Kinetic properties of Helicobacter pylori urease compared with jack bean urease

The urease proteins of the jack bean (Canavalia ensiformis) and Helicobacter pylori are similar in molecular mass when separated by non-denaturing gradient polyacrylamide gel electrophoresis, both having three main forms. The molecular mass of their major protein form is within the range 440-480 kDa with the other two lesser forms at 230-260 kDa and 660-740 kDa. These forms are all urease active; however, significant kinetic differences exist between the H. pylori and jack bean ureases. Jack bean urease has a single pH optimum at 7.4, whereas H. pylori urease has two pH optima of 4.6 and 8.2 in barbitone and phosphate buffers that were capable of spanning the pH range 3 to 10. The H. pylori Km was 0.6 mM at pH 4.6 and 1.0 mM at pH 8.2 in barbitone buffer, greater than 10.0 mM, and 1.1 mM respectively in phosphate buffer and also greater than 10.0 mM in Tris.HCl at pH 8.2. By comparison, the jack bean urease had a Km of 1.3 mM in Tris.HCl under our experimental conditions. The findings show that the urease activity of H. pylori was inhibited at the pH optimum of 4.6 in the phosphate buffer, but not in the barbitone buffer. This was shown to be due to competitive inhibition by the sodium and potassium ions in the phosphate buffer, not the phosphate ions as suggested earlier. Jack bean urease activity was similarly inhibited by phosphate buffer but again due to the effect of sodium and potassium ions.     

Charge heterogeneity of the folate binding protein in normal human leukocytes

The DEAE-Sepharose CL-6B chromatographic profile of supernatant from homogenized normal human leukocytes containing large amounts of folate binder revealed two peaks of binding activity. A minor binder (I) eluted with the equilibrating buffer (1 mM sodium phosphate of pH 6.0), while the major binder (II) first eluted after the initiation of a linear phosphate gradient with 200 mM sodium phosphate of pH 7.6 as the limiting buffer. Binder II was thus a more acidic protein since it required elution by a salt-pH gradient. Binding of [3H] folate to binder II was of a high-affinity type (K = 10(10) M-1) and displayed positive cooperativity.     

Enthalpy of acetylcholine hydrolysis by acetylcholinesterase

Direct microcalorimetric measurements were made of the reaction between acetylcholine chloride and acetylcholinesterase (EC 3.1.1.7) that was extracted from electric eel (Electrophorus electricus) and purified by affinity chromatography. Tris-HCl, sodium phosphate and potassium phosphate were used as buffers and sources of ions for the reaction. At pH 7.2 and in 0.1-0.2 M phosphate buffer, the delta H for acetylcholine hydrolysis was found to be -0.107 kcal/mol (under buffered conditions) and -0.931 kcal/mol under unbuffered conditions (water). At pH 8.0 in 0.1 M Tris-HCl buffer, values greater than -2.5 kcal/mol were obtained, with the highest value of -9.2 kcal/mol being seen with bovine erythrocyte acetylcholinesterase. Tris-HCl buffer at 4 X 10(-2) M enhanced the reaction velocity by 51.2% over that of 4 X 10(-3) M buffer. Enzyme purity, pH and ionic milieu of reaction mixture, and substrate concentration affected the measured delta H value.     

