to chemical catalystbased cells due to its renewability and better operation at low temperature. However, electrical biocatalysts have a low activity and electrical power. For increasing electrical properties of biocatalyst, a novel mixed buffer (phosphate and 3-morpholinopropanesulfonic acid (MOPS)) system was applied to an enzyme-based biofuel cell with microperoxidase (MP-11)-modified Au electrode. The cathodic electrical properties were increased by the phosphate and MOPS-mixed buffer solution. It was identified that the novel mixed buffer system obtained stronger ionic strength from phosphate buffer and better enzyme activity from MOPS buffer. The highest results of cyclic voltammetry were obtained when the proportion of phosphate to MOPS was nearly 1:1 and the pH was 7.0∼7.3. In addition, the novel mixed buffer led to the maximum power density (ca. 62.7 μW/cm²) in a basic enzymatic fuel cell (EFC).     

Optimal detection of cholinesterase activity in biological samples: Modifications to the standard Ellman’s assay

Ellman’s assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1 M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman’s assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412 nm. Indeed, this modification of Ellman’s method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman’s method into two successive steps that may be convenient for some applications.     

The ultrastructural ionic fixation technique localizing acetylcholinesterase activity, reveals simultaneously acetylcholine-like cation in the synaptic vesicles of the motor nerve terminals

A new cytochemical technique is proposed for side by side localization of acetylcholine and of acetylcholinesterase activity of motor end-plate at ultrastructural level. The technique is based on the simultaneous "ionic fixation" of vesicular acetylcholine and of histochemical copper thiocholine precipitate with phosphomolybdic acid: the molybdic heteropolyanion forms insoluble salts with these two quaternary ammonium cations, providing in situ "acetylcholine phosphomolybdate" and "copper thiocholine phosphomolybdate". Both of them are osmium resistant; the electron dense precipitates allow for a fine localization of acetylcholine and acetylcholinesterase activity at electron microscopic level.     

The type 2 copper of ascorbate oxidase

Ascorbate oxidase, dissolved in Hepes or sodium phosphate buffers, was analyzed by EPR and activity measurements before and after storage at −30°C and 77 K. The specific activity was somewhat higher in the phosphate buffer, about 3500–3700 Dawson units compared to about 3100 units of the enzyme dissolved in Hepes buffer. After storage at −30°C the activity fell to 1400–2000 units in the phosphate buffer but only to 2600–2800 units in the Hepes buffer. Large changes occurred in the EPR spectrum of enzyme dissolved in the phosphate buffer after storing at −30°C suggesting an alteration of the type 2 copper site. These changes were, however, reverted when the samples were thawed and rapidly frozen at 77 K. Copper analysis showed that about 50% of the total copper was EPR detected. The type 2 Cu²⁺ EPR intensity was in most samples close to 25% of the total EPR intensity. This low contribution of type 2 Cu²⁺ could not be changed if the enzyme was completely reduced and reoxidized, treated with Fe(CN)₆³⁻, large excess of NaF, addition of 50% (v/v) ethylene glycol or dialyzed against 0.1 M Mes buffer (pH 5.5). Since the crystal structure shows that there are one each of types 1 and 2 copper in the monomers there must be another species with an EPR signal rather different from these two copper species. This signal is proposed to originate from some trinuclear centers. The EPR simulations show that it is possible to house a broad unresolved signal under the resolved type 1 and 2 signals so that the total integral becomes 50% of the total copper in the molecule.     

Molecular properties of yeast glyceraldehyde-3-phosphate dehydrogenase in the presence of ATP and KCl.

The influence of ATP and KCl on the quaternary structure and the enzymatic activity of D-glyceraldehyde-3-phosphate dehydrogenase from yeast(Y-GAPDH) has been studied by ultracentrifugation, gel chromatography and standard optical tests. In 0.1 M imidazole buffer pH 7.0, at low temperature (0°C) both complete deactivation and dissociation to dimers occur in the presence of 2 mM ATP and 0.1 M 2-mercaptoethanol. In 0.067 M phosphate buffer pH 7.0, containing 2 mM ATP and 1 mM dithiothreitol, only slight deactivation paralleled by minor changes of the native quaternary structure is observed. In this same buffer, increasing temperature leads to stabilization of both the tetrameric state and the catalytic activity of the enzyme. Deactivation and dissociation in the presence of 0.15 M KCl (in 0.2 M glycine buffer 9.1 ≥ pH ≥ 8.0) is a function of pH rather than electrolyte concentration; at neutral pH the enzyme is stabilized in its native state. Contrary to earlier assumptions in the literature, ATP and KCl under the above experimental conditions do not appear to play an important role in the $\text{in vivo}$ regulation of Y-GAPDH.     