The Donnan effect in tobacco mosaic virus and its components

Osmotic pressure studies were carried on tobacco mosaic virus (TMV) and its components, protein and RNA, as well as on bis(3,3′-aminopropyl)amine, reported to be present in TMV preparations. Solvents were phosphate and barbital buffers at different values of pH and ionic strength. Measurements were made at room temperature. The Donnan effect was exhibited by TMV protein in phosphate buffer of 0.01 ionic strength at pH values ranging between 5.8 and 7.5. The observed values of the Donnan effect at pH 5.8 and 5.97 were in reasonable agreement with theoretical values calculated from the charge obtained by hydrogen ion titration. TMV-RNA in phosphate buffer at pH 7.5 and ionic strength 0.01 did not exhibit more than 1% of the expected Donnan effect. This is explained tentatively as the result of firm binding of metal ions. Negative values of osmotic pressure were observed with bis(3,3′-aminopropyl)amine. Similar anomalous osmosis was sometimes observed with TMV protein and with TMV. In agreement with earlier observations, TMV did not exhibit the Donnan effect in phosphate buffer of 0.01 ionic strength at pH values ranging from 5.5 to 8.0. However, TMV dialysed extensively in the presence of EDTA at pH 8.5 and TMV produced by reconstitution of purified protein and RNA did exhibit the Donnan effect in both phosphate and barbital buffers. The magnitude was of the same order as that calculated from the net charge determined by hydrogen ion titration. When reconstituted TMV, which did exhibit Donnan effect, was treated with calcium ions, the effect was abolished.     

Studies on alkaline phosphatase: Inhibition by phosphate derivatives and the substrate specificity

1. The kinetics of inhibition of calf-intestinal alkaline phosphatase by inorganic phosphate, fluorophosphate, inorganic pyrophosphate, beta-glycerophosphate and adenosine 5'-triphosphate in the range pH8-10 were investigated. The reference substrate was 4-methylumbelliferyl phosphate. 2. The inhibitions were ;mixed' in that both K(m) and V were affected, but the competitive element was by far the stronger. 3. In each case the pH profile for the competitive K(i) was similar to the pH profile for K(m). Since the K(m) and K(i) values both change 100-fold over the pH range 8-10, it is concluded that the inhibitors compete with the substrate for the same active site. 4. It was also found that the enzyme preparation hydrolysed fluorophosphate, pyrophosphate and adenosine 5'-triphosphate as readily as it hydrolysed 4-methylumbelliferyl phosphate and beta-glycerophosphate. Each pH-activity curve, however, had a different shape, but with the exception of pyrophosphate the activity approached the same maximum value at high pH. 5. Attempts to separate the phosphomonoesterase and pyrophosphatase activities by column chromatography were not successful, and the results of other experiments listed suggest that the two activities are a property of the same enzyme. 6. The effect of Mg(2+) ions is briefly mentioned: the phosphomonoesterase activity is enhanced whereas the pyrophosphatase and adenosine triphosphatase activities are strongly inhibited in the presence of excess of Mg(2+) ions.     

The soybean vegetative storage proteins VSP alpha and VSP beta are acid phosphatases active on polyphosphates.

The soybean vegetative storage protein genes (vspA, and vspB) are regulated in a complex manner developmentally and in response to external stimuli such as wounding and water deficit. The proteins accumulate to almost one-half the amount of soluble leaf protein when soybean plants are continually depodded and have been identified as storage proteins because of their abundance and pattern of expression in plant tissues. We have shown that purified VSP homodimers (VSP alpha and VSP beta) and heterodimers (VSP alpha/beta) possess acid phosphatase activity (alpha = 0.3-0.4 units/mg; beta = 2-4 units/mg; alpha/beta = 7-10 units/mg). Specific activities were determined by monitoring o-carboxyphenyl phosphate (0.7 mM) cleavage at pH 5.5 (VSP alpha) or pH 5.0 (VSP alpha/beta and VSP beta) in 0.15 M sodium acetate buffer at 25 degrees C. These enzymes are active over a broad pH range, maintaining greater than 40% of maximal activity from pH 4.0 to 6.5 and having maximal activity at pH 5.0-5.5. They are inactivated by sodium fluoride, sodium molybdate, and heating at 70 degrees C for 10 min. These phosphatases can liberate Pi from several different substrates, including napthyl acid phosphate, carboxyphenyl phosphate, sugar-phosphates, glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, ATP, ADP, PPi, and short chain polyphosphates. VSP alpha/beta cleaved phosphoenolpyruvate, ATP, ADP, PPi, and polyphosphates most efficiently. Apparent Km and Vmax values at 25 degrees C and pH 5.0 were 42 microM and 2.0 mumol/min/mg, 150 microM and 4.2 mumol/min/mg, and 420 microM and 4.1 mumol/min/mg, for tetrapolyphosphate, pyrophosphate, and phosphoenolpyruvate, respectively.     

Effect of microenvironment pH of swellable and erodable buffered matrices on the release characteristics of diclofenac sodium

The aim of this work is to design pH-dependent swellable and erodable-buffered matrices and to study the effect of the microenvironment pH on the release pattern of diclofenac sodium. Buffered matrix tablets containing diclofenac sodium, physically mixed with hydrophilic polymer (hydroxypropyl methylcellulose [HPMC]) and pH-dependent solubility polymer (Eudragit L100-55) were prepared with different microenvironment pHs. The release of diclofenac sodium from the buffer matrices was studied in phosphate buffer solutions of pH 5.9 and 7.4. The swelling and erosion matrices containing only HPMC and Eudragit L100-55 were studied in phosphate buffer solution of pH similar to the microenvironment pHs of the matrices. Drug release from matrices was found to be linear as a function of time. Amount of drug released was found to be higher in the medium of pH 7.4 than that of pH 5.9. The rate of drug release increased with the increase of the microenvironment pH of the matrices as determined from the slope. The pattern of drug release did not change with the change of microenvironment pH. The swelling and erosion occurred simultaneously from matrices made up of HPMC and Eudragit L100-55. Both extent of swelling and erosion increased with increase of the medium pH. It was concluded from this study that changing the pH within the matrix influenced the rate of release of the drug without affecting the release pattern. Fax: Not Forwarded     

COUNTERACTION OF THE INHIBITING EFFECTS OF VARIOUS SUBSTANCES ON NITELLA

When living cells of Nitella are first exposed to (1) phosphate buffer mixture, or (2) phosphoric acid, or (3) hydrochloric acid, or (4) sodium chloride, or (5) sodium borate, and are then placed in a solution of brilliant cresyl blue made up with a borate buffer mixture at pH 7.85, the rate of penetration of the dye into the vacuole is decreased as compared with the rate in the case of cells transferred directly from tap water to the same dye solution. When cells exposed to any one of these solutions are placed in the dye solution made up with phosphate buffer solution at pH 7.85, the rate of penetration of dye into the vacuole is the same as the rate in the case of cells transferred from the tap water to the same dye solution. It is probable that this removal of the inhibiting effect is due primarily to the presence of certain concentration of sodium and potassium ions in the phosphate buffer solution. If a sufficient concentration of sodium ions is added to the dye made up with a borate buffer mixture the inhibiting effect is removed just as it is in the case of the dye made up with the phosphate buffer mixture. The inhibiting effect of some of these substances is found to be removed by the dye containing a sufficient concentration of bivalent cations, or by washing the cells with salts of bivalent cations. The inhibiting effect and its removal are discussed from a theoretical standpoint.     

Alkali cation effect on carbonyl-hemoglobin's and -myoglobin's conformer populations when exposed to freeze-concentration of their phosphate-buffered aqueous solutions

Freeze-concentrated aqueous phosphate-buffered (pH 6.8) solutions of carbonyl-hemoglobin (HbCO) and -myoglobin (MbCO) were investigated by Fourier-transform infrared spectroscopy for the effect of alkali cation on the population of conformers. When using sodium phosphates as buffer components, HbCO was transformed from conformer III (at approximately 1951 cm-1) which is the dominant form at ambient temperatures, into conformer IV (at buffer concentration at a given temperature. The conformational changes started slightly below the temperature where ice began to crystallize and the remaining solution became freeze-concentrated, and they were reversible for HbCO. For MbCO in 0.5 M sodium phosphate buffer solution, however, they were irreversible and MbCO denatured completely. When potassium phosphate salts were used for preparing the buffer at the same pH of 6.8, little or no transformation of conformer III into conformer IV was observed. The conformational changes induced by sodium salts are attributed to a decrease in pH and it is shown by infrared spectroscopy that during freeze concentration drastic changes in composition of the two buffer components H2PO4-/HPO(2)4- occur, the acid component increasing strongly relative to the base component. Supersaturation is also important because change from conformer III to IV requires a minimum concentration of sodium salts: whereas 0.1 M sodium phosphate buffer concentration shows a strong effect, 0.03 M concentration does not and therefore behaves like a potassium phosphate buffer.     