THE EFFECT OF ACETATE BUFFER MIXTURES,ACETIC ACID,AND SODIUM ACETATE,ON THE PROTOPLASM,AS INFLUENCING THE RATE OF PENETRATION OF CRESYL BLUE INTO THE VACUOLE OF NITELLA

When living cells of Nitella are exposed to a solution of sodium acetate and are then placed in a solution of brilliant cresyl blue made up with a borate buffer mixture at pH 7.85, a decrease in the rate of penetration of dye is found, without any change in the pH value of the sap. It is assumed that this inhibiting effect is caused by the action of sodium on the protoplasm. This effect is not manifest if the dye solution is made up with phosphate buffer mixture at pH 7.85. It is assumed that this is due to the presence of a greater concentration of base cations in the phosphate buffer mixture. In the case of cells previously exposed to solutions of acetic acid the rate of penetration of dye decreases with the lowering of the pH value of the sap. This inhibiting effect is assumed to be due chiefly to the action of acetic acid on the protoplasm, provided the pH value of the external acetic acid is not so low as to involve an inhibiting effect on the protoplasm by hydrogen ions as well. It is assumed that the acetic acid either has a specific effect on the protoplasm or enters as undissociated molecules and by subsequent dissociation lowers the pH value of the protoplasm. With acetate buffer mixture the inhibiting effect is due to the action of sodium and acetic acid on the protoplasm. The inhibiting effect of acetic acid and acetate buffer mixture is manifested whether the dye solution is made up with borate or phosphate buffer mixture at pH 7.85. It is assumed that acetic acid in the vacuole serves as a reservoir so that during the experiment the inhibiting effect still persists.     

Kinetics and mechanism of the acid transition of the active site in plastocyanin

Exchange on the microsecond time scale between the protonated and deprotonated forms of His92 in the copper site of reduced plastocyanin from the cyanobacteria Anabaena variabilis was monitored using 15N NMR relaxation measurements. On the basis of the dependence of the kinetics on pH and phosphate buffer concentration, we propose a two-step model for the protonation of the copper site in agreement with previous crystallographic studies. It is shown that the proton transfer is the rate-limiting step in the reaction at low buffer concentrations, whereas at high buffer concentrations, another step becomes rate-limiting. We suggest that the latter step is a concerted dissociation of His92 from the Cu(I) ion and a 180 degrees rotation of the imidazole ring, which precede the protonation. The first-order rate constant for the dissociation of His92 from the Cu(I) ion is estimated to be 2.4 x 10(4) s(-1). Also, a cooperative effect of the protonation of the remote His61 on the protonation of His92 and the redox properties of the protein was investigated by substituting His61 with asparagine. The mutation causes a modest change in both the pKa value of His92 and the redox potential of the protein.     

Polymerization studies on protein from the dahlemense strain of tobacco mosaic virus: Light scattering and related studies

Light-scattering and related studies on protein of Dahlmense strain of tobacco mosaic virus (DTMV) show that its polymerization characteristics are considerably different from those of TMV protein. At pH 6.0 in phosphate buffer (I = 0.1), the extent of polymerization of DTMV protein is greater than that of TMV protein, they are nearly the same at pH 6.25, and that of DTMV protein is less than that of TMV protein at pH 6.5. At pH 7.0 and 7.5, DTMV protein polymerizes more readily than TMV protein. Similar studies in phosphate buffer (I = 0.05) show that the extent of polymerization for DTMV protein is less than that of TMV protein at pH 6.0 and almost negligible at pH 6.25. Acid-base titration studies show that, upon temperature-mediated polymerization, about 2 H⁺ ions are bound per monomer of DTMV protein at pH 6.O.Electron microscope studies show that DTMV protein exists at room temperature as double discs and polymerized rods in phosphate buffer at pH 7.5, I = 0.1; at pH values below 6.5, DTMV protein is entirely in the form of polymerized rods. Velocity sedimentation studies of DTMV protein at room temperature are in agreement with these findings. At low temperatures, except at pH 7.5, most of the material sedimented with an s value of around 25 S. Thus, at low temperatures, except at pH 7.5, DTMV protein in solution is in the form of particles the size of double discs with an $\text{M}\text{?}{}_{\mathrm{<mtext>r</mtext>}}$ of 596,000 g/mole or even larger. Therefore, temperature-mediated polymerization of DTMV protein at pH values below 6.5 in phosphate buffer (I = 0.1) and below 6.25 in phosphate buffer (I = 0.05) involves particles at least as large as double discs as the starting material.     