Effect of ions on antibacterial activity of human beta defensin 2

Human beta defensin 2 (HBD-2), the most recently discovered human defensin, has been considered to work as a host defense substance against microbial infection. Using Escherichia coli ATCC 25922, we investigated how some cations and anions influenced the antimicrobial activity of HBD-2. This activity, measured in 10 mM phosphate buffer at a concentration of 20 microg/ml, reduced significantly in the presence of 100 and 150 mM sodium or potassium chloride. The reduction was not significantly different when the total amounts of sodium and potassium ions were equal. The kind and the valence of anions (chlorine and sulfate ions) did not affect the bactericidal activity as long as the concentrations of sodium ions were equal. Divalent ions (calcium and magnesium ions) added to 10 mM of Tris buffer significantly inactivated HBD-2 at much lower concentrations (more than or equal to 0.01 mM and 0.05 mM, respectively) than the monovalent ions did. These findings suggest that HBD-2 kills the bacteria through at least two phases, which are affected independently by either monovalent or divalent ions and unaffected by anions.     

Studies on the thermotropic behavior of aqueous phosphatidylethanolamines

Transport of phosphate has been studied in subconfluent monolayers of LLC-PK1 cells. It was found that this transport system shows similar characteristics to those observed in the kidney. Uptake of phosphate is mediated by a Na+-dependent, substrate-saturable process with an apparent Km value for phosphate of 96 +/- 15 mumol/l. Kinetic analysis of the effect of Na+ indicated that at (pH 7.4) two sodium ions are cotransported with one HOP4(2-) ion (Hill coefficient 1.5) with an apparent Km value for sodium of 56 mmol/l. Pi uptake is inhibited by metabolic inhibitors (ouabain and FCCP). In the pH range of 6.6 of 7.4 Pi uptake rate does not change significantly, indicating that both the monovalent and the divalent form of phosphate are accepted by the transport system. It is suggested that phosphate is transported by LLC-PK1 cells together with sodium (2 Na+:1 HPO4(2-) in an electroneutral manner down a favourable sodium gradient.     

Binding of polycyclic aromatic hydrocarbons to DNA of cells in culture: a rapid method for its analysis using hydroxylapatite column chromatography.

A rapid procedure to study the interaction of carcinogens with DNA in cultured cells has been developed. The cells, which are labeled with 7,12-[3H]dimethylbenz[a] anthracene ([3H]DMBA), are lysed with 0.24 M phosphate buffer (pH 6.8), 1% sodium dodecyl sulfate (SDS), 8 M urea and 0.01 M ethylenediamine-tetraacetate (EDTA) and sonicated. The cell lysates are fractionated on columns of hydroxylapatite. Proteins and RNA are removed with 8 M urea in 0.24 M phosphate buffer (pH 6.8). DMBA-bound DNA is eluted with 0.4 M phosphate buffer (pH 6.8). DMBA-DNA isolated by this procedure is virtually free from proteins and RNA. Thermal stability, ultraviolet spectra and the density of DNA is not altered by DMBA binding. The uptake of DMBA by mouse epidermal cells is rapid and the binding of DMBA to DNA is linear for the first 8 h of exposure. DMBA binds to DNA in all phases of the cell cycle. However, the highest binding occurs immediately following maximum DNA synthesis.     

Proton transport inside the biofilm limits electrical current generation by anode-respiring bacteria

Anode-respiring bacteria (ARB) in a biofilm anode carry out an oxidation half-reaction of organic matter, producing an electrical current from renewable biomass, including wastes. At the same time, ARB produce protons, usually one proton for every electron. Our study shows how current density generated by an acclimated ARB biofilm was limited by proton transport out of the biofilm. We determined that, at high current densities, protons were mainly transported out of the biofilm by protonating the conjugate base of the buffer system; the maximum current generation was directly related to the transport of the buffer, mainly by diffusion, into and out of the biofilm. With non-limiting acetate concentrations, the current density increased with higher buffer concentrations, going from 2.21 +/- 0.02 A m(-2) with 12.5-mM phosphate buffer medium to 9.3 +/- 0.4 A m(-2) using a 100-mM phosphate buffer at a constant anode potential of E(anode) = -0.35 V versus Ag/AgCl. Increasing the concentration of sodium chloride in the medium (0-100 mM) increased current density by only 15%, indicating that ion migration was not as important as diffusion of phosphate inside the biofilm. The current density also varied strongly with medium pH as a result of the buffer speciation: The current density was 10.0 +/- 0.8 A m(-2) at pH 8, and the pH giving one-half the maximum rate was 6.5. A j-V curve analysis using 100 mM phosphate buffer showed a maximum current density of 11.5 +/- 0.9 A m(-2) and half-saturation potential of -0.414 V versus Ag/AgCl, a value that deviated only slightly from the standard acetate potential, resulting in small anode-potential losses. We discuss the implications of the proton-transport limitation in the field of microbial fuel cells and microbial electrolytic cells.     

High-speed gel filtration of polypeptides in sodium dodecyl sulfate

The separation of polypeptides treated with SDS was studied using G3000SW packing prepared from silica for high-speed gel filtration. The peaks of ovalbumin, chymotrypsinogen A, cytochrome c, aprotinin, and insulin B chain were completely separated in the presence of 0.1% SDS and 0.05 M sodium phosphate buffer (pH 7.0). A plot of the logarithm of molecular weight of polypeptides versus Kd was linear over a molecular weight range of 3,000 to 50,000 at the above concentrations of SDS and sodium phosphate. The slopes of the plots of log molecular weight versus Kd depend to a significant extent on the concentration of the sodium phosphate buffer (pH 7.0).     

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。     

Pig kidney phosphofructokinase. Purification to homogeneity and qualitative basic characterization.

Phosphofructokinase has been purified from pig kidney by extraction with phosphate buffer at pH 8, followed by alcohol treatment, affinity chromatography on matrix-bound Cibacron blue F3G-A, and gel chromatography on Sepharose 6B. Using sodium dodecyl sulphate electrophoresis the enzyme was found to be homogeneous and to have a specific activity of about 80 units/mg protein. Like other phosphofructokinases, at pH 7.0 the enzyme exhibits a sigmoidal dependence in its activity on the fructose 6-phosphate concentration and is strongly inhibited by ATP. The degree of citrate inhibition is influenced by the concentration of the two substrates. ATP strengthens and fructose 6-phosphate relieves the inhibition by citrate. AMP and cAMP are able to overcome the ATP inhibition. The ADP activation curve is biphasic. The molecular weight of the subunit of pig kidney phosphofructokinase was determined to be 88 000 by means of sodium dodecyl sulphate electrophoresis.     

Application of ultrafiltration method to measurement of catecholamines in plasma of human and rodents by high-performance liquid chromatography

In order to develop a reliable, simple and routine method using small sample volume to determine norepinephrine (NE) and epinephrine (E) concentrations in plasma of humans and rodents, we utilize the ultrafiltration (UF) method by Ultrafree-MC filter device and a high-performance liquid chromatography equipped with electrochemical detector (HPLC-ECD) to detect NE and E. Optimum UF and HPLC conditions were as follows: the filter nominal molecular weight limit size is 30,000, the pH of added phosphate buffer to each plasma sample for UF is 3.0, and the mobile phase is 0.1M phosphate buffer (pH 3)/acetonitrile (98:2) containing 0.05% sodium disulfite and 0.001% EDTA 2Na. The plasma samples and 1.0M phosphate buffer (pH 3) containing 3,4-dihydroxybenzylamine (DHBA), as an internal standard, was mixed and poured into the UF units. After the centrifugation for 60 min at 13,000 x g at 4 degrees C, the filtrate was directly injected into HPLC. The calibration curve of NE and E was linear for the concentrations studied (20-400 pg) with a correlation coefficient of >0.999. Intra-assay coefficients of variation for NE and E using this method were less than 3%. The method also correlated well with the well-established alumina method (r=0.954). The present findings suggest that a newly-developed UF method with HPLC-ECD would apply successfully to measure plasma NE and E concentrations in humans and rodents.     