Monitoring of enzymatic activity in situ by EPR.

A novel approach using an EPR method for the determination of activity of enzymes, whose substrates or products are low molecular weight compounds containing SH-groups, is proposed. The approach is based on thiol-disulfide exchange of stable nitroxyl biradical containing a sulfur-sulfur bond with low molecular weight thiol. The method is applied to determine activity of acetylcholinesterase in a larvae bollworm (from the formation of thiocholine from acetylthiocholine). The method is characterized by high sensitivity (up to 2 x 10(-12)m thiocholine) and makes possible measurements in optically unclear (scattering and coloured) media and determination of enzymatic activity (ca. 1 min) directly in the homogenate obtained from the heads of individual larvae. Thus, the method can be recommended for the fast monitoring of many enzymatic systems (including glutathione-dependent) directly in biological tissues of warm-blooded animals and insects.     

苹果多酚氧化酶的纯化与表征

运用丙酮浸漬干燥、磷酸盐缓冲液提取、低温离心、硫酸铵沉淀、DEAE-Sephadex(A-50)、Sephadex(G-75) 和DEAE-celluse(DE-52)层析等方法从苹果中分离获得一种新的含铜酶蛋白,该酶被命名为多酚氧化酶Ⅱ(polyphenol oxidase Ⅱ, PPOⅡ),纯化倍数是215,纯化收率是23%.PAGE、SDS-PAGE和MALDI-TOF 等技术用于测定所获的酶的纯度和分子量.在PAGE和SDS-PAGE 均显示一条带,表明PPOⅡ只由一个亚基组成,且已达到单一组分(MALDI-TOF的结果更证实了这一点).SDS-PAGE 和 MALDI-TOF 的结果都表明PPO的分子量为 38204 Da.pH值对酶活性和稳定性研究的结果显示,从pH值4.0～7.0随着pH值的增加,酶活性也不断增加;从pH值 7.0～11.0, 酶活性不断降低.PPOⅡ的最适pH值为6.6最适温度为30℃.     

Correlation of enzymatic activities and aggregation state in chicken liver fatty acid synthase

The relationships between the aggregation state and the enzymatic activities of chicken liver fatty acid synthase have been explored by monitoring the changes in light scattering, fluorescence, and the overall, beta-ketoacyl synthase, beta-ketoacyl reductase and enoyl reductase activities during dissociation and reassociation of the enzyme. The data obtained indicate that the enzyme dissociates at low temperature in both 0.1 M potassium phosphate (pH 7.0), 1 mM EDTA, and 5 mM Tris(hydroxymethyl)aminomethane, 35 mM glycine (pH 8.3) and 1 mM EDTA, but the extent of dissociation is less in the phosphate buffer. The assay conditions influence the assessment of the degree of dissociation and association: high temperatures, phosphate (high salt), NADPH and acetoacetyl-coenzyme A promote association of the monomeric enzyme, whereas dilution in the Tris-glycine buffer (low salt) and low temperature promote dissociation. Both the rate and extent of association and dissociation are altered by substrates. The monomeric enzyme does not possess beta-ketoacyl synthase and beta-ketoacyl reductase activities. Results obtained with the 1,3-dibromo-2-propanone cross-linked enzyme, which lacks beta-ketoacyl synthase activity, indicate that the NADPH-binding site of beta-ketoacyl reductase is disrupted at low ionic strength. In contrast, changes in ionic strength have little effect on the enoyl reductase activity. The dimer is stabilized by both electrostatic and hydrophobic interactions, with the former being of special importance for maintenance of the beta-ketoacyl reductase active site. site.     