Sensitive high-performance liquid chromatographic quantitation of gabapentin in human serum using liquid-liquid extraction and pre-column derivatization with 9-fluorenylmethyl chloroformate

Most of the published methods for analysis of gabapentin, an antiepileptic agent, in human serum are based on the same approach, involving o-phthaldialdehyde derivatization of deproteinized serum samples. The present paper however, describes a new, simple and sensitive high-performance liquid chromatographic method for determination of gabapentin in human serum using liquid-liquid extraction and 9-fluorenylmethyl chloroformate (FMOC-Cl) as pre-column labeling agent. The drug and an internal standard (azithromycin) were extracted from serum by salting-out approach using a mixture of dichloromethane-2 propanol (1:1, v/v) as the extracting solvent. The extracted analytes were subjected to derivatization with FMOC-Cl in the presence of phosphate buffer (pH 7). A mobile phase consisting of methanol-0.05 M sodium phosphate buffer (73/27, v/v; pH of 3.9) containing 1 ml/l triethylamine was eluted and chromatographic separation was performed on a Shimpack CLC-C18 (150 mm x 4.6 mm) column. The standard curve was linear over the range of 0.03-20 microg/ml and limit of quantification was 0.03 microg/ml. The performance of analysis was studied and the validated method showed excellent performance in terms of selectivity, specificity, sensitivity, precision and accuracy. No interferences were found from commonly co-administered antiepileptic agents.     

Effect of depletion of phosphate and bicarbonate ions on insulin action in rat adipocytes

The effect of insulin on rat adipocytes was studied in isotonic buffers (pH 7.4) containing NaCl, CaCl2, MgSO4, KCl, and bovine serum albumin but no phosphate or bicarbonate anions. In phosphate- and bicarbonate-free buffers the dose-response curve to insulin is shifted to the right, the effects of the hormone on hexose uptake, glucose metabolism, and inhibition of lipolysis being observed at much higher (nearly 2 orders of magnitude) concentrations of insulin. The insulin binding capacity of the cells is only slightly changed. The dose-response curve for isoproterenol which stimulates lipolysis in the same cell type is almost the same in both Krebs-Ringer bicarbonate buffer and phosphate- and bicarbonate-free buffers. The dose-response curves for agents that mimic the action of insulin such as wheat germ agglutinin or vanadate ions are also shifted to the right. The dose-response curve to insulin can be returned to "normal" by readdition of either bicarbonate or phosphate. Almost complete recovery is obtained at either 10 mM bicarbonate or 24 mM phosphate, respectively. External Ca2+ ions which are not required for the proper action of insulin in fat cells maintained in Krebs-Ringer bicarbonate buffer, become essential for insulin action in bicarbonate-free buffer. The study indicates that depletion of bicarbonate and, to a lesser extent, phosphate anions, interferes with an essential insulin-dependent post-binding event. Also, in bicarbonate-free medium, external Ca2+ ions are essential for insulin-mediated processes. The implications of this study to the mode of action of insulin, and to physiological and clinical states of insulin desensitization are discussed.     

Buffers for the reconstitution of aspartate aminotransferase

The cofactor activation of the apoenzyme of pig heart cytosolic aspartate aminotransferase was studied in various buffers. Cationic buffers are shown to allow maximal reconstitution in the pH range of 5.0 to 9.0. Anionic buffers made up of mono- and dicarboxylates are found to affect reconstitution in a pH-dependent manner. At low pH, the carboxylates strongly inhibit reconstitution, but at high pH, they show less effect. In contrast, the more potent inhibitor Pi shows the opposite pH profile. Dicarboxylates are considerably more inhibitory than monocarboxylates. Substantial protection against inhibition by a number of carboxylates may be achieved by the addition of sodium chloride.