Influence of inorganic phosphate and organic buffers on cephalosporin production by Streptomyces clavuligerus

A high concentration of potassium phosphate (75–100 mM) stabilized pH and supported extensive growth of Streptomyces clavuligerus in a chemically defined medium; such a concentration also inhibited cephalosporin production. Although Tris buffer was found to have detrimental effects on growth and antibiotic production, 3-(N-morpholino)-propane sulfonate (MOPS) or 2-(N-morpholino)-ethane sulfonate (MES) buffer provided a nontoxic buffering system. In the presence of MOPS buffer, cephalosporin production was optimal at 25 mM phosphate, whereas higher concentrations of phosphate progressively inhibited antibiotic production up to 85% without modifying the pH pattern. MOPS buffer can be used to conduct fermentations at a relatively constant pH value in shake flasks.List of Non-Common Abbreviations MOPS 3-(N-morpholino)propane sulfonic acid - MES 2-(N-morpholino)ethane sulfonic acid     

Histochemical Demonstration of Endogenous Dehydrogenase Systems by Supravital Perfusion

Endogenous dehydrogenase activity is demonstrated in fresh, intact organs by supravital perfusion with a tetrazolium solution. The animal is first injected intravenously with 1.5 mg Heparin/100 gm body weight. It is then anesthetized and a fine polyethylene cannula (PE50, Intramedic) is inserted into a major artery and secured with a ligature. An initial perfusion with warm (37°C) M/20 phosphate buffer (pH 7.6) to remove the blood from the tissues is followed by a 10 min perfusion with the same kind of buffer to which has been added 0.25% neotetrazolium chloride (Dajac Laboratories). The tetrazolium solution is delivered to the tissue at the rate of 1 ml/minute. A final perfusion with 10% formalin in warm phosphate buffer (pH 7.6) flushes and fixes the tissues. Frozen sections can then be cut and mounted in glycerol jelly. Fine, colored formazan crystals are deposited at the sites of enzyme activity. The method is simple and yields excellent histochemical preparations.     

磷酸缓冲液对丁酸梭菌A69酶活力及产氢量的影响

分别在不同浓度pH7.0的磷酸缓冲液中培养Clostridium butyricum A69,随着磷酸盐缓冲液浓度的增高,培养过程中pH值下降减慢,放氢氢酶较长时间地维持在较高的酶活水平上。而吸氢氢酶活力基本保持不变,因而导致氢气产量有较大幅度的提高。在10mmol和70mmol磷酸盐缓冲液中,最终氢气产量几乎相差一倍,说明控制溶液的pH能大幅度地提高氢气产量。     

A spectrophotometric study of the secondary structure of ribonucleic acid based on a method for diminishing single-stranded base-`stacking'' without affecting multihelical structures

1. On the basis of studies with model compounds it was concluded that in 8m-urea-m-potassium chloride (or 4m-guanidinium chloride) in 0.01m-potassium phosphate buffer, pH7.0, multi-helical structures have about the same stability as in 0.1m-potassium phosphate buffer, pH7.0, whereas the tendency of base residues to ;stack' along a single polynucleotide chain is much decreased. 2. Base-pairing was eliminated whereas base-;stacking' persisted after RNA in 1% formaldehyde-0.1m-potassium phosphate buffer, pH7.0, was heated to 95 degrees . 3. From a study of the thermal denaturation of unfractionated transfer RNA from Escherichia coli and of RNA from the fractionated sub-units of rabbit reticulocyte ribosomes in 8m-urea-m-potassium chloride (or 4m-guanidinium chloride) in 0.01m-potassium phosphate buffer, pH7.0, it was inferred that ;stacked' residues may account for up to 25% of the increase in E(260) found on heating RNA in solvents such as 0.1m-potassium phosphate buffer, pH7.0. 4. Changes in the spectrum with temperature were analysed on the basis of the assumptions that (a) the polynucleotide chain is amorphous on denaturation (which is probable in 8m-urea-m-potassium chloride-0.01m-potassium phosphate buffer, pH7.0) and that (b) the polynucleotide chain adopts a single-stranded ;stacked' conformation on denaturation (which is probable when ordinary solvents such as 0.1m-potassium phosphate buffer, pH7.0, are used).     

Phosphoramidate and phosphate prodrugs of (-)-beta-D-(2R,4R)-dioxolane-thymine: synthesis, anti-HIV activity and stability studies

A series of phosphoramidate and phosphate prodrugs of DOT were synthesized via dichlorophosphate or H-phosphonate chemistry and evaluated for their anti-HIV activity against LAI M184V mutants in PBM cells as well as for their cytotoxicity. The antiviral and cytotoxic profiles of the prodrugs were compared with that of the parent compound (DOT), and it was found that four aryl phosphoramidates 5, 18, 20, and 26 showed a significant enhancement (8- to 12-fold) in anti-HIV activity without cytotoxicity. Chemical stability of these prodrugs was evaluated in phosphate buffer at pH values of biological relevance (i.e., pH 2.0 and 7.4). Enzymatic hydrolysis was also studied in esterase or lipase in buffer solution. Chemical stability studies indicate that the phosphoramidates have good chemical stability at pH 2.0 and at pH 7.4 phosphate buffer. Phosphoramidate prodrugs were hydrolyzed in vitro by esterase or lipase and found to be better substrates for lipases than for esterases. 1,3-Diol cyclic phosphates showed potent anti-HIV activity without increasing the cytotoxicity compared with that of DOT and have good chemical and enzymatic stability. Long-chain lipid phosphates, although showed potent anti-HIV activity, exhibited increased cytotoxicity.     

Methods to extract NAD+-malate dehydrogenase efficiently from white spruce needles

The spectrophotometrically-determined activity of NAD⁺-malate dehydrogenase (MDH, EC 1.1.1.37) from white spruce [ Picea glauca (Moench) Voss] needles was assayed with NADH and oxaloacetate. Activity was very low when extracted with only acetate buffer (pH 5.4), phosphate buffer (pH 6.8), or Tris-HCl buffer (pH 8.0). However, activity increased from 1 to over 200 μmol (g dry weight)^-1 min^-1 with the addition of polymers such as polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG) and the detergents, Tween 80, Tergitol 15-S-9 and Triton X-100. Best activity was observed when extracted in a buffer at pH 6.8 and with 1% (v/v) for the three detergents and PEG, and 6% (w/v) for PVP.
MDH activity decreased with age of the needles on the tree. Six-year-old needles contained only about one-fifth of the activity of current year, fully-expanded needles. The main decrease in enzyme activity was observed in one-year-old needles. Protein content obtained from needles extracted with just phosphate buffer (pH 6.8) was very low, but increased greatly when the above chemicals were added to the buffer. In contrast with needles, extracts of vegetative buds contained much higher levels of MDH and protein when extracted with only phosphate buffer (pH 6.8). Although MDH activity in needle extracts declined with storage of the extracts at 4°C in the dark for 6 days, the decrease was least for buffers containing a combination of different protective agents.     

Electrophysiological identification of antennal pH receptors in the ground beetle Pterostichus oblongopunctatus

Abstract. Electrophysiological responses of antennal taste bristles to 100 mm acetate and phosphate buffers were tested at pH 3–11 in the ground beetle Pterostichus oblongopunctatus (F.) (Coleoptera, Carabidae). Additionally, responses of these sensilla to 10 and 100 mm phosphate buffers were compared with each other. Generally, in response to these stimulating solutions, two sensory cells, classified as a salt cell (cation cell) and a pH cell, respectively, showed action potentials distinguished by differences in their amplitudes and polarity of spikes. The firing rate of the cation cell increased with increasing buffer concentration, and was influenced by buffer pH in a complicated way. The best stimulus for the second cell (pH cell) was pH of the stimulating buffer solution. As the pH of the stimulus solution increased, higher rates of firing were produced by the pH cell. For example, the number of action potentials elicited by 100 mm phosphate buffer at pH 11.1 was approximately 16-fold higher compared with that at pH 8.1, and firing rates during the first second of the response were 27.9 and 1.7 imp/s, respectively. The pH cell did not fire or fired at very low frequency (first second response below 5 imp/s) at pH 3–6. This level of acidity probably represents the pH preferences of this ground beetle in its forest habitat and hibernating sites. By contrast to the cation cell, the pH cell responded to increases in buffer concentration by decreasing its firing rate.     

Characterization of a Xenopus laevis skin peptidylglycine alpha-hydroxylating monooxygenase expressed in insect-cell culture.

The C-terminal amide structure of peptide hormones and neurotransmitters is synthesized via a two-step reaction catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N-C lyase. A Xenopus laevis PHM expressed in insect-cell culture by the baculovirus-expression-vector system was purified to homogeneity and characterized. Using a newly established assay system for PHM, the kinetic features of this enzyme were investigated. As expected, the enzyme required copper ions, L-ascorbate and molecular oxygen for turnover. Salts like KI and KCl, and catalase stabilized the enzyme in the presence of L-ascorbate. The optimum pH value for the enzyme reaction was around six when Mes buffer was used and around seven when phosphate buffer was used under the same assay condition. Below pH 6, acetate, iodide and chloride ions activated the reaction. The kinetic analysis is consistent with a ping-pong mechanism with respect to peptide and L-ascorbate, and the peptide showed substrate inhibition. The substrate specificity of the enzyme at the penultimate position was examined by competitive assay using tripeptides with glycine at the C-termini and the inhibitory potency of these peptides in descending order was methionine > aromatic > non-polar amino acids.